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LSU Doctoral Dissertations Graduate School

2009

Evidence that the Boundary Element-Associated Factors Evidence that the Boundary Element-Associated Factors

BEAF-32A and BEAF-32B affect chromatin structure in Drosophila BEAF-32A and BEAF-32B affect chromatin structure in Drosophila

melanogaster melanogaster

Matthew Kenneth Gilbert Louisiana State University and Agricultural and Mechanical College

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Recommended Citation Recommended Citation Gilbert, Matthew Kenneth, "Evidence that the Boundary Element-Associated Factors BEAF-32A and BEAF-32B affect chromatin structure in Drosophila melanogaster" (2009). LSU Doctoral Dissertations. 1491. https://digitalcommons.lsu.edu/gradschool_dissertations/1491

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EVIDENCE THAT THE BOUNDARY ELEMENT-ASSOCIATED FACTORS BEAF-32A AND BEAF-32B AFFECT CHROMATIN STRUCTURE IN DROSOPHILA

MELANOGASTER

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Doctor of Philosophy

in

The Department of Biological Sciences

by

Matthew Kenneth Gilbert B.S. Louisiana State University, 2000

May 2009

ii  

Dedication

I dedicate this dissertation to Chandrasekhar Reddy Komma, 1976-2007. Yours would have been a great one, my friend.

iii  

Acknowledgements

I would like to acknowledge and thank my advisor Dr. Craig Hart for his thoughtful and

energetic guidance over the last several years. I would also like to thank Dr. Pat DiMario, Dr.

Mark Batzer, Dr. Randall Hall, and Dr. Marcia Newcomer for there scientific contributions to

my dissertation and personal words of encouragement.

I would like to very much thank Dr. Newcomer in particular, who, during a very difficult

time in my personal life demonstrated compassion, leadership, and sincerity towards me in a

manner that was truly extraordinary. She helped me to heal more then she realizes.

Several scientists have given me opportunities simply out of a desire to appease my

curiosity while knowing that they had little to gain from me scientifically. From these people I

learned to appreciate the importance and responsibility of being a scientific mentor. Dr. Bruce

McEwen and Dr. Chenjian Li literally took me off the streets of New York City and gave me the

undeserved opportunity that started my career. Dr. Thomas Moore and Dr. Richard Bruch at

LSU and Dr. Lawrence Reagan, Dr. Karen Bulloch, and other members of the McEwen have all

been invaluable sources of scientific instruction, friendship and support.

Several undergraduates have contributed to the collection of data in our lab. Alayna

Rockenschuh was exceptional among them for her hours of polytene squashes, general lab help,

and (her most enjoyable contribution) a great sense of humor. In addition, Jordan Roberts did

fantastic work with the fluorescent proteins and Ankit Patel was a nuclei-collecting and data

entry phenomenon. Fellow graduate student Nan Jiang provided much of the ChIP data in

Chapter 3, as well as many doses of fun conversation.

Everything that I have today is because of the love and support my parents have always

shown to me. They taught me that hard work, integrity, loving my family, and faith in God are

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what matters in life. I love them both very much and can’t wait to see what wonderful

grandparents they will be.

Finally, to my wife Kathy and unborn daughter Marley, everything I ever do in life is

because of you. You are my entire world and I love you both more than you could ever know.

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Table of Contents

Dedication………………………………………………………………………………………...ii

Acknowledgements……………………………………………………………………………...iii

Abstract………...……………………………………………………………………………….vii

Chapter 1: Literature Review of Chromatin Structure and Boundary Elements……….…..1 Introduction……………………………………………………………………………….1 Historical Perspective……………………………………………………………………..3 Gene Regulation and Chromatin Structure………………………………………………..5 Higher-Order Chromatin Structure………………………………………………………10 The Domain Model………………………………………………………………………11 The Discovery and Characterization of the Boundary Element-Associated Factors….…14 Models of Insulator Function……………………………………………………….……16 Research Objectives………………………………………………………………….…..23 References……………………………………………………………………………..…25

Chapter 2: The Drosophila Boundary Element-Associated Factors BEAF-32A and BEAF-32B Affect Chromatin Structure ……………………………………………………………...34 Introduction………………………………………………………………………………34 Materials and Methods…………………………………………………………………...36 Results…………………………………………………………………………………....40

Discussion………………………………………………………………………………..52 References………………………………………………………………………………..56

Chapter 3: Analysis of Chromatin Structure in a BEAF Knockout Line…………………..60

Introduction………………………………………………………………………….…..60 Materials and Methods……………………………………………………………….…..62 Results……………………………………………………………………………….…...67

Discussion………………………………………………………………………………..83 References………………………………………………………………………………..88

Chapter 4: Utilizing Fluorescently-Tagged BEAF-32A and BEAF-32B to Analyze BEAF Localization and Dynamics In Vivo ..……………………………………...…..………………92

Introduction………………………………………………………………………………92 Materials and Methods…………………………………………………………………...94 Results……………………………………………………………………………………95

Discussion…..…………………………………..………………………………………..99 References………………………………………………………………………………101

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Chapter 5: Conclusion………………………………………………………………………...103

Appendix: Permission to Include Copyrighted Material…………………………………...108

Vita……………………………………………………………………………………………..118

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Abstract

The Boundary Element-Associated Factors, BEAF-32A and BEAF-32B bind to hundreds

of loci on Drosophila chromosomes. These proteins function as insulators; they can prevent

promoter activation by an enhancer when placed between them and protect transgenes from

chromosomal position effects. To gain insight into BEAF function we designed and expressed a

transgene encoding a dominant-negative form of BEAF. This peptide, BID, consists of the BEAF

self-interaction domain. We demonstrate here that this peptide interferes with BEAF’s ability to

bind DNA and prevents it from functioning as an insulator. In addition, expression of BID leads

to a global disruption of polytene chromosome structure. Subsequent work using a fly line with a

null mutation in the BEAF gene (BEAFAB-KO) also demonstrates a perturbation to polytene

chromosome structure, although it is limited to the X-chromosome. Using Micrococcal nuclease

and DNase I we analyzed hypersensitive site alterations in the BEAFAB-KO line, and observed

alterations that are consistent with the shifting of positioned nucleosomes. This effect appears

limited to regions near promoters. Finally, using fluorescently-tagged BEAF-32A and BEAF-

32B we attempt to characterize the localization and behavior of these proteins. We find that they

localize very differently on polytene chromosomes, that BEAF-32B disassociates from mitotic

chromosomes while BEAF-32A remains associated, and FRAP experiments indicate different

recovery dynamics. This data is consistent with a model that BEAF-dependent insulators

function by affecting chromatin structure or dynamics.

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Chapter 1: Literature Review of Chromatin Structure and Boundary Elements

Introduction

The extensive amount of DNA found inside a somatic nucleus requires complex and

highly structured packaging and compaction of the chromatin. This must be maintained and

organized such that genes remain

accessible to regulatory proteins and

respond quickly to activation or

silencing. Early models have proposed

that one component of this organization

is comprised of boundary elements;

nucleoprotein complexes periodically

placed in the genome that function to

divide the genome into independently

regulated domains (UDVARDY et al. 1985; WEISBROD 1982). This model was supported by early

evidence that regulatory elements in one “domain” could not influence expression from a

transgene in a neighboring domain when a known boundary element was placed between them

(KELLUM and SCHEDL 1992; ROSEMAN et al. 1993).

Currently much effort is aimed at determining how boundary elements function in vivo.

As will be discussed in more detail, models of boundary element function range from dramatic,

such as the formation of chromatin into large 3-dimensional loop structures throughout the

genome (BYRD and CORCES 2003) and the use of alternative DNA secondary structures (WONG

et al. 2007) to more subtle models, such as the use of covalent histone modifications (EMBERLY

et al. 2008) or the incorporation of histone variants into chromatin (NAKAYAMA et al. 2007).

While all of these models are useful for consideration, it is likely that insulators function via a

Figure 1-1 The Boundary Element-Associated FactorsBEAF-32A and BEAF-32B (both labeled red) bind tohundreds of sites on polytene chromosomes (DNA labeledblue).

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variety of mechanisms, perhaps in very distinct manners from each other. Our goal here is to

describe the discovery and relevant data regarding how the Boundary Element-Associated

Factors BEAF-32A and BEAF-32B function, and discuss them in the context of existing models

for boundary element function.

BEAF-32A and BEAF-32B are functionally-defined boundary element binding proteins

(also called insulators) found in Drosophila melanogaster. They are localized to several hundred

sites on interphase polytene chromosomes (Figure 1-1) and are ubiquitously expressed

throughout all tissues and developmental stages examined (unpublished data). The DNA

sequences that BEAF-32A and BEAF-32B recognize function as insulators; they can prevent an

enhancer from activating a promoter when located in between, and when bracketing a transgene,

they can insulate it from repressive chromatin effects or positive regulatory sequences.

Previous work on the BEAF proteins focused on characterizing the sequences that they

bind to in vivo and the consequences of their binding on gene expression. It has been established

that BEAF proteins bind to a repeated CGATA motif and other uncharacterized sequences, and

the BEAF proteins interact with each other. It is also known that BEAF can protect a promoter

from nearby regulatory or positional effects and that this has implications for genome-wide

patterns of gene expression (HART et al. 1997; KELLUM and SCHEDL 1992; ROY et al. 2007b;

ZHAO et al. 1995). Here, we intend to contribute evidence for a model aimed at describing a

mechanism of how the BEAF proteins might perform these functions in vivo. In 2004, at the start

of this research, there was no available model system with mutant BEAF alleles to facilitate these

studies. Because of this, as will be described in Chapter 2, we expressed a truncated form of the

BEAF proteins designed to interact with the endogenous BEAF self-interaction domain, forming

complexes lacking one or more DNA binding domains and diminishing its DNA-binding

capacity. We demonstrated that the expression of this BEAF-interaction domain (BID) prevents

3  

BEAF from binding to DNA and prevents BEAF-dependent insulators from functioning as

boundary elements. Importantly, the expression of BID had a severe effect on the morphology of

polytene chromosome structure. In subsequent publications this recombinant protein provided a

useful tool for studying BEAF interactions in vivo (ROY et al. 2007b) and was used in a

publication attempting to define a characteristic BEAF-binding site (EMBERLY et al. 2008).

In 2006 a fly line was constructed in our lab with null mutations in the beaf gene that

precludes the translation of a functional protein (ROY et al. 2007a). Here, Chapter 3 will focus on

characterizing the chromatin structure at putative BEAF-binding sites in this beaf knockout line

(BEAFAB-KO). We compare DNase I and Micrococcal nuclease sensitivity in wildtype and

BEAFAB-KO and propose a model that differs from previous speculation that BEAF functions by

forming loop structures. Rather, it is possible that BEAF may function by regulating chromatin

structure by influencing nucleosome dynamics and, perhaps indirectly, histone modifications.

Furthermore, rather than supposing (as earlier models have) that insulators are passive

nucleoprotein complexes that are periodically positioned to “divide the genome”, it may be more

accurate to consider them as active components of the transcription apparatus that participate in

the complex remodeling processes occurring at transcription start sites.

Finally, Chapter 4 will discuss our use of fluorescently tagged BEAF-32A-GFP and

BEAF-32B-RFP proteins to partially characterize BEAF protein dynamics in vivo. The two

proteins demonstrate surprisingly different patterns of localization and dynamics on polytene

chromosomes, implicating that they have related, yet distinct roles in regulating gene expression.

Historical Perspective

The foundations of modern cellular and molecular biology were established during a

scientific renaissance in the late 19th century. It was during this time that Theodor Schwann and

Matthias Schleiden established our modern cell theory - that the cell is the fundamental unit of an

4  

organism. Louis Pasteur’s simple broth experiments disproved the theory of spontaneous

generation and demonstrated that life must come from life. It was also during this time that

Charles Darwin’s voyage on the Beagle inspired his famous theory of evolution by natural

selection, and in 1865 Gregor Mendel demonstrated his evidence of inheritable “elements” being

passed along generations in a predictable manner, although these experiments would not be truly

appreciated for another 35 years (DAHM 2005); (MENDEL 1866).

It was also during this time that a young Friedrich Miescher working at his first

postdoctoral position in Germany described the presence of a phosphorous-rich substance that he

had isolated from the nuclei of leukocytes (OLINS and OLINS 2003). Miescher had the simple and

perhaps ambiguous goal of determining the basic chemistry of the cell. Utilizing the unfortunate

source material of pus-soaked gauze bandages from a nearby surgical clinic, Miescher

empirically devised methods for obtaining purified leukocyte nuclei. His most successful

protocol involved digesting his leukocytes with pepsin and gentle washing with alcohol to

remove cytosolic lipids until he determined microscopically to have obtained pure nuclei.

Treating the nuclei with a hypotonic salt solution, then acetic acid, he obtained, according to his

own description, “precipitates that were insoluble in water…. consequently, could not belong to

any of the known albuminoid substances. Where did this substance come from?” Subsequent

analysis revealed that Miescher had successfully completed the first known DNA precipitation

(DAHM 2005; MORRISON and WEISS 2006). Later, W. Flemming, inspired by the work of

Miescher, stained nuclei with dyes and termed the “substance in the cell nucleus which is

readily stained” as “chromatin”, from the Latin for “colored things” (OLINS and OLINS 2003).

Subsequent findings during the early 20th century by Avery et al. in 1944 and the famous 1952

Hershey-Chase experiments made it apparent that deoxyribonucleic acid is the transferable,

inheritable, colored component of the cell (AVERY et al. 1944; HERSHEY and CHASE 1952).

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Gene Regulation and Chromatin Structure

The fundamental unit of DNA structure, the nucleosome, was probably first discovered

using electron microscopy as early as 1973 by Christopher Woodcock (WOODCOCK et al. 1976)

(OLINS and OLINS 2003), although these and other early micrographs were frequently greeted

with great criticism (SWIFT 1974). Interestingly, Woodcock’s initial proposal that the

chromosome is comprised of a “self-assembling 70A unit” was rejected from publication in

Nature because as one reviewer stated, it was “naïve” in its simplicity and “would necessitate

rewriting the textbooks” (OLINS and OLINS 2003). Regardless, electron micrographs (EVERID et

al. 1970; OLINS and OLINS 1972) and other biochemical studies (KORNBERG and THOMAS 1974;

SAHASRABUDDHE and VAN HOLDE 1974) in the 1970’s revealed the basic repeating structure of

chromatin to be the nucleosome. The nucleosome is comprised of 146 base pairs of double-

stranded DNA wrapped around pairs of H2A/H2B dimers and H3/H4 dimers (LUGER 2006;

THOMAS and KORNBERG 1975). This octamer of proteins together with DNA forms the core

nucleosome. The amino-terminal tails of the histone proteins protrude from the interior of the

nucleosome and are subject to being post-translationally modified by specific enzymes. Lysine

residues can be ubiquitinated, SUMOylated, acetylated and methylated, serine and threonine

residues can be phosphorylated and arginine residues can be methylated. A feature of methyl

additions is that multiple additions may occur to a single residue. For example, lysine 9 of

Histone 3 may be mono-, di-, or tri- methylated, although the ramifications of this are still

largely unknown. As will be discussed below, these modifications then serve to regulate gene

transcription through a variety of mechanisms.

There are two generally agreed upon descriptions of how histone modifications affect

gene expression. The first model suggests that these modifications alter the electrostatic

properties of the histone tails, thus either moderating its affinity for and positioning within the

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attached DNA or moderating nucleosome-nucleosome interactions (LANDSBERGER and WOLFFE

2001). There are several instances where this has been demonstrated, one example being the

small 5S rRNA genes. This gene has a positioned nucleosome that covers the transcription start

site and the promoter sequence that binds the general transcription factor TFIIIA. Acetylation of

the nucleosome is thought to sufficiently weaken the DNA-nucleosome interaction to allow

TFIIIA binding, in turn recruiting additional transcription proteins (SIMPSON and STAFFORD

1983). Another example is the hsp26 gene in Drosophila, noted as having the “first positioned

nucleosome” (LANDSBERGER and WOLFFE 2001). Precise positioning of this nucleosome in the

5’ regulatory region of the gene is influenced by the GAGA-binding sites that bracket the

nucleosome. When proper positioning occurs distal regulatory elements are brought into the

correct orientation with proximal elements to then facilitate the initiation of transcription

(THOMAS and ELGIN 1988). It has been suggested that the heat-shock induction mechanism at

this locus is dependent on subtle perturbations in nucleosome-DNA interactions caused by the

acetylation of histone 3, lysine 9. More specifically, this perturbation caused by the modification

allows additional transcription factors to bind the TATA box to initiate gene induction (ZHAO et

al. 2007).

Another model, originally termed the “histone code hypothesis” states that the

modifications also serve as binding sites for gene regulatory proteins. With the discovery and

prevalence of chromodomains and bromodomains this model has become widely accepted

(STRAHL and ALLIS 2000). The chromodomain consists of approximately 60 amino acids and

depending on which protein it is associated with can bind selectively to specific methylated

lysines. For example, if it is part of heterochromatin protein 1 (HP1), then it binds to methylated

histone 3, lysine 9 (TSCHIERSCH et al. 1994). It is thought that HP1 then recruits the histone

methyltransferase Su(Var)3-9, resulting in more methylation, a repetition of which results in the

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active spreading of heterochromatin (LOMBERK et al. 2006; PARO and HOGNESS 1991). In

contrast, if the chromodomain is part of the polycomb group complex protein Pc, then it

recognizes methylated histone 3 lysine 27. This complex is important for silencing genes such as

the developmentally relevant HOX class of genes (SCHWARTZ and PIRROTTA 2007). These

examples illustrate instances of a chromodomain binding to methylated lysines as part of a

repressive mechanism, however chromodomains have been found in a wide array of complexes,

including gene stimulatory proteins such as the histone aceyltransferase MOF (HILFIKER et al.

1997), and ATP-dependent histone remodeling complexes such as the chromodomain helicase

DNA binding family (EISSENBERG 2001; MARFELLA and IMBALZANO 2007).

The bromodomain, alternatively, binds to acetylated lysines and are found in nearly all

histone acetyltransferases (HATS) in higher eukaryotes (ZENG and ZHOU 2002). This domain

was originally discovered in the protein brahma, a subunit of the ATP-dependent nucleosome

remodeling complex, BRM. Since acetylated lysines are generally found in regions of active

transcription, it is not surprising that complexes with bromodomains are also associated with

transcriptional activation. Brahma, for example, has been shown to localize with RNA

polymerase on polytene chromosomes (ARMSTRONG et al. 2002) and is necessary for the

transcription of several developmentally important genes (VAZQUEZ et al. 2008). Other notable

examples of bromodomain-containing transcription complexes include the SAGA, Gcn5, and

RISC complexes in yeast, TATA-associated factor 250 in humans, and brahma and TBP-

associated factor (a subunit of TFIID) in Drosophila (FLAUS and OWEN-HUGHES 2004).

As already mentioned, nucleosome remodeling complexes are ATP-dependent enzymes

that either displace or disrupt nucleosomes as part of a gene regulatory mechanism. The

complexes purified to date have been classified according to the ATPase domain sequence into 4

main families: the SWI/SNF family, the ISWI family, the INO80 family and the CHD/Mi-2

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family. The SWI/SNF family in Drosophila consists of 2 distinct complexes, both of which share

the ATPase protein brahma. They share many other subunits as part of the complex, but differ in

their localization on polytene chromosomes suggesting they regulate their own subsets of genes

(MOHRMANN et al. 2004). The ISWI-containing complexes in Drosophila are comprised of

NURF (Nucleosome remodeling factor), CHRAC (chromatin accessibility complex), and ACF

(ATP-utilizing chromatin assembly and remodeling factor). The Mi-2/CHD family contains a

unique ATPase remodeling subunit and subunits responsible for histone deacetylase activity. The

basic model proposed for the Mi-2/CHD family is that remodeling occurs by these complexes to

provide access for the repressive modification of histones to occur (TONG et al. 1998; ZHANG et

al. 1999). The INO80 family has been studied primarily in the context of nucleosome remodeling

during double strand break repair processes

(OSLEY and SHEN 2006). Finally, and perhaps

not surprisingly, there are also prokaryotic

homologs of the SWI/SNF family

(SUKHODOLETS and JIN 1998).

The molecular mechanisms describing

how these enzymes function have not been

fully determined. Both the ISWI and

SWI/SNF complexes are able to disrupt DNA

phasing on the nucleosome thereby affecting

DNA-histone interactions. However, the

SWI/SNF complex is able to bind naked DNA with high affinity whereas the ISWI complex is

dependent on intact nucleosomes for binding (COTE et al. 1994; GEORGEL et al. 1997). A

beautiful set of experiments including electron micrographs published in 1999 of SWI/SNF

Figure 1-2 An electron spectroscopic image of anucleosome and SWI/SNF remodeling complex(black) interacting to form a loop of DNA. Inset inupper right depicts a drawing of the structure. Themagenta dots are a superimposed phosphourousimage. (Used with permission; BAZETT-JONES etal. 1999)

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complexes revealed that this complex can bind to DNA simultaneously in two distant locations

forming large or small loops of chromatin ranging from a few base pairs to >200bp (Figure 1-2)

(BAZETT-JONES et al. 1999). Another observation was that one of the loops of DNA that was

wrapped around the histone core may come off, and other structures resembled DNA that is

“peeling off” of the nucleosomes. In any case, additional experiments revealed that the histones

remain intact. Furthermore, a GAL4 binding site was present and it was demonstrated by DNase

I digestion that the addition of the GAL4 proteins was a requirement for the remodeling of

nucleosomes to occur (BAZETT-JONES et al. 1999).

How, then, are transcription factors, histone modifications, chromatin remodeling

complexes, nucleosome positions, and other proteins coordinated to regulate transcription?

While the examples seem as numerous as there are genes, some classic paradigms have been

established. One example is at the PHO5 gene in yeast, where the remodeling of 4 positioned

nucleosomes precedes activation of the gene (ALMER and HORZ 1986). Under repressive

environmental conditions these 4 nucleosomes span the promoter region, covering the binding

site for the transcription factor TFIID and other regulatory proteins (BARBARIC et al. 1996). The

nucleosomes immediately adjoining the promoter are enriched in acetylated lysines, however the

inclusion of the TFIID binding sites in the nucleosome helps maintain the gene in a repressed

state. Upon the induction of transcription a nucleosomal shift of 2-3 base pairs exposes the

TATA box, thus permitting the binding of TFIID and acetylated lysines become enriched down

the length of the gene (MARTINEZ-CAMPA et al. 2004). It is also worth mentioning that many of

the details of this mechanism, despite careful scrutiny for nearly 30 years have yet to be

determined. For example, data indicates that there are several independent pathways for

transcription to be induced at this promoter, with some depending on the acetylation of lysines

on histone 4 and the bromodomain containing factor bdf1 to recruit TFIID, and other

10  

mechanisms being independent of these factors (MARTINEZ-CAMPA et al. 2004). More recently a

genetic screen that methodically deleted yeast remodeling complexes has verified this; that the

displacement of nucleosomes in this promoter occurs via a variety of mechanisms (BARBARIC et

al. 2007).

Another example of nucleosome remodeling is the activity of NURF that has been

demonstrated at the hsp70 promoter and at the GAL4 trascription binding sites in yeast.

Evidence has suggested that site specific transcription factors, GAGA factor and HSF in the case

of hsp70, and GAL4 in the case of the GAL4-E4 promoter, behave in a cooperative manner with

the NURF remodeling complex to reposition nucleosomes away from the promoter region

(HAMICHE et al. 1999). In the case of GAL4, in vitro nucleosome remodeling experiments have

demonstrated that the presence of the transcription factor can direct NURF to “slide” the

nucleosome to provide access to the upstream activating sequence (KANG et al. 2002). In

summary, the significant observations from these experiments that are relevant for our purposes

is that chromatin remodeling is often dependent on the presence of site-specific transcription

factors for their proper activity, that the remodeling often consists of very subtle (often only a

few base pairs) alterations to DNA-histone interactions, and these alterations are detectable by

nuclease sensitivity.

Higher-Order Chromatin Structure

The compaction afforded DNA by means of incorporation into nucleosomes cannot

account for the degree of compaction that must occur in vivo, thus considerable efforts have been

focused on defining “higher orders” of chromatin structure (VAN HOLDE and ZLATANOVA 1995).

It has become widely accepted that the nucleosomal array can further coil into a 30 nm fiber,

although the details of this structure are not yet resolved. Some evidence advocates that

chromatin assumes a simple tube-shaped helical structure (FINCH and KLUG 1976; RATTNER and

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HAMKALO 1979; THOMA et al. 1979), while others advocate a more complex zigzag structure

(WOODCOCK et al. 1984; WORCEL et al. 1981). More recent electron microscopy (HOROWITZ et

al. 1994; MCDOWALL et al. 1986), X-ray scattering data (WIDOM and KLUG 1985), and

theoretical models (KOCH 1989) advocate for a “roughly” helical, more irregular structure that is

tightly compacted but not helical in a repetitive pattern (VAN HOLDE and ZLATANOVA 1995).

The formation of chromatin into large looped structures, anchored to a nuclear protein

scaffold by sequences called “scaffold attachment regions” or SARs, have been implicated as an

additional order of structure (HART and LAEMMLI 1998; MIRKOVITCH et al. 1984). The

eukaryotic nucleus lacks a regular array of scaffold-specific structures to mediate dynamic

chromatin and protein activities, such as actin filaments or microtubules. (An exception to this is

lamin, which primarily localizes to the surface of the inner nuclear membrane.) Rather, evidence

suggests that scaffold attachment regions (SARS) of A-T rich DNA associate with a largely

uncharacterized complex of RNA and proteins. These proteins include Histone H1,

topoisomerase II, and the matrins family of proteins, but may differ depending on the isolation

protocol used. Thus, the components and even the acceptance of this biochemical fraction as a

“structure” remains controversial (NAKAYASU and BEREZNEY 1991; NICKERSON 2001;

PEDERSON 2000). However, the regulation of SAR-associated proteins represents another order

of chromatin dynamics (HENG et al. 2004), and some evidence exists that insulator proteins may

associate with this nuclear matrix (ISHII et al. 2002; NABIROCHKIN et al. 1998; YUSUFZAI and

FELSENFELD 2004). As will be discussed later, BEAF-32B has been isolated with other SAR

proteins, implicating yet another possible mechanism of insulator function. (PATHAK et al. 2007).

The Domain Model

Despite the microscopic and biochemical evidence for the existence of 30 nm solenoid

fibers and nuclear matrix-associated loops, it remains necessary to explain even higher orders of

12  

chromatin organization. The domain model describes a scenario by which 30 nm solenoid fibers

are divided into domains, or chromomeres, by intermittently spaced nucleoprotein complexes.

These nucleoprotein complexes would then be anchored to the nuclear matrix or to an undefined

protein complex comprising the chromosomal scaffold (WEISBROD 1982). Furthermore, these

domains have been described as defining functional “genetic units” which could be exclusively

decondensed when transcriptionally activated (UDVARDY et al. 1985; WEISBROD 1982).

In a 1985 publication, A. Udvardy, working under Paul Schedl at Princeton University

attempted to identify the borders of a putative chromomere domain. The region they selected for

study was the hsp70 heat shock locus in Drosophila. This region is comprised of two divergently

transcribed genes that can be activated by heat-induced stress to Drosophila larvae, forming a

distinct and characteristic “puffing” in polytene chromosomes. The cytological location and

activation of these genes had been well characterized (GAUSZ et al. 1981; LEIGH BROWN and

ISH-HOROWICZ 1981; MILLER and ELGIN 1981), and the unique morphology of the activated

gene suggested to Schedl and his colleagues that this region likely adheres to the hypothesized

domain model.

Using in situ hybridization with a combination of probes at varying distances from the

promoters of the hsp70 transcribed genes they measured the distance between probes with and

without puff formation. They concluded, first, that upon heat shock induction the puffed region

of DNA decompacts to equal the length of a single nucleosomal array, and further speculated

that a “specialized chromatin structure” must flank the outside of the hsp70 puffed region and

functionally define the borders of the puffed region. Using cell culture and embryonic extracts

they then conducted a series of indirect end-labeling experiments examining the hsp70 locus

using a variety of nucleases. They examined the region with and without heat shock treatment

and after salt treatment. The results of these experiments showed two distinct regions found

13  

outside the hsp70 genes, each characterized as having two nuclease-sensitive regions

surrounding a nuclease resistant region. They show that the presence of these hypersensitive

regions are dependent on the presence of proteins and that subtle alterations occur to these

hypersensitive sites upon the induction of heat shock. The results indicated that two dynamic

nucleoprotein complexes exist that define the “boundaries” of the activated region. They named

these two complexes scs and scs’, acronyms for specialized chromatin structures. Descriptions of

exactly how these complexes function or which proteins were involved remained a matter of

speculation.

Subsequent publications, also by members of the Schedl lab, demonstrated that the scs

and scs’ sequences could function in regulating gene transcription. They reasoned that a

nucleoprotein complex functioning to separate domains should be expected to insulate gene

elements in one domain from the effects of regulatory elements in neighboring domains. In 1991

Kellum and Schedl devised a “Position-Independent Expression” assay in adult Drosophila to

demonstrate this (KELLUM and SCHEDL 1991). Using transgenic fly lines they reported that when

scs and scs’ bracket a reporter gene that expression from that gene is not influenced by

regulatory elements near the site of transgene insertion. It was further demonstrated that this

insulating effect can prevent the silencing of a promoter-gene construct and also prevent the

activation of a promotor-less construct.

A subsequent publication by Kellum and Schedl using a lacZ reporter gene also

expressed in Drosophila adult flies demonstrated that the presence of the scs or scs’ sequence

between an enhancer and lacZ promoter blocked activation of the lacZ transcript. These two

assays, position-independent expression and enhancer blocking, are the two main assays used to

detect boundary element function. A number of additional experiments clarified that the

14  

observed effects were not due to silencer or promoter interference activity, were not promoter

specific, and were not an artifact of spacing between the elements (KELLUM and SCHEDL 1992).

The Discovery and Characterization of the Boundary Element-Associated Factors

In 1995 Keji Zhao and Craig Hart of the Laemmli lab purified an scs’ binding factor and

identified the motifs within scs’ that are primarily responsible for both protein binding and the

enhancer blocking function of scs’ (ZHAO et al. 1995). Gel shift and footprinting analysis

revealed a fragment of scs’ comprised of three copies of a CGATA motif as binding with high-

affinity to a protein complex the authors termed the BEAF (Boundary Element-Associated

Factor) Complex. An additional cluster of three CGATA motifs is found 200 bp upstream of the

high affinity site where the BEAF complex binds with a lower affinity. Enhancer blocking assays

utilizing chromosomally integrated constructs demonstrated that the CGATA motifs are needed

for boundary element function, and significantly, that enhancer blocking activity does not occur

in transient transfection assays. This suggests that scs’ enhancer blocking activity involves

chromatin structure or dynamics as the expression vector in transient assays does not form a

regular nucleosomal array.

Using this newly identified BEAF binding sequence, DNA-affinity chromatography was

used to purify BEAF. Peptide fragments of BEAF purified from nuclear extracts were sequenced

and low-degeneracy probes were constructed against the peptides to screen an embryonic cDNA

library. They identified a cDNA clone encoding a novel 31.6 kDa protein, termed BEAF-32, that

demonstrated the same migration on SDS-PAGE gels, gel shift mobility, and immunoreactivity

as the previously observed BEAF complex. The authors concluded that BEAF-32 is one

component of the BEAF complex, although subsequent results implicated the presence of

additional proteins.

15  

Zhao and Hart also examined BEAF immunoreactivity on polytene chromosomes from

Drosophila third instar larvae. Polytenized chromatin is DNA that has undergone several rounds

of replication without mitotic division. In healthy organisms this results in approximately 1000

copies of DNA arranged side-by-side which allows relatively easy microscopic visualization of

the DNA at readily available magnifications. Polytene chromosomes in Drosophila exhibit

intermittent and alternating regions of condensed, darkly stained regions termed “bands” and

decondensed, lightly stained regions termed “interbands”. Bands represent condensed,

chromatin-rich regions and interbands are regions of extended, open chromatin. Immunostaining

these chromosomes with an anti-BEAF antibody revealed that BEAF is primarily restricted to

the interband regions, with some staining appearing on the borders of the band/interband regions

and at the borders of “puffed regions” such as scs’ after heat shock (ZHAO et al. 1995).

DNase I footprinting experiments indicated that other components of the BEAF complex

remained to be determined. Furthermore, these experiments had demonstrated that the BEAF

complex binds to both single and palindromic CGATA motifs found at scs’. In contrast, the same

experiments with bacterially expressed BEAF-32 showed that it does not bind to the palindromic

repeat and uniquely binds to a direct repeat of TCACG separated by 40-50 bp. In 1997 Hart et al.

described re-screening an embryonic library and identifying several clones that were partially

homologous to the BEAF-32 clone (HART et al. 1997). They identified another 32kDA protein

that differs from the original BEAF-32 only in the 80 N-terminal amino acids. The initial protein

was renamed BEAF-32A and the second protein was named BEAF-32B. It was subsequently

determined that that both proteins are derived from the same gene, presumably by alternate

promoters. Immunoprecipitation from cell extracts has demonstrated that BEAF-32A (herein

called 32A) and BEAF-32B (herein called 32B) proteins interact with each other. This

interaction can alter the DNA-binding affinity of the complex such that there are subtle

16  

differences in the gel shift of the high affinity binding site when bound to 32A, 32B or nuclear

extract heterocomplexes. A 1998 study further verified the importance of clustered CGATA

motifs when several additional genomic sequences identified as BEAF-32B target sequences also

contained this sequence (CUVIER et al. 1998).

Three domains were defined in the BEAF proteins and the N-terminal domain

differentiates these two proteins. Gel-shift analysis and DNase I footprinting has established the

N-terminal domains are responsible for DNA binding. The shared C-terminal domain is

comprised of a putative leucine zipper domain and BESS domain. Yeast-two hybrid experiments

and co-immunoprecipitation assays with truncated 32B constructs has demonstrated that the C-

terminal domain is responsible for 32A-32B interactions in vivo (HART et al. 1997). It has also

been shown in yeast reporter assays that the C-terminal portion alone is sufficient for protecting

genes against the spreading and silencing of heterochromatin (ISHII et al. 2002; ISHII and

LAEMMLI 2003). Finally, deletion of the center portion of the protein (between the C-terminal

and N-terminal domains) has no discernable effect on either heterocomplex formation or DNA

binding (HART et al. 1997). However, it has been reported that in yeast reporter assays the

middle domain of BEAF, when bound to DNA without other portions of BEAF, may act as a

transcription activator (ISHII et al. 2002; ISHII and LAEMMLI 2003).

Models of Insulator Function

Boundary elements have traditionally been defined by having two characteristics; they

interfere with enhancer activation of a promoter when placed between the enhancer and

promoter, and they protect transgenes from chromosomal position effects (ISHII and LAEMMLI

2003; LABRADOR and CORCES 2002). While it has been demonstrated that BEAF fulfills the

above criteria (CUVIER et al. 2002; GILBERT et al. 2006; HART et al. 1997; ROY et al. 2007a), a

comprehensive description of how BEAF mediates these functions has remained elusive. In

17  

order to create a model for BEAF it is necessary to explore both general models that describe

other boundary element mechanisms and the available evidence regarding BEAF function. It is

possible, perhaps likely, that various components from these “distinct” models will eventually be

needed to accurately describe BEAF function.

According to U. Laemmli, descriptions of boundary element function can be grouped into

two general non-exclusive models (ISHII and LAEMMLI 2003). The first is a “dynamic-

modifying” model by which boundary elements act by mediating an active process such as

histone modifications or transcriptional processes to either enhance or suppress gene expression.

Notable evidence of these processes include work by D. Donze in yeast whereby function of the

HMR tRNAThr boundary is affected by mutations in the SAS2 or GCN5 genes encoding histone

acetyltransferases and the Sir2 gene encoding a histone deacetyltransferase (DONZE et al. 1999;

DONZE and KAMAKAKA 2001). In this region the active spreading of heterochromatin from the

HMR locus is stopped by the acetylation and activation occurring at the neighboring tRNAthr

gene. A similar observation is made at the subtelomeric anti-silencing regions, where

transcription factor binding infers barrier activity, even without transcription (FOUREL et al.

2001). In both cases it is possible that a dynamic equilibrium between acetylation and

methylation may occur to prevent the spreading of heterochromatin (DORMAN et al. 2007). A

variation on this theme is the insulator protein CTF-1, which contains a H3 binding domain

sufficient for conferring insulator activity. In yeast this protein binds to histone H3, preventing

enzymes from associating with it (FERRARI et al. 2004). Gene activation by histone acetylation

and repression by histone methylation has also been implicated as a boundary element controlled

mechanism in mammals at the chicken β-globin locus (LITT et al. 2001a; LITT et al. 2001b).

Other dynamic chromatin processes associated with boundary element function include

the removal and replacement (turnover) of the entire Histone H3 protein with the Histone variant

18  

H3.3. H3.3 has been shown to incorporate preferentially into active genes in a transcription-

dependent process (SCHWARTZ and AHMAD 2005), and is more highly associated with active

chromatin marks than the H3 histone (MCKITTRICK et al. 2004). One model of a boundary

element mechanism describes this H3.3 turnover event as a barrier that maintains active

chromatin marks at a specific nucleosome, therefore preventing centromeric heterochromatin

from spreading into regions of active euchromatin and silencing reporter genes. Another example

of H3.3 turnover has been observed at the Drosophila Fab-7 boundary elements. In Drosophila

the three genes of the bithorax complex dictate parasegment development in adult Drosophila.

The expression of these genes is controlled by an upstream regulatory region consisting of nine

regulatory sub-regions, and the regulatory-gene interactions are moderated by a complex

interaction of boundary elements (MAEDA and KARCH 2006). The boundary elements within this

region that contain a GAGA binding site have been found to utilize the H3.3 turnover

mechanism (NAKAYAMA et al. 2007).

A final example of chromatin-dependent insulator function is the case of CCCTC-binding

factor (CTCF). The CTCF-dependent insulators in mammals at many loci has been shown to

depend on the helicase protein CHD8 (ISHIHARA et al. 2006), a SNF2-like ATPase/Helicase and

chromodomain protein that is thought to function by mediating DNA-nucleosome interactions

(ITO et al. 1997; WOODAGE et al. 1997). The lack of CHD8 has been shown to increase CpG

methylation and decrease histone acetylation at regions near CTCF binding sites, and a

knockdown of CHD8 results in a loss of CTCF enhancer blocking function (ISHIHARA et al.

2006). The D. melanogaster homologue of CTCF (dCTCF) was found to associate with one of

the boundary elements in the bithorax complex described above and contribute to its insulator

function (GERASIMOVA et al. 2007).

19  

Another model to describe insulators involving the interference of transcriptional

processes is termed the “promoter decoy” model. This describes a scenario by which an

enhancer-blocking insulator can associate with components of the transcriptional complex, thus

allowing it to mimic a bona fide promoter. This decoy promoter could then interact with

enhancers and compete for their influence (GEYER 1997). This model is supported by evidence

that CTCF insulators at the H19 imprinting control locus have been observed to co-localize with

enhancers (YOON et al. 2007), and by evidence indicating that the effectiveness of an insulator is

dramatically altered depending on the affinity of nearby promoters for a particular enhancer.

Promoters with a high affinity for an enhancer are blocked less effectively (CAI et al. 2001).

As previously mentioned, there are also models to describe boundary element function

that adhere to a “passive-physical” model whereby two distant boundary element sequences are

brought together via protein interactions forming a loop of DNA (aka, the loop model)

(BLANTON et al. 2003; DORMAN et al. 2007; KUHN and GEYER 2003). A variety of this

mechanism hypothesizes that boundary element sequences are tethered to a nuclear structure,

thus passively forming a physical block to heterochromatin propogation (HART and LAEMMLI

1998).

Perhaps the most characterized Drosophila insulator protein, and one that is proposed to

adhere to the loop model is Suppressor of Hairy-wing [(Su(Hw)]. The Su(Hw) gene was

discovered (and named) because mutations at this loci suppress multiple phenotypes caused by

other mutations at distant loci (MODOLELL et al. 1983; PARKHURST and CORCES 1985;

RUTLEDGE et al. 1988). It was also observed that a disproportionately high number of X-ray

induced mutations were subject to suppression by Su(Hw) mutations (LINDSLEY and GRELL

1968). This lead to speculation that the X-ray induced mutations were not a result of simple

sequence alterations but likely a result of mobile elements – and these mobile elements somehow

20  

interacted with the Su(Hw) protein. It was eventually confirmed that the mutations were caused

by the insertion of the gypsy retrotransposon (MODOLELL et al. 1983). Initially researchers

believed this was due to Su(Hw) interfering with transcription factors at the affected loci or that

Su(Hw) affected the “superhelicity” of the regions in which the gypsy element was inserted

(FREUND and MESELSON 1984; PARKHURST and CORCES 1985). A model gene in Drosophila

used to study the gypsy-Su(Hw) phenomenon is the y gene. The y2 allele is a mutant allele with a

gypsy element inserted between the enhancer and promoter (GEYER and CORCES 1992)

(HOLDRIDGE and DORSETT 1991), and Su(Hw) binds to defined sequences within this element

which prevents a distal enhancer from activating the y2 promoter (GEYER and CORCES 1992).

While the original discoverers of this phenomenon continued to hypothesize that this was a

transcription-factor related event, this was actually a demonstration of enhancer blocking by a

boundary element. It was subsequently demonstrated, like scs and scs’, that the Su(Hw) binding

site could insulate a transgene from positive or negative chromosomal position effects and isolate

a gene from the inhibitory effects of heterochromatin (ROSEMAN et al. 1993).

As stated previously, Su(Hw) is thought by some to function by the loop model. An early

observation was made by immunostaining diploid cells with anti-Su(Hw) antibodies and utilizing

a GFP-tagged Su(Hw) protein with 3D deconvolution microscopy that large “insulator bodies”

form around the periphery of the nucleus, and individual insulator bodies contain Su(Hw)

binding sequences located at chromosomally distant loci (GERASIMOVA et al. 2000). These same

observations were made with the Mod(mdg4)2.2 protein, which was previously shown to

associate with Su(Hw) (GERASIMOVA et al. 1995). Figure 1-3 is an artist’s interpretation of

insulator bodies. Early biochemical evidence supporting this model demonstrated that Su(Hw)

and mod(mdg4)2.2 both remain a component of the nuclear matrix even after salt removal of

most nuclear proteins.

21  

The Su(Hw) loop model has recently evolved into a more dynamic model with evidence

suggesting that the insulator bodies are RNA mediated complexes. Chromosomal Protein 190

(CP190) is a another component of the Su(Hw) and mod(mdg4)2.2 complex (PAI et al. 2004),

and co-immunoprecipitation experiments have revealed RNA-dependent interactions between

CP190 and proteins involved in RNAi pathways. Additionally, RNase treatment has been shown

to disrupt the visualization of insulator bodies in vivo. A

model inclusive of this data proposes that an RNA helicase

protein, Rm62, mediates insulator protein-nuclear matrix

interactions thereby modulating chromatin domain dynamics

(LEI and CORCES 2006).

The looping model used to describe SuHw function is

not a universally accepted explanation. A vast majority of the

evidence described above for the existence of insulator bodies

is a result of individuals from the same laboratory interpreting microscopic images after various

fixation and harsh chemical or salt treatments, potentially introducing experimentally induced

artifacts (BYRD and CORCES 2003; GERASIMOVA et al. 2000; LEI and CORCES 2006). Perhaps the

most damning evidence is a recent finding that certain mutations in the mod(mdg4) protein will

prevent its association with “insulator bodies” yet the gypsy insulator still functions normally in

insulator assays. Other mutations in mod(mdg4) allow the association with insulator bodies but

prevent the gypsy insulator from functioning. Thus, the aggregation of proteins to form bodies in

the nuclear periphery is not a requirement of their function as the models would require them to

be (GOLOVNIN et al. 2008). In addition to insulator bodies there are many subnuclear

compartments comprised of protein aggregates that have unknown or only partially understood

function (HANDWERGER and GALL 2006).

Figure 1-3 Artistic rendering ofchromatin loops. Proteins such asSu(Hw) bind to chromatin andnuclear lamina or nuclear matricassociated proteins to physicallyseparate regions of DNA. (Usedwith permission; GERASIMOVAet al. 2000)

22  

There have been experimental attempts to demonstrate that the loop model applies to the

scs and scs’ boundary elements. The Zeste-white 5 (Zw5) protein binds to the scs sequence

which, together with the BEAF binding site at scs’, bracket the hsp70 genes. Zw5 has also

demonstrated insulating properties in enhancer blocking assays (GASZNER et al. 1999). The same

laboratory that produced these results also claim that Zw5 and BEAF interact in vivo and in

vitro. The sequences pulled down in ChIP assays using an antibody against Zw5 were enriched

in the scs sequence, as expected. However, these same pulldowns were also mildly enriched in

the BEAF-binding scs’ sequences. The authors suggest that crosslinking occurred between Zw5,

BEAF, and scs’ because they are in close proximity with each other and potentially interact.

Furthermore, in Glutathione-GST pulldown assays, a GST tagged BEAF-32A construct co-

precipitated with the Zw5 protein, and immunoprecipitation of nuclear extracts with an anti-Zw5

antibody will co-precipitate with GST-BEAF. Interestingly, the authors mention that an anti-

BEAF antibody recovers BEAF-32A-GST but does not co-precipitate Zw5 (BLANTON et al.

2003).

A genetic interaction between Zw5 and BEAF has also been shown. Expression of the

BEAF-interfering domain (BID, described above) or the over-expression of only BEAF-32A

results in an observable distortion in eye development termed a rough-eye phenotype. In the

latter case a tissue-specific yeast transcription factor GAL4 drove the over-expression of BEAF-

32A, possibly resulting in a dominant negative effect. The introduction of mutated Zw5 alleles

into an individual fly expressing the BID protein or ectopically expressed BEAF-32A enhanced

the rough eye phenotype, indicating a genetic interaction in vivo (BLANTON et al. 2003; ROY et

al. 2007b). This assay, however, is limited in interpretation. The results can also be explained by

the fact that both Zw5 and BEAF bind to many sites on polytene chromosomes, potentially

influencing the expression of many downstream factors. Additionally, the eye-screen assays

23  

using the BEAF-interfering domain has demonstrated that BEAF genetically interacts with

several genes, many of which encode transcription factors (ROY et al. 2007b). Thus, introducing

mutations in Zw5 might alter the transcription of proteins that then in turn interact with BEAF

causing a rough-eye phenotype.

Additional studies aimed at determining a mechanism for BEAF function involve the

biochemically defined nuclear matrix. The isolation of nuclear matrix associated proteins is

accomplished by a variety of methods, but essentially consist of proteins that remain associated

in the nucleus after protein and DNA removal via high salt treatments and DNase I treatment.

One group claims that MALDI-TOF and mass spectrometry identified BEAF-32B as remaining

associated as part of the nuclear matrix. They also claim that immunostaining for Zw5 and

BEAF-32B on slide-mounted nuclear matrix preparation shows a high degree of overlap in their

localization, implicating this as evidence that BEAF-32B associates with Zw5 and the nuclear

matrix to stabilize loop formation (PATHAK et al. 2007).

There are several aspects of the loop model that require further explanation. For example,

it remains unclear how the formation of loops could prevent the activation of a promoter. The

formation of loops could bring regulatory regions into closer proximity with the promoter than in

the absence of loops, thereby activating transcription instead of preventing it. This has been

described as a mode of activation for genes such as hsp26 and other loci (BUCK et al. 1987;

THOMAS and ELGIN 1988), and has been supported by electron microscopy (BAGGA et al. 2000).

Research Objectives

The study of the BEAF proteins as insulators was previously comprised of two aspects.

First, extensive work characterized the BEAF proteins biochemically, including detailed

description of the scs’ binding site. Second, genetic studies of scs’ and the BEAF proteins

demonstrated that scs’ is a BEAF-dependent insulator. Thus it is known that BEAF has effects

24  

on genes, but how this occurs has remained elusive. Our ultimate goal throughout this project has

been to investigate how BEAF-32A and BEAF-32B function to regulate gene expression.

To address these questions we first expressed a protein designed to interfere with

endogenous BEAF activity. This is described in Chapter 2. This protein, termed BID (BEAF-

self-interacting domain) is the C-terminal half of the BEAF proteins, which includes the BEAF

self-interacting domain but not the DNA-binding domain. We demonstrate here that this protein

interacts with endogenous BEAF proteins and prevents them from binding to DNA. This

prevents BEAF dependent insulators from functioning in enhancer blocking and position-

independent expression assays, and also prevents BEAF from inhibiting the spread of

heterochromatin from centromeres. The lack of BEAF activity also has a severe effect on

polytene chromosome structure, providing evidence that BEAF functions by influencing

chromatin structure or dynamics.

The third chapter describes work utilizing a fly line with a null mutation in the BEAF

gene; the BEAFAB-KO line. Using Micrococcal nuclease and DNase I assays we attempt to

characterize changes to chromatin structure occurring at BEAF binding sites when BEAF is not

present. This correlates with changes to hypersensitive sites and nucleosome positioning near

promoters, but not in bulk chromatin preparations or in regions not near promoters. Furthermore,

we will demonstrate that at select genes histone modifications have been perturbed or altered in

the BEAFAB-KO.

In the fourth chapter we describe experiments done with GFP and RFP tagged BEAF

proteins to study protein dynamics in vivo. Fluorescence recovery after photobleaching (FRAP)

and fluorescence microscopy indicate significant differences between BEAF-32A and BEAF-

32B. The proteins have very different localization patterns on polytene chromosomes and display

different recovery kinetics in preliminary FRAP experiments. Furthermore, it is evident from our

25  

analysis of mitotic brain cells that BEAF-32B comes off the chromosomes during prophase

while BEAF-32A remains associated with the DNA throughout mitosis.

Taken together, we will propose a model for BEAF function that is different from the

loop models previously proposed. Furthermore, we also propose that BEAF does not function as

a passive nucleoprotein complex that serves only to physically divide regions of the genome.

Rather, the data presented here and by others in our laboratory suggest that BEAF functions by

influencing the recruitment or activity of proteins associated with promoter complexes. This may

include nucleosome positioning or indirect effects on histone modifications at promoter regions,

which consequently affect gene expression levels.

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Chapter 2: The Drosophila Boundary Element-Associated Factors BEAF-32A and BEAF-32B Affect Chromatin Structure*

Introduction

Chromosomal DNA in the nucleus of a eukaryotic cell is tens of thousands of times

longer than the nuclear diameter. A high level of structural organization inside nuclei is required

to allow chromosomes to function properly in processes such as transcription and mitosis. The

first level of organization is the nucleosome, which is a 10 nm bead composed of 146 bp of DNA

wrapped 1.6 times around an octamer of histone proteins. High resolution crystal structures of

nucleosomes have been solved (LUGER et al. 1997; MUTHURAJAN et al. 2004). Higher levels of

chromatin structure are not well understood, progressing from 30 nm fibers to looped domains.

Communication between enhancers and promoters involves long range interactions, and is also

poorly understood (BULGER and GROUDINE 1999; DORSETT 1999). The physical organization of

chromatin likely plays a functional role in this communication.

Chromatin domain insulators (also known as boundary elements) help establish patterns

of gene expression by limiting possible interactions between regulatory elements and promoters

(KUHN and GEYER 2003; LABRADOR and CORCES 2002; WEST et al. 2002). In enhancer

blocking assays, insulators interfere with enhancer-promoter communication only when

positioned between the enhancer and promoter. When located upstream or downstream, they

have no effect. Transgenes bracketed by insulators are protected from chromosomal position

effects. After integration into most chromosomal loci, similar levels of transgene expression are

observed because expression is driven solely by regulatory elements in the transgenic construct

*Reprinted from Genetics, Vol. 173, Gilbert, M.K., Tan, Y.Y., Hart, C.M., The Drosophila Boundary Element-Associated Factors BEAF-32A and BEAF-32B Affect Chromatin Structure, pages 1365-1375, copyright 2006, with permission.

35

(position-independent expression assays). It is likely that endogenous insulators divide

chromosomes into functional domains such that regulatory elements and promoters can only

interact if they are in the same domain. If this functional organization plays a role in the physical

organization of chromosomes in nuclei, then insulators are candidate elements for linking

chromatin organization and dynamics to gene regulation.

The boundary element-associated factors BEAF-32A and BEAF-32B bind to the scs’

insulator as well as to hundreds of other sites on polytene chromosomes (HART et al. 1997; ZHAO

et al. 1995). The BEAF binding sites in scs’ are essential for its insulator activity, and other

genomic BEAF binding sites that have been tested also function as insulators (CUVIER et al.

1998). Thus BEAF-utilizing insulators are common in Drosophila. BEAF-32A and 32B are 32

kDa proteins derived from the same gene (HART et al. 1997). They differ at their amino termini,

which have different BED finger DNA-binding domains (ARAVIND 2000). The carboxy-

terminal two-thirds of these proteins are identical. A BESS domain is found near the carboxy

termini (BHASKAR and COUREY 2002; DELATTRE et al. 2002), and is preceded by a potential

leucine zipper domain. BEAF monomers interact with each, presumably via interactions

between BESS domains or leucine zippers or both. Evidence suggests BEAF binds DNA as

trimers, although larger complexes could also be involved (HART et al. 1997). No other proteins

copurify with BEAF, indicating that BEAF only forms stable complexes with itself.

To gain insight into the role of the BEAF proteins in Drosophila, we constructed a

transgene encoding the BEAF self-interaction domain (BID) but lacking a DNA binding domain.

This design is based on the Drosophila Emc and vertebrate Id proteins (CAMPUZANO 2001;

NORTON et al. 1998). These proteins lack DNA binding domains, and so inhibit DNA binding

by their partner transcription factors by forming dimers that lack one DNA binding domain. The

BID protein should similarly inhibit DNA binding by BEAF. The BID transgene is under GAL4

36

UAS control, allowing expression to be driven in different patterns by different GAL4 driver fly

lines (BRAND et al. 1994). We demonstrate that the BID protein inhibits BEAF activity, and

provide evidence that BEAF function influences chromatin structure or dynamics.

Materials and Methods

DNA Constructions: Four P-element plasmids were used to establish transgenic fly

lines in this study: pUAS-BID, pC4-gBF, pC4-YG4 and pM2. To construct pUAS-BID, a 700

bp BamHI fragment was isolated from a plasmid containing the 32A cDNA (ZHAO et al. 1995).

This encodes the carboxy-terminal 141 amino acids of BEAF. This fragment was ligated into a

modified pUAST-HN plasmid. pUAST-HN, kindly provided by JA Simon (University of

Minnesota), has sequences encoding an HA epitope tag and an SV40 NLS located upstream of

the EcoRI site of pUAST (BRAND and PERRIMON 1993). This was further modified by placing a

BglII linker into the EcoRI site so that the BamHI fragment would fuse BEAF sequences in the

correct reading frame.

BEAF sequences were PCR-amplified from genomic DNA and cut with BglII, resulting

in a 5 kb fragment. The 5’ end is located in the first intron of the divergent CG10155 gene, 2.5

kb upstream of the putative 32A transcription initiation site. The 3’ end is located in the final

exon of the convergent knot gene, 250 bp downstream of the putative BEAF poly-adenylation

site. This BglII fragment was ligated into the BglII site of pUC19 which had been modified by

the insertion of a BglII linker into the SacI site, resulting in pUC-gBF. For germline

transformation of flies, the 5 kb BglII gBF fragment from pUC-gBF was ligated into the BamHI

site of pCaSpeR4 (PIRROTTA 1988), upstream of the mini-white gene. In the resulting pC4-gBF

plasmid, the BEAF gene is transcribed in the same direction as mini-white.

The transformation vector pC4-YG4 has GAL4 coding sequences under the control of the

yellow wing and body enhancers. This plasmid has a 2.9 kb PCR fragment from pCaSpeR4-

37

yellow (kindly provided by V. Pirrotta, Rutgers University) that encompasses the yellow wing

and body enhancers and promoter to +65, a 2.9 kb PCR fragment from pCL1 (Clontech) that

encompasses the GAL4 coding sequences, and a 490 bp HincII fragment from pCaSpeR4-yellow

that encompasses the yellow poly-adenylation region. The assembled yellow-GAL4 gene

construct was cloned into the NotI site of pCaSpeR4.

The mini-white position-independent expression vector pM2 is described in CUVIER et al.

(1998).

Fly Stocks and Germline Transformation: Flies were maintained at 25°C or 18°C on

standard cornmeal, yeast, and sugar medium with Tegosept. The following yellow enhancer-

blocking lines, described in KUHN et al. (2004), were used: 2scs’ inserted at 19D; scs inserted at

60A; gypsy inserted at 25C. The ey-GAL4/TM6b line was kindly provided by TE Haerry

(Florida Atlantic University). Lines from the Bloomington Drosophila Stock Center were da-

GAL4 (BSC-8641); ey-GAL4/CyO (5535); salivary-gland-GAL4 (1824 and 1967); CNS-

GAL4/TM3 (3742); UAS-GFP.S65T (1521 and 1522); and wm4h (isolated from 6234). The y

variegating line KV20, located at 39-40H of chromosome arm 2R, was kindly provided by GH

Karpen (UC Berkeley). Transgenic flies were generated by co-injecting plasmids (0.4 μg/μl)

with the pπ25.7wc helper plasmid (0.1 μg/μl) into pre-blastoderm y1 w67c23 embryos (SPRADLING

1986). Names of fly lines generated in this study refer to the relevant transgene, followed by a

designation of the chromosome the transgene is inserted onto and a letter for each independent

line.

Immunoprecipitations: To prepare nuclear extracts, embryos were homogenized in

nuclear isolation buffer (3.75 mM Tris [pH 7.4], 0.05 mM spermine, 0.125 mM spermidine, 0.5

mM EDTA [pH 7.4], 20 mM KCl, 0.5% thiodiglycol, 0.05% Empigen BB, 0.1 mM PMSF, 2 mg

of aprotinin per ml) using a Dounce homogenizer and A and B pestles. 300 μl buffer was used

38

per 100 mg embryos. Nuclei were filtered through Miracloth (Calbiochem) and pelleted by

centrifugation at 2,000xg for 10 min in a refrigerated microfuge. The supernatant was saved as

cytoplasmic extract. The nuclei were washed twice in nuclear isolation buffer, then resuspended

in 80 μl of nuclear extraction buffer (10 mM HEPES [pH 7.6], 360 mM KCl, 3 mM MgCl2, 0.1

mM EDTA, 1 mM dithiothreitol, 10% glycerol, 4 mg of aprotinin per ml, 0.2 mM PMSF, 5 mg

each of leupeptin, antipain, pepstatin A, and chymostatin per ml) per 100 mg of embryos and

incubated for 30 min at 4°C with gentle agitation. Extracts were centrifuged at 16,000xg for 30

min in a refrigerated microfuge. The supernatant was aliquoted, flash frozen and stored at -80°C.

Affinity-purified antibodies against BEAF were previously described (HART et al. 1997;

ZHAO et al. 1995). 40 μl of extract was incubated with 2 μl anti-32A or 2 μl anti-32B antibodies

for 2 hr at 4°C. Immunoprecipitates were recovered with protein A-agarose beads (Roche),

washed five times with 350 mM NaCl, 10 mM Hepes [pH 7.6], 0.1% Tween-20, and proteins

were eluted with SDS sample buffer. After 10% SDS-PAGE and transfer to nitrocellulose,

proteins were detected using anti-BEAF antibody (1:2,000) followed by horseradish peroxidase-

conjugated goat anti-rabbit antibody (1:10,000) (Biorad). Signals were developed using an ECL

detection kit (Amersham).

Scanning Electron Microscopy: Flies were fixed in FAA (16% formaldehyde, 5%

acetic acid, 45% ethanol) for at least 24 hours, then put through a dehydration series of ethanol

(10 min each 75%, 87%, 94%, 97%, 4x 100%) followed by 2x 30 min in 100%

hexamethyldisilazane. Flies were dried overnight in a hood and stored in a dessicator. Flies

were sputter coated and observed in a Cambridge Stereoscan 260 SEM.

Immunostaining Polytene Chromosomes: Polytene chromosomes were prepared from

salivary glands of healthy, wandering 3rd instar larvae and immunostained as previously

described (ZHAO et al. 1995). Affinity purified rabbit anti-BEAF antibody was used at a 1:50

39

dilution, and Texas Red-conjugated goat anti-rabbit antibody (Jackson) was used at a 1:500

dilution. Chromosomes were stained with 100 ng/ml DAPI. Slides were viewed with a Ziess

Axioskop microscope equipped with a Spot RT Slider CCD camera.

PEV Assays: wm4h females were crossed to ey-GAL4/CyO; BID.3A/BID.3A males and

the eyes of wm4h; ey-GAL4/+; BID.3A/+ males were compared to those of their wm4h; CyO/+;

BID.3A/+ male siblings. Because the ey-GAL4 and BID.3A transposons are marked with mini-

white, crosses were conducted to determine the eye pigmentation of wm4h; ey-GAL4/+; +/+

males, w-; ey-GAL4/+; BID.3A/+ males, w-; +/+; BID.3A/+ males, and w-; ey-GAL4/CyO; +/+

males. To determine the effect of an extra copy of the BEAF gene, gBF.3C/gBF.3C males were

crossed to wm4h females or y1 w67c23 females and the eyes of wm4h males, wm4h; gBF.3C/+ males

and w-; gBF.3C/+ males were compared. Four to five day old males were etherized and

photographed using darkfield illumination with a 4x objective on a Ziess Axioskop microscope

equipped with a Spot RT Slider CCD camera. To quantitate the pigment, heads of 20 males

were homogenized in 200 μl 0.1% ammonium hydroxide, extracted once with chloroform, and

the OD480 was determined (ASHBURNER 1989).

For y variegation, KV20 males were crossed to females of the genotypes indicated in Fig.

7 and abdomens of two to three day old males were photographed with a dissecting scope.

Mitotic Analysis: Brains were dissected out of wandering third instar larvae, fixed in

3.7% formaldehyde in PBS for 30 min, transferred to 45% acetic acid for 3 min, and squashed in

60% acetic acid (BONACCORSI et al. 2000). Chromosomes were stained with 100 ng/ml DAPI.

In most cases at least 50 fields per brain were scored for mitotic figures, where a field was

defined as the region visible at 100x magnification with a 1x eyepiece on a Zeiss Axioskop

microscope. For two brains, 25 fields were scored. The mitotic index was calculated as the total

mitotic figures divided by the total number of fields scored, and the average field was estimated

40

to have around 300 cells. Mitotic figures from UAS-GFP/+; CNS-GAL4/BID.3B larvae, UAS-

GFP/UAS-GFP; CNS-GAL4/TM3 larvae and y1 w67c23 larvae were compared. There was no

difference in mitotic figures or mitotic index between UAS-GFP/UAS-GFP; CNS-GAL4/TM3

larvae and y1 w67c23 larvae.

Results

Rationale for the Design of the BID Transgene: The BEAF gene encodes two 32 kDa

proteins, BEAF-32A and 32B. There are

no mutations available in this gene. To

circumvent the lack of mutations, we

designed an inducible transgene that

should inhibit DNA binding by the BEAF

proteins (BID, for BEAF interaction

domain). Expression of the BID

transgene is under GAL4 UAS control

(Fig. 1A). Many driver lines are available

that express GAL4 transgenes in different,

known patterns. When crossed to these

driver lines, BID expression will be driven

in the same pattern as that of GAL4. The

BID protein has part of the common

portion of the BEAF proteins. This part

of the BEAF proteins is involved in

interactions with other BEAF molecules,

but lacks a DNA binding domain (Fig. 2-

Figure 2-1. Design of the BEAF-interaction-domain(BID) protein. (A) The carboxy-terminal half of theBEAF coding sequences were joined in frame tosequences encoding an HA epitope tag and SV40 NLS(black box). The BID sequence is in pUAST (BRAND andPERRIMON 1993), and so is under GAL4 UAS control(ovals) and has a downstream SV40 polyadenylation site(not shown). (B) BEAF-32A and 32B have uniqueamino-terminal DNA binding domains of 80 amino acids(hatched boxes). The rest of the proteins are identical,being derived from the same exon. The identical portionincludes a 120 amino acid central region of unknownfunction (open box) and an 80 amino acid carboxy-terminal domain that mediates interactions between BEAFproteins (gray box) (HART et al. 1997). The BID proteinhas an amino-terminal HA epitope tag and SV40 NLSjoined to the carboxy terminal half of BEAF. (C)Evidence suggests that BEAF forms trimers, and trimerformation occurs independently of DNA binding (HART etal. 1997). The BID protein should form complexes with32A and 32B, inhibiting DNA binding by BEAFcomplexes.

41

1B; HART et al. 1997). No other proteins co-purify with BEAF, and evidence from

immunoprecipitations and gel filtration columns indicates that BEAF subunits stably interact in

solution; DNA binding is not necessary. Data indicate that BEAF forms trimers (Fig. 2-1C),

although cooperative binding to two sites separated by 200 bp suggests larger BEAF complexes

also form, at least transiently (HART et al. 1997). The design of BID is based on the Drosophila

Emc protein (CAMPUZANO 2001) and the vertebrate family of Id proteins (NORTON et al. 1998).

Emc and Id lack DNA binding domains and form heterodimers with certain DNA-binding

transcription factors. The lack of one DNA binding domain prevents stable binding to DNA.

Thus these proteins are dominant negative antagonists of their transcription factor partners. This

plays an important role in developmental processes such as sensory organ development,

myogenesis and differentiation of blood cells. The BID protein should similarly act as a

dominant negative by eliminating DNA binding domains from BEAF complexes, thereby

drastically reducing their affinity and specificity for DNA (Fig. 2-1C).

Developmental Effects of BID Expression: Six transgenic fly lines containing single

inserts of the BID transgene were generated by P-element mediated germline transformation of

microinjected embryos. As an initial test of the effects of BID expression, these flies were

crossed to a da-GAL4 line to drive ubiquitous expression during embryogenesis. Both

transgenes were heterozygous in the resulting embryos. This resulted in embryonic lethality with

two BID fly lines, with a few embryos giving rise to first instar larvae (Table 1). For a third BID

line, adult females eclosed but had a rough eye phenotype. Males died as pharate adults. This

could indicate an effect on dosage compensation, which involves chromatin modifications that

double the activity of expressed genes on the single X chromosome in males. For the other three

BID lines, viable adults were obtained. One of these three lines exhibited a rough eye phenotype.

The different phenotypes observed could be due to chromosomal position effects that affect the

42

Table 1: Phenotypes of flies expressing the BID transgene

BID fly line Crossed to da-GAL4 Crossed to ey-GAL4

BID.2A male lethal (pupae); rough eyes rough eyes

BID.3A lethal (embryo/larvae) rough eyes

BID.3B lethal (embryo/larvae) rough eyes

BID.3C viable mild rough eyes

BID.4A viable; rough eyes rough eyes

BID.4B viable mild rough eyes

The BID transgene is under GAL4 UAS control. Six independent fly lines were generated and tested by crossing to GAL4-producing driver lines. Line names indicate the chromosome the transgene is on followed by a letter for each independent insertion found; each line has a single insert. da-GAL4: GAL4 protein produced under control of the daughterless promoter, ubiquitous expression. ey-GAL4: GAL4 protein produced under control of the eyeless promoter, eye imaginal disc expression. The phenotypes correlate with the level of BID expression, as determined by semi-quantitative Westerns. level of GAL4-mediated activation of the BID transgene in the different lines. In support of this,

the levels of BID protein detected on Western blots of embryo protein extracts correlated with

the severity of the phenotypes (data not shown).

It has previously been reported that over-expression of a BEAF-32A transgene in eye

imaginal discs results in a rough eye phenotype (YAMAGUCHI et al. 2001). In those experiments,

it would be expected that 32A-utilizing insulators would be functional but those whose function

relies on the 32B DNA binding activity would be impaired. In our experiments, the function of

all BEAF-utilizing insulators should be impaired. When our BID lines were crossed to ey-GAL4

lines to drive expression in eye imaginal discs, all lines exhibited a rough eye phenotype (Table 1

and Fig. 2-2). As expected, the severity of the rough eye phenotype increased when the BID

chromosome was homozygous in the presence of an ey-GAL4 driver. This was done for the

three lines that were lethal in the presence of the da-GAL4 driver (BID.2A, BID.3A and BID.3B).

Both ey-GAL4 lines we used in these experiments were recessive lethal; one had ey-GAL4

43

balanced over CyO and the

other had it balanced over

TM6B. The resulting flies

were sickly, and only the ey-

GAL4/CyO; BID.3A/BID.3A

line could be maintained as

a stock.

To address the

specificity of the BID protein

for BEAF, transgenic lines

were generated that contained

a 5 kb Bgl II fragment of

genomic DNA that spans the

BEAF gene. This DNA, which we refer to as gBF, includes portions of genes located upstream

(CG10155) and downstream (kn) of BEAF. Therefore it is likely that this region contains all

control elements necessary for proper expression of the BEAF gene. When these flies were

crossed to the ey-GAL4/CyO; BID.3A/BID.3A line, the gBF transgene rescued the rough eye

phenotype in the resulting heterozygous GAL4/+; BID.3A/gBF.3A or gBF.3C progeny (Fig. 2-2).

The ability of an extra copy of the BEAF gene to overcome the effects of BID expression

indicates that BID specifically interferes with BEAF function to result in the rough eye

phenotype.

BID Co-Immunoprecipitates with BEAF: To further explore in vivo interactions

between BID and BEAF proteins, we performed a co-immunoprecipitation assay. BID.3B males

were crossed to da-GAL4 females, resulting in ubiquitous expression of the BID transgene in

Figure 2-2. BID-dependent rough eye phenotype and rescue by gBF.Scanning electron micrographs show that the ey-GAL4/TM6B and BIDfly lines have wild-type eye morphology (A, B, D, F). The same is truefor the ey-GAL4/CyO line (not shown). Flies heterozygous for both ey-GAL4 and BID exhibit a rough eye phenotype (C, E, G). Thisphenotype is more extreme when BID is homozygous (H), and isrescued to near wild-type when a third copy of the BEAF gene isintroduced by a transgene (I, J). Two fly lines with the gBF rescuetransgene inserted at different locations rescued the rough eyephenotype.

44

embryos. Because this is a lethal combination, most of the embryos do not hatch. Embryos were

collected on grapejuice agar plates for 16 hours or less. Nuclear extracts were prepared from

these embryos as well as from y1 w67c23 embryos that did not have the BID transgene. In

addition, we tested the cytoplasmic fractions and found that some cytoplasmic extracts from da-

GAL4/+; BID.3B/+ embryos contained

BID but not BEAF protein. BID was

only immunoprecipitated if BEAF was

present in the extract (Fig. 2-3).

Therefore BID forms complexes with

BEAF in vivo.

BID Expression Interferes

with Scs’ Insulator Function: The

BID-dependent rough eye phenotype is

rescued by an extra copy of the BEAF

gene, and BID physically interacts

with BEAF in vivo. We next wanted to

determine if BID expression would interfere with scs’ function in a transgene assay. We used

two assays, a position-independent expression assay and an enhancer-blocking assay. For the

position-independent expression assay we generated transgenic fly lines in which the mini-white

gene was bracketed by the M2 derivative of scs’ (on the 5’ side) and scs (on the 3’ side) (CUVIER

et al. 1998). The M2 insulator has two copies of the high affinity BEAF binding site present in

scs’. The bracketed mini-white gene is insulated from most chromosomal position effects;

around 90% of fly lines have yellow or light orange eyes. In the absence of the 5’ insulator, less

than 50% of fly lines have such light eye pigmentation (CUVIER et al. 1998). Therefore if BID

Figure 2-3 BID interacts with BEAF in vivo. Nuclearextracts were prepared from y1 w67c23 embryos (lanes 2-4)or embryos heterozygous for da-GAL4 and BID.3B (lanes5-7). A cytosolic extract prepared from embryosheterozygous for da-GAL4 and BID.3B was found to haveBID protein but essentially no BEAF, and was used as anegative control (lanes 9-11). Immunoprecipitations wereperformed with antibodies specific for the unique aminotermini of 32A (lanes 3, 6, 10) or 32B (lanes 4, 7, 11).Proteins on the Western blots were detected with anantibody that recognizes both BEAF and BID proteins.BID co-immunoprecipitated with BEAF-32A (lane 6) and32B (lane7). BEAF proteins immunoprecipitated in theabsence of BID (lanes 3, 4), but BID did notimmunoprecipitate in the absence of BEAF (lanes 10, 11).Ec: 32B protein produced in E. coli used as a Westerncontrol; In: input extract; P: immunoprecipitated proteins.

45

interferes with BEAF function, about half of

the M2 lines will have darker eye

pigmentation in the presence of BID

expression. To test whether BID interferes

with insulator function, flies homozygous for

M2 insertions were crossed to the ey-

GAL4/CyO; BID.3A/BID.3A line. Eye

pigmentation in three day old female flies

heterozygous for GAL4, BID.3A and M2 was

compared to their siblings that were

heterozygous for CyO, BID.3A and M2. Two

of four M2 lines tested had significantly

darker eye pigmentation when BID expression

was driven by the ey-GAL4 driver, while very

little effect was observed in the other two

lines (Fig. 2-4A).

It is unlikely that the mini-white

transgenes in the BID and ey-GAL4

transposons account for the darker eye

pigmentation described above. Flies

heterozygous for the ey-GAL4 and the BID.3A

transposons together have light orange eyes,

indicating that only a low level of pigment is

produced. The only difference between the

Figure 2-4 BID interferes with scs’ insulatoractivity, but not with scs or gypsy insulatoractivities. (A) BID expression inactivates theBEAF-dependent M2 insulator, an scs’ derivative,in a position-independent expression assay.Panels show eyes of three to four day old femalesheterozygous for all indicated transposons. Seetext for details. (B) BID expression inactivates anscs’ dimer, but has minimal effects on the scs orgypsy insulators, in an enhancer-blocking assay intransgenic flies. Panels show abdomens of threeto four day old females heterozygous for theindicated transposons, with (Ins) or without[del(Ins)] the indicated insulator between theenhancer and promoter. See text for details.

46

flies compared in the assay was the presence or absence of the ey-GAL4 transposon, which by

itself results in a pale yellow eye color. Two of the M2 lines tested apparently are not subject to

chromosomal position effects, and the additive effect of the ey-GAL4 transposon on eye

pigmentation was slight. We conclude that the other two M2 lines tested are susceptible to

chromosomal position effects, and the BID protein interferes with M2 insulator function to result

in darker eye pigmentation. The enhancer blocking assay we employed utilized a yellow

transgene rather than mini-white. An scs’ dimer (2scs’), scs or gypsy insulator was located

between the yellow wing and body enhancers and the yellow gene. This allows insulators that do

not have BEAF binding sites to be tested. “Sibling” lines in which the insulator was removed by

the Cre recombinase were also used, as controls for the presence and absence of an insulator at

the same chromosomal locus. Using these fly lines, it was previously found that insulators do

not block the propagation of heat shock puffs in polytene chromosomes (KUHN et al. 2004). To

use this assay, an appropriate GAL4 driver line was required. For this purpose we made a

construct that has the yellow wing and body enhancers and promoter upstream of the GAL4

coding sequences, and the yellow polyA region downstream (hereafter referred to as YG4).

Transgenic flies with the YG4 construct were crossed to UAS-GFP flies to confirm that GAL4

protein was produced. A fly line homozygous for YG4 (on the X chromosome) and BID.3A (on

the third chromosome) was constructed and crossed to fly lines homozygous for the enhancer-

blocking constructs. As controls for body pigmentation, the enhancer-blocking lines and their

“siblings” lacking the insulators were crossed to y1 w67c23 flies. The resulting progeny were

heterozygous for all transposons that were present. The level of pigmentation in the dorsal

abdomen of 3 to 4 day old females was recorded. As shown in Fig. 2-4B, control flies with the

insulators had less pigment than their “sibling” lines without the insulators. BID expression

inactivated the scs’ dimer, resulting in pigmentation similar to that in flies lacking the insulator.

47

There was no effect of BID expression on the function of the gypsy insulator. The effect of BID

expression on the function of the scs insulator was less clear. The level of pigmentation

appeared to be intermediate between the insulated and uninsulated controls, suggesting some

effect on scs function. It has been shown that BEAF and Zw5, the scs’ and scs binding proteins,

can interact with each other (BLANTON et al. 2003). Perhaps this interaction accounts for the

effect observed. The main conclusion is that BID expression strongly interferes with scs’

function.

BID Expression Interferes with Polytene Chromosome Structure: Some models of

insulator function hypothesize that insulators affect chromatin structure or dynamics. To

determine whether BID expression affects chromatin structure, polytene chromosomes were

prepared from salivary glands of third instar larvae after crossing BID lines to lines that produced

GAL4 in salivary glands (SG-GAL4 driver). A BID-dependent global disruption of polytene

chromosome organization was observed (Fig. 2-5). In the presence of a SG-GAL4 driver,

animals heterozygous for BID had salivary glands with chromatin that easily fragmented. These

SG-GAL4; BID homozygous flies were crossed with flies containing the gBF transgene, resulting

in progeny that were heterozygous for the SG-GAL4 driver, BID and gBF. By introducing a third

copy of the BEAF gene in this way, the defect in polytene chromosome organization was largely

rescued (Fig. 2-5). This provides further evidence that the BEAF proteins are the main target of

BID.

Immunostaining indicated that animals heterozygous for SG-GAL4 and BID had reduced

levels of BEAF on their polytene chromosomes, and the BEAF banding pattern observed on

normal polytene chromosomes was absent. Chromosomes prepared from larvae homozygous for

SG-GAL4 and BID had virtually no BEAF staining, as well as having a more extreme

morphology (Fig. 2-5). In addition, there appeared to be a higher background level of staining.

48

Figure 2-5 Expression of BID in salivary glands leads to a global disruption of polytene chromosome structure, and a loss of the BEAF immunostaining pattern. Polytene chromosomes from salivary glands of a wild-type third instar larva have a well-defined banding pattern (A). Polytene chromosomes from salivary glands of third instar larva with two different salivary gland GAL4 (SG-GAL4) drivers and different BID transgenes lack this defined pattern and are easily over-stretched (B and C). Adding a third copy of the BEAF gene via a gBF transgene largely resuces the BID-associated defect in polytene chromosome organization (D). Immunostaining of wild-type polytene chromosomes for BEAF gives a characteristic banding pattern. BEAF binds to several hundred interbands and band/interband junctions (E: DAPI; F: BEAF; G: overlay). Immunostaining of polytene chromosomes from larvae heterozygous for 1967-SG-GAL4 and BID.3A show a reduced level of BEAF on the chromosomes and a lack of any defined banding pattern of BEAF (H: DAPI; I: BEAF; J: overlay). Immunostaining of polytene chromosomes from larvae homozygous for 1967-SG-GAL4 and BID.3A show an apparent lack of BEAF staining (K: DAPI; L: BEAF; M: overlay).

This could indicate a higher level of BID protein remaining on the slide after fixation, even

though it is a soluble protein (i.e. it is not chromatin bound). These results indicate that BID

49

interferes with the ability of BEAF to associate with chromatin in vivo, and the chromatin

structure of the resulting polytene chromosomes is globally affected.

BID Expression Does Not Affect Mitotic Chromosomes or Mitosis: Many proteins

participate in chromosome condensation during mitosis. The condensin complex clearly plays a

key role, although disruption of condensin only partially interferes with mitotic chromosome

condensation. Therefore condensin cannot determine all levels of compaction. BEAF remains

on mitotic chromosomes (HART et al. 1999). If interphase organization is utilized in a modified

form to produce highly condensed metaphase chromosomes, BEAF might also play a role in

mitotic chromosome organization. In that case, BID expression should affect chromosome

condensation. This hypothesis was addressed by observing mitotic cells in brain squashes from

third instar larvae.

The CNS-GAL4 driver line used in these experiments has a third chromosome insertion

that is sickly when homozygous. We generated a line with the CNS-GAL4 driver chromosome

balanced over TM3 with a homozygous UAS-GFP responder second chromosome. This allowed

identification of larvae that were producing GAL4 protein, and therefore also both GFP and BID,

after crossing these flies to BID flies. Both UAS-GFP/UAS-GFP; CNS-GAL4/TM3 and y1 w67c23

larvae were used as controls, with similar results. BID expression did not affect the health of

animals, and did not affect the size of the larval brain or the mitotic index of neuroblasts obtained

from DAPI-stained brain squashes (Table 2). In addition, we did not observe any defects in

chromosome condensation, premature sister chromatid separation, aneuploidy or anaphase

problems. We conclude that BEAF does not play a role in mitosis. It is possible that BEAF

remains on mitotic chromosomes to provide a molecular memory of the location of insulators.

BID Expression Affects PEV: To further explore the apparent link between BEAF

function and chromatin structure, we tested the effect of the BID protein on position effect

50

Table 2: BID expression does not affect the mitotic index in larval brains

Wild type BID.3B

Mitotic index 6.45 ± 0.79 6.60 ± 1.35

The mitotic index ± standard deviation is shown for two genotypes: Wild type = UAS-GFP/UAS-GFP; CNS-GAL4/TM3 and BID.3B = UAS-GFP/+; BID.3B/CNS-GAL4. Data for wild type are from 5 brains (50 fields/brain); data for BID.3B are from 6 brains (50 fields/brain for 4 brains, 25 fields/brain for 2 brains). variegation of the wm4h gene. Due to a chromosomal inversion on the X chromosome, this gene

is near pericentric heterochromatin. This rearrangement results in variegated expression in eyes,

which is detected as varying numbers of pigmented ommatidia (TARTOF et al. 1989). The level

of variegation is very sensitive to mutations that directly or indirectly affect chromatin

organization.

The wm4h assay is complicated by the presence of mini-white genes on the ey-GAL4 and

BID.3A chromosomes. The level of pigmentation in the presence of these heterozygous

chromosomes is relatively low and is not variegated (Fig. 2-6). Males heterozygous for the

BID.3A chromosome have more eye pigment than males heterozygous for the ey-GAL4

chromosome. Males heterozygous for both chromosomes have still more eye pigment, despite

having smaller, rough eyes. In a similar series of male flies hemizygous for wm4h, males of the

genotype wm4h; ey-GAL4/+; BID.3A/+ had the fewest red ommatidia and the lowest levels of eye

pigment (Fig. 2-6). The smaller, rough eyes cannot account for the lower pigment levels, since

ey-GAL4/+; BID.3A/+ flies had more pigment than ey-GAL4/+ or BID.3A/+ flies. This indicates

that BID expression enhances PEV, leading to a suppression of wm4h expression.

If BID enhances PEV via effects on BEAF, then overproduction of BEAF should

suppress PEV. This was tested with the gBF rescue transgene. Once again, the gBF transposon

is marked with a mini-white gene that results in a low level of eye pigmentation. Males of the

51

genotype wm4h; gBF/+ had more red

ommatidia and more eye pigment than wm4h

males (Fig. 2-6). While the increase in eye

pigment is in part due to the mini-white

gene associated with gBF, this is unlikely

to account for the entire increase. As

predicted based on the BID expression

results, we conclude that BEAF is a triplo-

suppressor of PEV. The involvement of

BID and BEAF in pericentric PEV suggests

that BEAF protects the wm4h gene from

being incorporated into heterochromatin,

perhaps by forming a barrier that limits

heterochromatin spreading.

We further tested the effect of BID

on PEV associated with a different reporter

gene and chromosome. For this purpose we

used the KV20 line which has a yellow

transgene inserted into the pericentric

heterochromatin of chromosome arm 2R (YAN et al. 2002). This circumvents the use of

variegated w expression in a background that introduces transgenic mini-white genes. Male flies

heterozygous for the KV20 transposon, and different YG4 drivers and BID responders exhibited

enhanced variegation relative to males heterozygous for only KV20. They had fewer dark spots

on their posterior abdominal segments (Fig. 2-7). Adding a third copy of BEAF via a gBF

Figure 2-6. Expression of BID in eye imaginal discsenhances wm4h variegation, while expression of gBFsuppresses wm4h variegation. Eyes of four to five dayold males of the following genotypes are shown: A:wm4h; ey-GAL4/+; BID/+; B: wm4h; CyO/+; BID /+; C:wm4h; ey-GAL4/+; +/+; D: w-; ey-GAL4/+; BID /+; E:w-; +/+; BID /+; F: w-; ey-GAL4/CyO; +/+; G: wm4h;+/+; +/+; H: wm4h; +/+; gBF/+; I: w-; +/+; gBF /+.Note that all transgenes are marked with mini-white,and result in yellow or light orange eyes whenheterozygous alone. J: Pigment was extracted byhomogenizing heads in 10 μl/head of 0.1% ammoniumhydroxide and extracting 1x with chloroform. TheOD480 values for the extracted pigment is shown in thesame order as the eye pictures.

52

transgene suppressed PEV of the y transgene, resulting in a larger number of dark spots. Thus

two PEV assays, using different reporter genes on different chromosomes, indicate that BEAF

interferes with the formation of pericentric heterochromatin.

Discussion

To gain insight into BEAF function, we designed a gene encoding the BEAF self-

interaction domain. The encoded protein should act as a dominant negative form of the BEAF

proteins by interfering with DNA binding. We have shown by co-immunoprecipitation that the

BID protein physically interacts with BEAF in vivo, and by immunostaining that it removes

BEAF from polytene chromosomes. Adding a third copy of the BEAF gene rescues the BID-

associated rough eye phenotype and disruption of polytene chromosome structure. Furthermore,

BID interferes with scs’ insulator function in both a position-independent expression and an

enhancer-blocking assay. We conclude that BID interferes with BEAF function by reducing the

level of chromatin-associated BEAF.

Could interactions between BID and proteins other than BEAF account for the effects of

BID? No proteins co-purify with BEAF, indicating that BEAF does not form stable complexes

with other proteins. However, interactions between BEAF and other proteins have been

Figure 2-7. Variegation of a y transgene located in the pericentromeric heterochromatin ofchromosome arm 2R is enhanced by BID expression and suppressed by a third copy of BEAF. The ytransgene is in the KV20 transposon. Abdomens of two to three day old males heterozygous for theindicated transposons are shown. See text for details.

53

reported. D1 is an abundant chromosomal protein that resembles mammalian HMGA (formerly

HMG-I) proteins, except it is larger (ASHLEY et al. 1989). Whereas mammalian HMGA proteins

have three AT-hook domains, D1 has ten (at least six of which should be functional). Although

D1 predominantly binds to AT-rich satellite DNA sequences (AULNER et al. 2002; LEVINGER

and VARSHAVSKY 1982), it can cooperatively bind to certain DNA sequences with BEAF

(CUVIER et al. 2002). The potential role of this in the effect of BEAF on PEV of the wm4h and

KV20 y alleles is discussed below. Another protein that interacts with BEAF is Zw5 (BLANTON

et al. 2003), a protein that binds to the scs insulator (GASZNER et al. 1999). This interaction

could account for the apparent weak effect of BID on scs insulator activity in the enhancer-

blocking assay. A protein interaction map derived from a high throughput yeast two-hybrid

screen identified five proteins that can interact with BEAF (GIOT et al. 2003)

(http://portal.curagen.com/cgi-bin/interaction/flyHome.pl). Four of these proteins are encoded

by conceptual genes, and no functional information is available. The fifth protein is Katanin-60,

the catalytic component of a microtubule severing complex. The two-hybrid screen did not

identify D1 or Zw5, and it is unknown if BEAF interacts with any of these five proteins in vivo.

We cannot formally rule out the possibility that interactions with these or other proteins

contribute to the effects of the BID protein. But the effect of BID on the activity of the scs’

insulator, the lack of effect on the gypsy insulator, the minimal effect on the scs insulator, and

the rescue of the rough eye and polytene chromosome phenotypes by a third copy of the BEAF

gene suggests that BEAF is the major target of BID.

Ubiquitous expression of BID during embryogenesis is lethal, indicating that the BEAF

proteins are essential during development. It was previously shown that expression of a BEAF-

32A transgene in eye imaginal discs led to a rough eye phenotype associated with increased

apoptosis (YAMAGUCHI et al. 2001). 32A over-production should affect the function of

54

insulators that require 32B DNA binding activity, but not those that only require 32A. The BID

protein should affect all BEAF-dependent insulators. Based on the proposed role of BEAF in

insulator function, we hypothesize that many genes are misregulated when BEAF insulator

function is perturbed. This misregulation could in part be due to the transcription factor DREF

(HIROSE et al. 1996). Originally proposed to regulate DNA replication-related genes, it has more

recently been proposed that DREF functions as part of a core promoter selectivity factor for

TRF2-utilizing promoters (HOCHHEIMER et al. 2002; OHLER et al. 2002). There is evidence that

BEAF and DREF compete for binding to certain DNA sequences (HART et al. 1999); removing

BEAF would facilitate binding by DREF to these sites. We propose that a breakdown in gene

regulation disrupts the developmental program in the developing eye, resulting in a rough eye

phenotype. In the developing embryo, this breakdown is lethal.

BEAF and the D1 protein can cooperatively bind to DNA (CUVIER et al. 2002).

However, their patterns of immunolocalization on polytene chromosomes are largely distinct.

D1 binds an AT-rich sequence and largely immunolocalizes to heterochromatin, especially the

AT-rich 1.672 and 1.688 g/cm3 satellites (AULNER et al. 2002; RODRIGUEZ ALFAGEME et al.

1980). These satellites are found in the pericentromeric heterochromatin of the X and Y

chromosomes and of chromosome 4 (LOHE et al. 1993). BEAF binds to several hundred sites in

euchromatin (ZHAO et al. 1995). Despite their largely distinct chromosomal distributions, BEAF

and D1 likely interact at the bases of the X, 2L and 2R chromosome arms, where several hundred

dispersed copies of a sequence (BE28) that has both BEAF and D1 binding sites are found

(CUVIER et al. 2002; CUVIER et al. 1998). The wm4h gene is located near the base of the X

chromosome, and the 1.688 g/cm3 satellite is a component of the pericentromeric

heterochromatin in this region. Interfering with D1 function suppresses wm4h variegation

(MONOD et al. 2002). The BE28 repeats could be locations where BEAF and D1 normally

55

interact to create a transition zone that is checkered with heterochromatin and euchromatin

islands. Perhaps BID enhances PEV of wm4h by allowing D1-associated heterochromatin to

spread further, while extra BEAF blocks the spread. This could also occur for the KV20 y

transgene, although 2R does not have high concentrations of the 1.672 or 1.688 g/cm3 satellites.

Alternative possibilities include direct suppression of variegation by BEAF by some other

currently unknown mechanism, or indirect suppression by affecting the activity or gene

expression of other chromatin proteins that directly affect variegation.

The mechanism leading to disruption of polytene chromosome structure by BID is not

known. It is possible that the D1 protein is involved, although as pointed out above D1 is mainly

associated with satellite heterochromatin and BEAF is mainly found on euchromatin.

Furthermore, the chromosomes look puffy, not condensed like heterochromatin. It is possible

that under-replication of the chromosomes could be involved, but that cannot account for the loss

of banding patterns. Also, no effect on replication was apparent in our examination of mitotic

figures in larval brain squashes. It has been shown in vertebrates and yeast that covalent histone

modifications can differ on either side of insulators or barrier elements (LITT et al. 2001a; LITT et

al. 2001b; NOMA et al. 2001). Perhaps impairing BEAF function allows these modifications to

spread further in a stochastic manner. Then individual chromosomes in the polytene bundle

could have different patterns of histone modifications over the same sequences, causing a loss of

banding and coherence between chromosomes. Similar phenotypes are observed in the presence

of mutations known to affect proteins that act on chromatin. Examples include the JIL-1 histone

H3 Ser10 kinase (WANG et al. 2001), the chromatin remodeling factor ISWI (DEURING et al.

2000), SU(VAR)2-10 (HARI et al. 2001), and the Z4 interband-specific protein (EGGERT et al.

2004). In all cases, the cause of the loss of polytene chromosome morphology remains

unknown.

56

Some models propose that insulators limit communication between regulatory elements

and promoters located in different domains by affecting chromatin structure or dynamics (KUHN

and GEYER 2003; LABRADOR and CORCES 2002; WEST et al. 2002). Inhibiting the ability of

BEAF to associate with chromatin leads to a global disruption of polytene chromosome

structure, and enhances PEV of the wm4h and KV20 y alleles. These results provide strong

support for a role of chromatin structure or dynamics in BEAF-dependent insulator function.

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Chapter 3: Analysis of Chromatin Structure in a BEAF Knockout Line

Introduction

Insulators, or boundary elements, have traditionally been defined by their ability to

protect transgenes from the influence of chromosomal position, prevent the spread of

heterochromatin into regions of active transcription, and prevent enhancer activation of a

promoter when placed between them (ISHII and LAEMMLI 2003; LABRADOR and CORCES 2002).

This definition is limited, however, in that it describes the result of their presence on gene

expression but is not inclusive of how these mechanisms occur in vivo. It is likely that the

various boundary elements discovered to date function via a variety of mechanisms. For

example, evidence suggests that one of the most studied insulators, Suppressor of Hairy-wing

(Su(Hw)), may form chromatin into a loop-like structure with the anchoring of the loops to the

nuclear lamina (BYRD and CORCES 2003). This could physically segregate genes into

independently regulated domains. However, details of how looping provides enhancer blocking

are experimentally lacking. Other sequences with boundary element function, such as certain

tRNA genes in yeast or CTCF-binding sites in Drosophila and humans appear to be associated

with chromatin dynamics such as influencing histone modifications or other chromatin

remodeling processes (DONZE et al. 1999; ISHIHARA et al. 2006; NAKAYAMA et al. 2007; WONG

et al. 2007).

Previous work in our laboratory and others has demonstrated that BEAF-32A and BEAF-

32B are major regulatory components of the Drosophila genome, influencing global patterns of

transcription (ROY et al. 2007b). Position independent expression assays and enhancer blocking

assays using our BEAF knockout (BEAFAB-KO) line have demonstrated that the BEAF proteins

are vital for the boundary element activity of the scs’ boundary element. Furthermore, when

BEAF protein levels are perturbed, processes such as ovary development, eye development, and

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chromatin structure of polytene chromosomes are affected (ROY et al. 2007a; YAMAGUCHI et al.

2001). BEAF immunolocalizes to hundreds of locations on polytene chromosomes. There are

three additional previously characterized BEAF-binding sequences named BE28, BE51, and

BE76. Immunoprecipitation assays have demonstrated that these sequences have clustered

CGATA motifs and associate with BEAF-32B in vitro. It has also been demonstrated that BE28

and BE76 function as boundary elements in position independent expression assays in vivo

(CUVIER et al. 2002; CUVIER et al. 1998).

Elucidating a mechanism of how BEAF functions as an insulator protein is the topic of

this study. Much of the previous work in this area has been based on the hypothesis that BEAF

functions similarly to the Su(Hw) boundary element by forming loops (BLANTON et al. 2003).

Blanton and Schedl produced evidence that the Zeste-white 5 protein (which binds to the

insulator sequence scs) may interact with BEAF to form chromatin loops. Pathak et al. claim that

that this complex in turn interacts with the nuclear matrix, anchoring the complex and forming

loops visible by fluorescent and confocal miscroscopy (PATHAK et al. 2007). Others, however,

have reported that using RNAi in Drosophila S2 cell culture to knockdown BEAF levels leads to

an increase of H3K9 tri-methylation at BEAF-binding sites (EMBERLY et al. 2008).

Here we use DNase I and MNase hypersensitive site mapping, nucleosome positioning

data, and ChIP analysis to investigate the chromatin structure at different genomic locations and

at BEAF binding sites, and to compare this information in wildtype and BEAFAB-KO lines. Our

work will demonstrate that a lack of BEAF causes a subtle disruption to nucleosome-DNA

interactions and nucleosome placement at BEAF-binding sites in gene regulatory regions, but

causes no noticeable effects at a BEAF-binding site that is not in a gene regulatory region.

Furthermore, we observe a perturbation to histone modifications in the BEAFAB-KO line, further

suggesting a role in altering chromatin dynamics.

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Materials and Methods

Immunostaining Polytene Chromosomes: Polytene chromosomes were prepared from

salivary glands of healthy, wandering third instar larvae and immunostained as previously

described (GILBERT et al. 2006). For this purpose, a fly line with the BEAFAB-KO allele over a w+

CyO GFP balancer was created. Homozygous BEAFAB-KO larvae derived from this line were

identified by the lack of green fluorescent protein. Affinity-purified rabbit anti-BEAF antibody

was used at a 1:50 dilution. Rabbit antibodies against the X chromosome dosage compensation

complex components MOF, MLE, MSL-1, MSL-2, and MSL-3 were kindly provided by M. I.

Kuroda (Howard Hughes Medical Institute and Harvard Medical School) and J. C. Lucchesi

(Emory University) and were used at 1:500 dilutions (except MSL-2: 1:250). Rabbit anti-histone

H4-acetyllysine 16 was purchased from Upstate Biotech (07-329) and used at a 1:400 dilution.

Texas Red or FITC-conjugated goat anti-rabbit secondary antibodies were used at 1:400

dilutions (Jackson, West Grove, PA). Chromosomes were stained with 100 ng/ml DAPI. Slides

were viewed with a Zeiss Axioskop microscope equipped with a Spot RT Slider CCD camera.

For viewing GFP fluorescence, salivary glands were fixed for 1 min with 3.7%

paraformaldehyde in PBS plus 5% Triton X-100, stained 20 min with 100 ng/ml DAPI in PBS

plus 2% Triton X-100, and washed 2 min in 50% glycerol. The chromosomes were then gently

spread in a fresh drop of 50% glycerol and viewed immediately.

Evaluating Nucleosome Positioning in BEAF-Binding Regions: Sixty-one genomic

regions on chromosome 2L were identified by ChIP-chip experiments (Jiang and Hart, in

preparation) as binding with high affinity to BEAF-32B. These sequences also contained

multiple CGATA motifs that are known to bind to the BEAF proteins (ZHAO et al. 1995).

Therefore we refer to these sequences as “BEAF-binding regions” (Bbr). Nucleosome

positioning data are available from the Penn State Cartography project (http://atlas.bx.psu.edu/)

63  

(MAVRICH et al. 2008). In that study genomic DNA from Drosophila embryos was digested with

micrococcal nuclease and hybridized to Affymetrix tiling arrays to identify the undigested

sequences protected by inclusion in a nucleosome. The genomic locations of the CGATA motifs

in the 61 BEAF-binding regions and the location of the mapped nucleosomes were entered into

Microsoft Excel, which was used for calculations and analysis. The fraction of motifs inside and

outside the nucleosomes was determined (presented as “actual”) as was the expected number of

the motifs if they were distributed randomly (“expected”). The expected ratios were calculated

by determining the percentage of base pairs within nucleosomes, and presuming that a random

distribution would result in a similar percentage of motifs to be within nucleosomes. The length

of inter-nucleosomal regions was determined by tabulating the number of inter-nucleosomal

regions that fall into the appropriate categories (i.e. 0-19 bp, 20-39 bp, etc). The same tabulation

was done for the BEAF-binding regions and the values are expressed as a ratio of the BEAF-

binding regions fraction to all of chromosome 2L, illustrating the difference between the BEAF-

binding regions and chromosome 2L.

Probe Synthesis for Southern Blot Analysis: The DNA sequences used for probe

construction were obtained by PCR amplification from either genomic DNA (adh, light, su(f)) or

by restriction digestion from plasmids containing the desired sequences (BE76, BE28, scs’ and

yellow). The PCR or restriction digestion products were then gel isolated and quantified using a

GeneQuant spectrophotometer. Incorporation of radioactive deoxyadenosine 5’-triphosphate [α-

32P] into Southern blot probes was done using a random-primed DNA labeling kit from Roche

(Ca# 1004760) following the manufacturer’s instructions. Briefly, 50 ng of DNA encoding the

desired probe sequence was heated to 95°C for 10 minutes, then random hexamers, dCTP,

dGTP, dTTP, 50µCi of 32P-ATP, and (exo-) Klenow polymerase (NEB) was added. The labeling

reaction occurred for 30 minutes at 37°C and stopped by the addition of 10 mM EDTA. The

64  

resulting probe was ethanol precipitated twice using 500 mM Tris and radioactive incorporation

was measured with a Beckman scintillation counter.

Nuclear Isolation and Nuclease Digestion: Wandering 3rd instar larvae were collected,

cleaned in PBS, frozen in liquid N2, and stored at -80°C. BEAFAB-KO larvae where identified as

described above. To isolate nuclei, 50 wildtype or 50 BEAFAB-KO larvae were thawed on ice and

ground in 500 µL Buffer A (15 mM Tris pH 7.4, 60 mM KCl, 8 mM NaCl, 0.1% NP40, 13 mM

EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM DDT, 0.05 mM PMSF)

using a Kontes micro pestle (Ca# K749520). If the nuclei were to be digested with DNase I then

only 1.0 mM EDTA was used in Buffer A. The homogenized larvae were then ground in 15 ml

Buffer A using 15 ml Kontes glass homogenizers (Ca# 885302-15) and pestle B. The

homogenized larvae were then filtered through a 125 micron monofilament nylon cloth and 5 ml

of Buffer AS (15 mM Tris pH 7.4, 60 mM KCl, 8 mM NaCl, 0.3 M sucrose, 0.5 M DTT, 0.5

mM PMSF) was added. The nuclei were pelleted by centrifugation at 3,000 g for 5 minutes and

the supernatant was discarded. The process of homogenization, filtering and centrifugation was

then repeated. For nuclei subjected to serial MNase digestion, the resulting nuclei were

resuspended once in 1.0 ml of MNase buffer (15 mM Tris pH 7.4, 60 mM KCl, 15 mM NaCl, 6

mM CaCl2, and 250 mM sucrose), transferred to a microtube, pelleted at 3,000 g for 5 minutes,

then resuspended a second time in 500 µL MNase buffer. A 5 µL aliquot was stained with DAPI

and viewed under a microscope to verify the presence of nuclei. A serial dilution of 50 ODU/µl

MNase (Sigma N5386) was made (1:160, 1:320, 1:640, 1:1280, 1:2,560) in MNase buffer. For

each MNase digestion, 90 µL of nuclei and 10 uL of diluted MNase was combined and digested

for exactly 5 minutes at room temperature. For DNase I digestions, DNase I (Sigma, D5025)

stock solution was diluted to 1.0 mg/ml according to the manufacturer’s instructions. A serial

dilution of DNase I was made using 1:6.25, 1:12.5, 1:25, 1:50 and 1:100 dilutions. For nuclei

65  

subjected to serial DNase I digestion, 90 uL nuclei were rinsed and resuspended in DNase I

buffer (50 mM Tris, 100 mM NaCl, 6 mM MgCl2, 1 mM DTT, 0.2 mM PMSF) and added to 10

uL of the appropriate DNase I dilution for 5 minutes. For MNase and DNase I, the reaction was

stopped by adding Stop Solution (20 mM Tris pH 7.4, 200 mM NaCl, 40 mM EDTA, 2% SDS)

and 50 µg of Proteinase K and digested overnight at 37°C. Following 3 phenol:chloroform

extractions and ethanol precipitation, the resulting pellet was digested with 15 ug RNase A for

1.5 hr at 37° C. This was followed by phenol:chloroform extraction, ethanol precipitation, and

resuspension in 15 µL TE.

Southern Blot Analysis and Indiret-End labeling: The digested DNA was resolved

over night at 4°C in a 15 cm long, 1.2% TBE agarose gel at 15V. If the DNA was to be viewed,

then the gel was stained with 0.5 µg/ml ethidium bromide and the DNA was imaged under ultra-

violet light. For Southern blot or indirect-end labeling the transfer of the DNA to a positively

charged nylon membrane was done as described (SAMBROOK and RUSSELL 2001). Briefly, 0.4 N

NaOH perfused through the agarose gel by upward capillary transfer and deposited DNA onto

the membrane. The membranes were rinsed at 65°C in 2X SSC for 5 min and blocked for 45

minutes with 50 µg of salmon sperm DNA in pre-hybridization buffer (2X SSC, 0.5% SDS.

0.5% Denhardt’s). Radioactive probes where then added and rotated overnight at 65°C. The

membranes were washed 3 times in 2X and twice in 0.3X SSC at 65°C, exposed to film and

developed.

Nucleosome-Scanning Assay: Nuclei from wildtype and BEAFAB-KO were isolated as

described above. The nuclei were then suspended in Cavalli’s cross-linking buffer (1.8%

formaldehyde, 50 mM HEPES, 1.0 mM EDTA, 0.5 mM EGTA, 100 mM NaCl pH 8.0)

(CAVALLI et al. 1999) for 10 minutes at room temperature. Cross-linking was stopped with 125

mM glycine. The nuclei were rinsed and resuspended in MNase Buffer. To obtain

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mononucleosomal size DNA fragments, 80% of the intact nuclei were then digested with a 1:160

dilution of 50 ODU/ul MNase for 5 minutes at room temperature, stopped with 12 mM EDTA,

and a 5 µL aliquot was viewed on an agarose gel to verify that mononucleosome size fragments

were obtained. Twenty percent was left undigested. The samples were then digested with 50 µg

proteinase K overnight at 37° C. The samples were then chloroform:phenol extracted 3 times and

ethanol precipitated. The DNA was quantified on a Nanodrop system, and qPCR reactions were

set-up on 96-well plates. For qPCR, each reaction consisted of 10 ng DNA, a 1:10,000 dilution

of stock SYBR green (Molecular Probes) was added to each reaction, and the reaction was

monitored by an ABS Prism detection System. Ct values from individual DNA samples were

normalized to each other using Ct values from a positioned nucleosome at hsp26 (THOMAS and

ELGIN 1988). The ratio of undigested to digested DNA for a given genotype was determined and

expressed as 2(undigested-digested). These values are expressed relative to the center positioned

nucleosome of scs’.

Chromatin Immunoprecipitation: Nuclei from 3rd instar larvae were prepared as stated

above, except that the initial homogenization of larvae was done in 4% formaldehyde in

Cavalli’s crosslinking buffer at room temperature for 10 minutes. The nuclei were then digested

with micrococcal nuclease as described in the Nucleosome-scanning assay. The chromatin was

then sonicated 4 times for 5 minutes at 30% power on ice. Sarkosyl was added to 0.5% final, the

chromatin was flash frozen in liquid N2 and stored at -80ºC. Chromatin Immunoprecipitation

was done as described (CAVALLI et al. 1999). Briefly, the chromatin was pre-cleared for 30

minutes with protein G sepharose beads. Twenty percent of each sample was isolated and used as

“input” for the qPCR reactions. The chromatin for ChIP was incubated with 4 µg of the indicated

antibody overnight, rotating at 4ºC. The beads were then rinsed extensively and crosslinks were

reversed by incubation at 65º for 6 hours. After phenol:chloroform extraction and ethanol

67  

precipitation with 2 µg linear acrylamide as carrier, the resulting pellet was resuspended in 60 µL

of TE, and input DNA was resuspended in 120 µL TE, with 1uL being used per qPCR reaction.

qPCR was set-up as described above.

Gene Expression Analysis: RNA was extracted from BEAF knockout and wildtype

embryos using TRIzol reagent (Invitrogen). 50-100 mg thawed embryos were added to 1 mL

TRIzol solution and were homogenized. After incubation at room temperature for 5 min, 200 µL

chloroform was added and shaken vigorously for 15 seconds. After incubation at room

temperature for 2-3 min, samples were centrifuged at 12000 g for 15 min at 4º C. The aqueous

phase was transferred to a new tube and 500 µL isopropanol was added. After 10 min at room

temperature samples were centrifuged at 12000 g for 10 min at 4º C. After removing the

supernatant, the pellet was washed with 1 mL 70% ethanol and centrifuged at 7200 g for 5 min at

4º C. After drying, the pellet was dissolved in 20 mL H2O and placed at 55-60 ºC for 10 min.

The RNA samples were stored at -80º C. Superscript III (Invitrogen) was used for cDNA

synthesis. For first strand synthesis, 1 µL of of 2 µM gene specific primers were added; 1 µL

RNA (2-3 µg); 1 µL 10 mM dNTP mix; H2O to total volume of 14 µL. Samples were heated at

65 ºC for 5 min and placed on ice for several minutes. After brief centrifugation, 4 µL 5× buffer

was added together with 1 µL 0.1 M DTT and 1 µL SuperScript III RT (200 units/µL). Samples

were incubated at 55 ºC for 30-60 min and 85 ºC for 5 min. One microliter of the product was

used in each quantitative PCR (described above). The gene expression levels tested from

different samples were normalized by the level of Trf mRNA.

Results

BEAF Mutations Perturb Male Polytene X Chromosome Morphology: Since the

expression of the BEAF-Interaction Domain described in Chapter 2 had a deleterious effect on

the structure of interphase chromatin structure, and because we hypothesize that BEAF functions

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by influencing chromatin structure, we prepared polytene chromosome squashes of 3rd instar

larvae salivary glands to view chromatin structure in the BEAFAB-KO line. The X-chromosome in

BEAFAB-KO males from heterozygous mothers shows a highly perturbed morphology (Figure 3-

1A). The chromosome appears decondensed and has lost the regular banding pattern of

condensed and decondensed regions of chromatin. The wildtype morphology of the chromosome

is restored by the introduction of a BEAF-GFP transgene (Figure 3-1B). The chromosome

morphology from the male and female offspring of BEAFAB-KO inter se crosses (therefore no

maternal contribution of BEAF present) displayed a range of perturbation on all chromosomes

(data not shown). Because of the unambiguous and consistent effect specifically observed on the

X-chromosome of males we looked at proteins associated with the dosage compensation

complex (DCC). The DCC proteins associate with the male X-chromosome and cause a two-fold

up-regulation of sex-linked genes (BONE et al. 1994; HAMADA et al. 2005). One component of

the DCC is MOF, which acts as a histone acetyltransferase and acetylates lysine 16 on histone

H4, a hallmark of the male X-chromosome. As can be seen in Figure 3-1A, MOF remains on the

X-chromosome despite the disrupted morphology. We also immunolocalized other protein

components of the DCC (MSL-1, MSL-2, MSL-3, and MLE) and found them to be present (data

not shown). In order to determine if the DCC functions normally in regulating X chromosome

transcription we conducted an assay utilizing expression from white alleles based on eye

pigmentation. The wa mutation normally shows dosage compensation (males and females have

similar eye pigment levels) and the we mutation does not (males have less eye pigment than

females) (LERACH et al. 2005). Eye-pigment levels were not affected by the BEAFAB-KO allele in

either line tested, indicating BEAF has no effect on dosage compensation (data not shown).

Thus, despite the apparent specificity for disruption of the X chromosome, the dosage

compensation complex appears to function normally.

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Polytene X chromosomes from BEAFA-KO males also had perturbed morphology (Figure

3-1C). However, the phenotype was less severe and more variable. The X chromosome

morphology ranged from normal or near normal to moderately perturbed. Thus flies lacking the

32A protein are not completely normal even though adults have no obvious phenotypes, are

healthy, and have normal fertility.

The BEAF-Binding Motif CGATA is Preferentially Located Between Nucleosomes

at BEAF-Binding Regions: Recent experiments in our laboratory (Jiang and Hart, in review)

used ChIP-chip to identify likely BEAF-binding regions in the Drosophila genome. Of the sites

identified, 61 regions gave particularly strong ChIP-chip results using an antibody against

BEAF-32B and contained several copies of the previously identified BEAF-binding motif

CGATA. Based on these two characteristics we decided to analyze these regions in more detail.

If BEAF functions by influencing chromatin structure at the level of nucleosome positioning,

then there may be a functionally relevant pattern or measurable correlation between the location

of BEAF-binding motifs and positioned nucleosomes. Nucleosome positioning data are available

from the Penn State Cartography Project (http://atlas.bx.psu.edu/). This database of nucleosomes

was obtained by crosslinking chromatin from embryos and digesting with micrococcal nuclease.

The remaining nucleosome-protected DNA was identified by hybridization to affymetrix genome

tiling microarrays (MAVRICH et al. 2008).

By determining the number of nucleosomes and CGATA/TATCG motifs within the 61

BEAF-binding regions we could calculate the expected fraction of motifs that should be found

inside nucleosomes given a random distribution. We found that 53% of the base pairs in BEAF-

binding regions are associated with nucleosomes. If BEAF-associated CGATA motifs are

distributed randomly then approximately 53% of them should also be nucleosomal. Our analysis

found that only 35% of the CGATA motifs are associated with nucleosomes (Figure 3-2A).

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Figure 3-1. BEAF mutations cause a disruption of male X polytene chromosome structure. (A) Salivary gland polytene chromosomes prepared from a wild-type male third instar larvae exhibit a normal banding pattern when the DNA is stained with DAPI. One chromosome arm of polytene chromosomes prepared from a BEAFAB-KO male has lost the banding pattern and appears shorter and broader. Indirect immunostaining with an antibody against MOF shows that it is the X chromosome that appears abnormal. (B) The GFBF transgene rescues the abnormal phenotype of the BEAFAB-KO male polytene X chromosome. (Top) Chromosomes stained with DAPI and gently spread in 50% glycerol without acid treatment to allow direct visualization of green fluorescent BEAF fusion proteins. (Bottom) Chromosomes that have undergone normal fixation, with the X chromosome identified by indirect immunostaining with an antibody against MOF. (C) Polytene chromosomes prepared from BEAFA-KO

males show a similar X chromosome phenotype, but it is less extreme and more variable. Note that 32B protein can be detected on these chromosomes by indirect immunofluorescence with an antibody against BEAF. The X chromosome is identified by indirect immunostaining with an antibody against MOF.

71  

Therefore, there is a higher than expected number of motifs in inter-nucleosomal regions. Chi-

squared analysis reveals these results are significant (p<0.0001). The evolutionary conservation

of this motif in inter-nucleosomal regions is consistent with the hypothesis that BEAF may

function by influencing chromatin at the level of nucleosome positioning or DNA-nucleosome

dynamics. In addition, BEAF-binding regions also contain a disproportionately high number of

large inter-nucleosomal regions (Figure 3-2b). We calculated the percentage of inter-

nucleosomal regions that fall into a given size category (i.e. 1-19 bp, 20-39 bp, etc.) This was

tabulated for chromosome arm 2L as a whole and for BEAF-binding regions. The results

expressed are the difference between what is observed for all of chromosome arm 2L and what is

observed among BEAF-binding regions, thus a value of 0 indicates there is no difference. The

‐1‐0.8‐0.6‐0.4‐0.2

00.20.40.60.81

log2

 (Bbr/Total)

inter-nucleosomal length

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53%

65%

47%

35%

0

10

20

30

40

50

60

70

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Inside

A

Figure 3-2. BEAF-32B is preferentially located in large inter-nucleosomal regions. Sixty-one regionsof chromosome arm 2L were identified by ChIP and sequence analysis as likely containing BEAF-32Bbinding sites (BEAF-binding regions). 331 CGATA/TATCG motifs were identified in these regions,and 215 nucleosome positions were mapped according to the Genome Cartography Project (see text).A) The BEAF-binding motifs CGATA/TATCG are preferentially located in inter-nuclesosomalregions. The Expected values were calculated as (# motifs)*(% of base pairs that are nucleosomal).(p<0.0001) B) The 61 BEAF binding regions contain a high proportion of large inter-nucleosomalregions relative to chromosome arm 2L. The length of all inter-nucleosome regions in the 61 BEAFbinding regions and all of chromosome arm 2L was determined. The y-axis is a log ratio of thepercentages of CGATA motifs that fall into the described categories. (y=log2[(fraction of regions of nsize in BEAF binding regions)/(fraction of regions of n size in 2L)] A value of 0 indicates no differencebetween BEAF binding regions and chromosome 2L. Bbr = BEAF binding region.

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data indicate there is a higher fraction of large inter-nucleosomal regions among BEAF-binding

sites. This is consistent with BEAF-binding regions being found in regions of low nucleosomal

occupancy, typically correlated with gene regulatory regions or active transcription.

Southern Blot Analysis of Several Chromosomal Regions Show a Normal

Nucleosomal Array: To further explore the relationship between BEAF and nucleosome

dynamics we hypothesized that the nucleosome-DNA interactions may be sufficiently perturbed

to observe in bulk nucleosome preparations, particularly on the X chromosome of males. Nuclei

from wildtype and BEAFAB-KO third instar larvae males were digested with micrococcal nuclease

and run on a 1.2% agarose gel with ethidium bromide. Under these conditions the size of the

various nucleosomal fragments (mono-, di-, tri-, etc) appear similar in wildtype and BEAFAB-KO,

indicating that genome-wide nucleosomes remain intact in the BEAFAB-KO line (Figure 3-3A). If

BEAF’s affect on DNA-nucleosome interactions are specific to a particular chromatin state or

chromosome then Southern blot analysis of various chromosomal regions should reveal this. The

digested chromatin in an agarose gel (as in Figure 3-3A) was transferred to a nylon membrane

overnight and probed with radioactively labeled DNA probes. The probes used were fragments

of genes representing various chromatin states. The gene alcohol dehydrogenase (adh) is found

in euchromatic regions of an autosomal chromosome; the light gene is found in heterochromatic

regions of an autosomal chromosome; the gene yellow is located in euchromatic regions of the X

chromosome; and the suppressor of forked (su(f)) gene is in heterochromatic regions of the X

chromosome. The results in Figure 3-3B demonstrate that, in all chromatin states examined, the

nucleosomal arrays appear unaltered (note: wildtype and BEAFAB-KO labels removed for clarity,

but are arranged in the same manner as Figure 3-3A).

If BEAF affects global patterns of gene regulation, then histone modifications may be

globally affected in either deposition levels or localization on chromosomes from the BEAFAB-KO

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line. This should be observable by immunostaining polytene chromosomes from 3rd instar larvae.

As mentioned above, previous reports have indicated that RNAi-mediated knockdown of BEAF

in S2 cell culture leads to a global increase in Histone H3, lysine 9 tri-methylation, a marker for

gene silencing. Immunostaining of Histone H1, Histone H3, lysine 4 mono-, di-, and tri-

methylation, lysine 9 di- and tri- methylation, lysine 9 acetylation, lysine 14 acetylation and

histone H4, lysine 16 acetylation has failed to demonstrate any global alteration of these histone

modifications (data not shown and Figure 3-3C). Furthermore, semi-quantitative western blot

analysis of these modifications has corroborated this finding; that no observable effects to global

expression patterns have emerged (data not shown).

Since BEAF binds to hundreds of sites on polytene chromosomes (ZHAO et al. 1995),

affects gene regulation (ROY et al. 2007b), and perturbs chromatin structure (GILBERT et al.

2006; ROY et al. 2007a), we considered it reasonable that there may be a global effect on

nucleosome-DNA associations and/or histone modifications. The data from this assay do not

support this hypothesis. Rather, global histone modifications and nucleosome positioning

remains unaltered and the phenotype observed on the male X-chromosome is likely a result of

the perturbation of a higher order chromatin structure.

Indirect-End Labeling of Known BEAF-Binding Sites Show Alterations to

Hypersensitive Sites Near Gene Regulatory Sequences in the BEAFAB-KO Line: Since global

nucleosome-DNA interactions were not visibly affected by MNase digestion in the regions

examined we focused our analysis on known BEAF-binding sites. Immunoprecipitation and

footprinting analysis has identified several genomic regions that BEAF binds to in vivo (CUVIER

et al. 1998). In addition to binding to BEAF, the sequences BE28, BE76, and scs’ also exhibit

the shared characteristic of multiple CGATA motifs and function as boundary elements in

transgene assays. One important distinguishing characteristic is that the BE28 sequence is a

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Figure 3-3 BEAFAB-KO does not show a global alteration in chromatin structure. A) Nuclei from wildtype and BEAFAB-KO 3rd instar larvae were isolated and the chromatin was digested with a serial dilution of micrococcal nuclease. After protienase K, samples were phenol-chloroform extracted and ethanol precipitated. Samples were then separated in a 1.2% agarose gel at 4°C. B) After nuclei isolation and MNase digestion as described in A the chromatin was transferred overnight to a nylon membrane and hybridized with a 32P-labeled probe for the euchromatic autosomal gene alcohol dehydrogenase (adh), the heterochromatic autosomal gene light, the euchromatic, sex-linked gene yellow, and the heterochromatic, sex-linked gene suppressor of forked (su(f)). C) Histone 3, lysine 9 tri-methylation immunostaining on polytene chromosomes has similar patterns in wildtype and BEAFAB-KO, being primarily localized to the centromeric heterochromatin. The exposure times of histone H3, lysine 9 tri-methylation and DAPI have identical ratios in wildtype and BEAFAB-KO, and no changes in expression levels are apparent in the BEAFAB-KO. multicopy element located near centromeric heterochromatin and is apparently not associated

with any genes or known gene regulatory elements. In contrast, BE76 is located near the

promoter of the raspberry IMPDH gene, and the scs’ sequence is in the center of two divergently

transcribed genes, aurora and CG3281. To examine chromatin structure in these regions we

conducted Southern blot analysis and indirect-end labeling using both micrococcal nuclease and

DNase I.

Indirect-end labeling of BE28 revealed no distinguishable alterations to hypersensitive

sites (HS) in BEAFAB-KO (Figure 3-4A). After digestion with MNase or DNase I and subsequent

isolation of the BE28 fragment with HindIII digestion, there are weak HS present in both

wildtype and BEAFAB-KO and the region appears to be generally sensitive to digestion. Digesting

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Figure 3-4. Indirect-end labeling and Southern blot analysis of the BEAF-binding region BE28 shows no effect in the BEAFAB-KO. Nuclei from 3rd instar larvae were isolated and subjected to a serial dilution of DNase I or MNase. A) The digested chromatin was then subjected to HindIII digestion to isolate the BE23 fragment. The DNA was separated by gel electrophoresis and transferred to a Nitrocellulose or Nylon membrane for blotting with a radioactive probe. The region indicated “a/b” contains the inverse CGATA motifs. W:wildtype, and K:BEAFAB-KO. B) Southern blot analysis using the same probe as in A, with no HindIII digestion. Samples are arranged the same as in A (labels removed for clarity).

 

the chromatin with MNase and using the same probe as in part A (with no HindIII digestion)

reveals a normal nucleosomal array pattern (Figure 3-4B).

The results are different when examining HS near gene regulatory regions. Digesting

chromatin with MNase or DNase I and isolating a BglII fragment containing scs’, or a PvuII

fragment containing BE76 does reveal subtle alterations to HS in the BEAFAB-KO line (Figure 3-

5A). For chromatin digested with MNase, the scs’ fragment shows the loss of a distinct HS

occurring at the BEAF-binding site labeled D, and the formation of a new HS a few base pairs

above it. The BEAF- binding site labeled “B” is adjacent to the CG3281 promoter. DNase I

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digestion shows the loss of a HS near this promoter at the EcoRI site in the BEAFAB-KO. This is

consistent with either subtle nucleosome remodeling in the vicinity, or alterations in protein-

binding that affect nuclease sensitivity.

The PvuII fragment containing the BE76 fragment also shows alterations to nuclease

sensitivity in the BEAFAB-KO line (Figure 3-5B). In the MNase digested sample, the HS that is

located near the BEAF-binding sequences labeled “b” becomes more defined in the BEAFAB-KO

and more hypersensitive to digestion relative to the triplicate of sites located above it. The three

sites located above the “a” and “b” binding sites become less sensitive in the knockout, and a

new HS forms even further upstream. For DNase I-treated chromatin, the HS near the “a” and

“b” binding site shifts upward. These results are consistent with the explanation that in the

absence of BEAF there is a shifting of nucleosomes that occurs, or in the case of BE76 that

nucleosomes in a given population of cells are altogether absent. Combining the results from

BE28, BE76, and scs’ it appears as though BEAF acts locally at regions near promoters in a

manner that influences nucleosome dynamics or other remodeling functions.

  A Nucleosome-Scanning Assay Reveals Changes to the Chromatin Structure at the

Scs’ BEAF-Binding Site: Since it appears that BEAF is affecting chromatin dynamics in a

subtle manner near its binding sites by promoters, we decided to focus on scs’ to elucidate in

better detail the possible effect to nucleosome dynamics caused by a lack of BEAF. To this end

we employed a nucleosome-scanning assay. Experiments similar to the nucleosome-scanning

assay have been used in humans, Drosophila, and yeast to determine the removal or shifting of

nucleosomes at promoter regions (MAVRICH et al. 2008; PETESCH and LIS 2008; SEKINGER et al.

2005).

Nuclei from 3rd instar larvae were isolated as described above and chromatin was

digested to obtain mononucleosome-size fragments. Quantitative PCR using an overlapping

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Figure 3-5 Indirect-end labeling and Southern blot analysis of the BEAF-binding regions scs’ and BE76 shows effects to HS location and sensitivity in the BEAFAB-KO. Nuclei from 3rd instar larvae were isolated and subjected to a serial dilution of DNase I or MNase. A) The digested chromatin was then subjected to BglII digestion to isolate the scs’-containing fragment. The DNA was separated by gel electrophoresis and transferred to a Nylon membrane for hybridization with a radioactive probe. The regions indicated “B” or “D” are previously identified HS sites. W:wildtype, and K:BEAFAB-KO. B) After Mnase or DNase I digestions the chromatin was digested with PvuII to isolate the BE76-containing fragment. After electrophoretic separation and transfer to a membrane the DNA was probed with the indicated radioactive fragment. Arrows indicate transcription start sites.

 

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series of primers that generate ~100 bp products were then used to determine which regions of

DNA are protected from digestion by inclusion in a nucleosome. Nineteen primer pairs that span

the region were used. These encompass the transcription start site (TSS) of CG3281, the BEAF-

binding regions in scs’ and the TSS for aurora (Figure 3-6A). The results indicate that subtle

alterations occur to DNA-histone interactions at scs’ in the BEAFAB-KO. The edge of the

nucleosome at the TSS of CG3281 is spanned by primer pair #2 (Figure 3-6B). This particular

primer pair shows a hypersensitivity to digestion in the BEAFAB-KO line. The same effect is

observed upstream at the left side of the center nucleosome, spanned by the 8th, 9th, and 10th

primer pairs. This is consistent with the chromatin peeling off of the edges of the nucleosome, as

has been observed to be caused by some nucleosome remodeling complexes (BAZETT-JONES et

al. 1999). The alterations observed at the aurora TSS demonstrate an effect consistent with the

nucleosome shifting to cover the TSS.

Since the scs’ region was analyzed by both indirect-end labeling and nucleosome

scanning assay the results from both experiments can be compared (Figure 3-6D). The

nucleosome at the TSS at CG3281 may have been evicted or shifted in the BEAFAB-KO line,

resulting in a smaller HS (“a”) and increased sensitivity to digestion of primer pair #2 (Figure 3-

6B). It is also likely that the presence or absence of transcription factors and other components of

the transcription complex could contribute to any perturbation of HS sensitivity, making the

interpretation of these results more complex.

The effects observed at the TSS of aurora is consistent with a scenario by which there are

two distinct cut sites near the D site (Figure 3-6D, labeled b and c), each one located at the edge

of the neighboring nucleosomes. In the BEAFAB-KO line the D site is more heavily digested

(perhaps due directly to a lack of BEAF binding) and the HS “c” near the aurora TSS

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Figure 3-6 The Nucleosome-scanning assay reveals changes in chromatin structure at the BEAF-binding locus scs’. Nuclei from wildtype and BEAFAB-KO 3rd instar larvae were isolated and 80% of each genotype was digested with micrococcal nuclease to obtain mononucleosome size DNA fragments (digested). DNA from the remaining 20% of nuclei was left intact (undigested). Quantitative PCR reactions were carried out using primers that scan the scs’ sequence (dashes, part A). The amplicons depicted as lines are 90-110 bp fragments with 20-40 bp between the 5’ primers. B) The values depicted are ratios of Ct values from undigested to digested DNA, indicating the relative amounts of DNA that was resistant to digestion. The positioned nucleosome at the hsp26 locus was used as a control to indicate digestion of the inter-nucleosomal regions. The Internal values indicate relative DNA enrichment using primers that amplify within the positioned nucleosome, and the External primers indicate enrichment when one primer is located ~20 bp upstream of the nucleosome. The results from 3 biological replicates are reported here. C) The scs’ region contains 3 nucleosomes and two divergently transcribed genes. The regions labeled B and D are previously characterized HS containing repeats of the BEAF-binding motif CGATA. D) The autoradiograph depicted in Figure 3-5A of scs’ treated with MNase repositioned for comparison. HS a-d are altered in the BEAFAB-KO samples (see text).

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disappears. The loss of the HS “c” and the resistance to MNase (Figure 3-6B, primers #16 and

#17) is consistent with nucleosome eviction or shifting towards the aurora TSS.

BEAF Affects Histone Modifications at Divergently Transcribed Genes: Since BEAF

has been reported to affect histone modifications and transcription levels of BEAF-associated

genes, we decided to investigate if histone modifications were altered in the BEAFAB-KO line. In

addition to scs’, three additional genomic regions were selected for ChIP analysis. Previous

ChIP-chip experiments have identified these genomic regions as major sites of BEAF binding

(N. Jiang, pers. communication) and contain several copies of the BEAF-32B binding motif

CGATA. Additionally, these motifs are between two divergent gene pairs. We have conducted

RT-PCR and ChIP analysis in these regions and in scs’. This allows us to examine alterations to

the histone modifications to histone H3 lysine 9 tri-methylation (H3K9me3) and histone H3

lysine 4 di-methylation (H3K4me2) and analyze this in the context of gene expression levels in

wildtype and BEAFAB-KO lines.

The results indicate that histone modification processes may be perturbed in the BEAFAB-

KO line, although the perturbations may vary from gene to gene. General observations are that

H3K4me2 is not necessarily correlated with increases in gene expression, and there are generally

lower levels of H3K9me3 in the BEAFAB-KO line. The RpS6/bys gene pair (Figure 3-7A) indicates

an increase in both H3K9 and H3K4 methylation correlated with an increase in gene expression.

The cpsf/Asx gene pair however demonstrates a decrease in H3K9me3 with decreased

transcription. Also exhibited at this gene pair is a high level of H3K4me2 at the cpsf gene not

correlating with higher gene expression levels (Figure 3-7B). The CG33671/Iswi also exhibits

lower levels of H3K9me3 with a decrease in transcription levels. At scs’, despite the evidence

for changes to nucleosome positioning, there are no observed effects on gene expression levels

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(Figure 3-7D). Consistent with this, there are no changes in H3K4me2 at the TSS, and again,

lower levels of H3K9me3.

 

Figure 3-7 ChIP analysis of histone 3 lysine 4 di-methylation and histone 3 lysine 9 tri-methylation at 4 putative BEAF-binding regions. Nuclei from wildtype and BEAFAB-KO 3rd instar larvae were isolated, crosslinked, and subjected to MNase digested to obtain mononucleosomes. ChIP was conducted to 80% of each preparation with antibodies against histone 3 lysine 4 di-methylation (H3K4me2) and histone 3 lysine 9 tri-methylation (H3K9me3). Primers were designed to amplify regions protected by nucleosomes (gray boxes). Quantitative PCR was then used to determine the relative enrichment of sequences associated with each nucleosome (labeled 1 through 5 or 1 through 3). Percentage enrichment was determined by comparing Ct values of ChIP chromatin to input, and the values expressed are ratios of percent encrichment of BEAFAB-KO to wildtype. A value of “0” indicates DNA sequences below detectable levels. Scale bar = 150 bp.

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Discussion

To help elucidate a mechanism for determining how BEAF functions in vivo we analyzed

the structure of chromatin in nuclei from wildtype and BEAFAB-KO Drosophila larvae. Alterations

to micrococcal nuclease sensitivity are observed in the BEAFAB-KO line, suggesting that BEAF

acts by influencing chromatin structure at the level of nucleosome positioning and/or dynamics.

Furthermore, these effects are not observed globally or to regions that are specific for a particular

chromatin state (such as heterochromatin or the X-chromosome) that do not have known BEAF-

binding sites (Figure 3-3).

The effects observed at hypersensitive sites in the BEAFAB-KO were limited to regions near

promoters. We have determined previously that BEAF binds to hundreds of sites on polytene

chromosomes, and additional data from our laboratory indicates that most of these binding sites

are near transcription start sites (pers. communication, N. Jiang) (ZHAO et al. 1995). Taken

together, this implies that BEAF may play a role in the recruitment, organization, or dynamics of

nucleosome positioning, which are known to influence transcriptional processes (SCHULZE and

WALLRATH 2007). Conceptually, this is in contrast to a model of BEAF forming an array of

nucleoprotein complexes that act to divide the genome into independently regulated domains

(UDVARDY et al. 1985).

While the scs’ sequence has traditionally been characterized as an insulator sequence, in

vivo it is located at the transcription start site of two genes, aurora and CG3281. It is noteworthy

that the B and D binding sites illustrated in Figure 3-6A are on opposite sides of a nucleosome,

therefore brought into close proximity to each other after wrapping around the histone core. It

has been demonstrated that BEAF-32B binds to both of these sequences and that both BEAF

32A and 32B interact in vivo via a C-terminal self-interaction domain. Finally, binding of 32B to

the D site greatly facilitates binding to the B site, indicating cooperative binding (HART et al.

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1997). With this evidence in mind, the subtle shifting of this center nucleosome observed in the

BEAFAB-KO line is consistent with a model by which the BEAF proteins are bound to the B and D

site, and interacting with each other, thus forming a complex to hold the nucleosome in place. A

loss of BEAF would then result in a susceptibility to digestion of the DNA at the edges of the

nucleosome as is observed in Figure 3-6B and D.

The BE76 sequences are within the transcribed sequences of the raspberry IMPD gene,

the rate-limiting enzyme in guanine nucleotide synthesis which has been implicated in cell-cycle

control. This gene has very complex expression patterns involving several developmentally

regulated transcripts (SLEE and BOWNES 1995). According to the online nucleosome positioning

maps, the CGATA motifs located at BE76 are located at the edge of the +4 nucleosome of the

raspberry locus, corresponding exactly to the location of the perturbed HS (“a” and “b”, Figure

3-5B and data not shown). It is possible that BE76, like scs’, is binding to a complex of BEAF

proteins that act to stabilize the +4 nucleosome. Although there are no readily identifiable motifs

on the other side of this positioned nucleosome (data not shown), we know that BEAF binds to

as yet uncharacterized sequences (N.Jiang, personal communication). Thus it is conceivable that

alterations identified in our HS mapping assays at BE76 are also a result of subtle nucleosome

repositioning (Figure 3-5 and Figure 3-6). In contrast, the lack of HS alterations at BE28 in the

BEAFAB-KO line may be attributable to the fact that it is located as part of a repeating element

found primarily near centromeric heterochromatin and not near any known genes (CUVIER et al.

2002).

Subtle nucleosome displacement or shifting at promoters has been observed as a common

and essential mechanism for regulation of several genes. Notable examples include the PHO5

gene in yeast where several independent pathways mediate the shifting of a nucleosome to

provide transcription factor TFIID access to the TATA box (MARTINEZ-CAMPA et al. 2004). The

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nucleosome remodeling and transcription that occurs there is dependent on binding of the trans-

acting regulatory factor PHO4 to the DNA. DNase I digestion assays of chromatin from yeast

with mutant pho4 alleles demonstrate subtle HS alterations at the site of nucleosome disruption

(FASCHER et al. 1990; SVAREN and HORZ 1995), very comparable to what is observed here in the

BEAFAB-KO line. Subsequent to the binding of PHO4, several factors participate in nucleosome

destabilization, leading to the recruitment and accumulation of SWI/SNF chromatin remodeling

complexes and general transcriptional machinery (ADKINS et al. 2007). Minor nucleosome

perturbation at promoters was also observed in a more recent genome-wide study in human cells.

An analysis of this type has demonstrated that nucleosome positioning at promoters in T cells

shift by an average of 30 base pairs upon gene induction (SCHONES et al. 2008). This is the

average distance between primers used in our nucleosome-scanning assay where an effect on

nucleosome positioning may be evident by only one primer pair (Figure 3-6B).

Histone modifications are a hallmark of transcriptional regulation and many histone

modifications at known enhancers are positively associated with DNase I hypersensitive sites in

the human genome (WANG et al. 2008). Thus our observations of HS alterations are consistent

with the possibility of alterations to histone modifications. Furthermore, it has been found that

histone tail modifications can influence sequence-directed translational positioning of a

nucleosome (YANG et al. 2007). We looked at H3K9me3 because of reports that this

modification is increased after RNAi-mediated knockdown of BEAF in Drosophila S2 cells

(EMBERLY et al. 2008). We did not find this to be the case in BEAFAB-KO larvae, both by

immunostaining polytene chromosomes and by ChIP analysis (Figure 3-3C, Figure 3-7, and data

not shown). In fact, the quantitative analysis of the ChIP experiments and a less quantitative but

thorough examination of immunostained polytene chromosomes and diploid brain cells suggest

that the opposite may actually be true. This may be attributable to the differences in experimental

86  

models (larvae vs cell culture) or due to the differences in the regions analyzed. Another

possibility is that S2 cells likely underwent many fewer rounds of DNA replication compared

with BEAFAB-KO larvae, leading to observations of a more direct effect in S2 cells. The BEAFAB-

KO larvae, however, have ostensibly undergone disruption to several developmental processes

leading to an observation of cumulated indirect effects. Furthermore, since lower levels of

transcription are commonly observed with decreased H3K9me3 in our data, it is possible that

other mechanisms are responsible for the decreases observed in transcription. This is reasonable

since H3K9 mono- or di- methylation are associated with euchromatic repression and H3K9me3

is associated with pericentric heterochromatin. Additionally, evidence suggests that different

methyltransferases are responsible for the different marks (RICE et al. 2003).

H3K4me2 is well documented as an activator of transcription, but our data has failed to

find this correlation at the genes studied (Figure 3-7). Our results found decreases in

transcription levels (RpS6, Asx, CG33671, Iswi) with no corresponding decrease in H3K4me2.

This suggests that H3K4me2 may occur at the genes examined, but a lack of BEAF still prevents

transcription from occurring.

A genome-wide analysis of nucleosome positioning in Drosophila has revealed different

nucleosomal architectures occurring at promoters depending on the presence of known

regulatory elements. Most highly expressing genes have a canonical nucleosomal array starting

downstream of the promoter and lack a TATA box, initiator sequence, or other core promoter

element to attract general transcription machinery, suggesting they are dependent on specific

transcription factors. Genes with these regulatory sequences have a diminished nucleosome-free

region near the promoter and a non-canonical array, implicating they may adopt gene-specific

chromatin architectures (MAVRICH et al. 2008). This assortment of nucleosomal architectures at

promoters could lead to the complexity of molecular consequences that is observed among genes

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in the BEAFAB-KO line, such as in the case of histone modifications (Figure 3-8).

Despite the lack of correlating H3K4me2 with activation and H3K9me3 with repression,

it is apparent that levels of the modifications are altered in the BEAFAB-KO line. In order to better

determine what is occurring at these sites it will be necessary to conduct further ChIP analyses.

First, ChIP analysis using an antibody against histone H3 will indicate general nucleosome

occupancy. We can then determine if our observations with H3K9me3 and H3K4me2 are due to

alterations in the modification or if the nucleosome occupancy has been changed. Second, other

evidence from our lab has indicated that BEAF is often found in conjunction with the

transcription elongation inhibitor NELF, which in turn is found commonly associated with

paused RNA polymerase (LEE et al. 2008). Interestingly, a depletion of NELF results in a subtle

shifting of nucleosomes near the paused polymerase as measured by micrococcal nuclease

sensitivity, similar to some of our observations in the BEAFAB-KO line (MAVRICH et al. 2008).

In further support of BEAF’s possible role in transcription, ChIP evidence from our lab

indicates that BEAF binding sites are found near transcription start sites, and in the case of 32B,

these sites are preferentially nucleosome-free regions (Figure 3-2). Also, the data presented here

indicates that BEAF-32B comes off of condensed chromatin during mitosis (see Chapter 4,

Figure 4-2), a common occurrence among transcription-related proteins and RNA polymerase II

(CHEN et al. 2005). Finally, while BEAF-32A and BEAF-32B can interact, and this interaction

influences DNA-binding capacity in band shift assays, fluorescence microscopy demonstrates

that BEAF-32B binds at many regions of decondensed chromatin on polytene chromosomes

forming very intense, discreet banding, often irrespective and sometimes without BEAF-32A

binding (see Chapter 4, Figure 4-1B) (HART et al. 1997). This punctated pattern of BEAF-32B

binding is reminiscent of known transcription factors such as DREF (data not shown).

In conclusion, our results suggest that BEAF may play a role in transcriptional processes

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occurring at promoter regions. This insight should entail a different approach to future

experimental designs regarding BEAF, because while its ability to function as an insulator has

been made evident, its possible role as a transcriptional regulator has not. Future work in general

should focus on refining BEAF’s role in this capacity, which in turn will help better elucidate the

broad set of mechanisms occurring at promoter complexes.

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linked to promoter-proximal pausing at many genes in Drosophila. Mol Cell Biol 28: 3290-3300.

LERACH, S., W. ZHANG, H. DENG, X. BAO, J. GIRTON et al., 2005 JIL-1 kinase, a member of the

male-specific lethal (MSL) complex, is necessary for proper dosage compensation of eye pigmentation in Drosophila. Genesis 43: 213-215.

MARTINEZ-CAMPA, C., P. POLITIS, J. L. MOREAU, N. KENT, J. GOODALL et al., 2004 Precise

nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1. Mol Cell 15: 69-81.

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PATHAK, R. U., N. RANGARAJ, S. KALLAPPAGOUDAR, K. MISHRA and R. K. MISHRA, 2007 Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix. Mol Cell Biol 27: 4796-4806.

PETESCH, S. J., and J. T. LIS, 2008 Rapid, transcription-independent loss of nucleosomes over a

large chromatin domain at Hsp70 loci. Cell 134: 74-84. RICE, J. C., S. D. BRIGGS, B. UEBERHEIDE, C. M. BARBER, J. SHABANOWITZ et al., 2003 Histone

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YAMAGUCHI, M., H. YOSHIDA, F. HIROSE, Y. H. INOUE, Y. HAYASHI et al., 2001 Ectopic

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boundary element-associated factor BEAF-32. Cell 81: 879-889.

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Chapter 4: Utilizing Fluorescently-Tagged BEAF-32A and BEAF-32B to Analyze BEAF Localization and Dynamics In Vivo

Introduction

It has been well established that BEAF-32A and BEAF-32B (herein referred to

individually as 32A and 32B, respectively) are capable of functioning as insulator proteins; they

can prevent enhancer activation of a promoter when placed between them and they can insulate a

transgene from chromosomal position effects (KELLUM and SCHEDL 1991; KELLUM and SCHEDL

1992; ROY et al. 2007). A mechanism of how BEAF accomplishes this has yet to be elucidated.

However, evidence presented here and elsewhere suggests that BEAF may function by

regulating chromatin structure or dynamics (EMBERLY et al. 2008; GILBERT et al. 2006; ROY et

al. 2007).

The functional relationship between 32A and 32B has been partially characterized.

These proteins are alternative transcripts of the same gene, likely a result of alternate promoters.

The proteins differ only at their amino-termini which both have atypical Zn-fingers termed BED-

finger domains (after the Drosophila proteins in which they were first found, BEAF and DREF)

(ARAVIND 2000). Yeast two-hybrid analysis of truncated BEAF proteins has determined that the

amino-termini of both proteins are responsible for DNA binding and the identical carboxy-

termini are necessary and sufficient for the formation of BEAF complexes. This region has a

potential leucine zipper and a BESS domain, one or both of which are probably responsible for

BEAF-BEAF interactions (HART et al. 1997).

The hsp70 genes in Drosophila are bracketed by the scs and scs’ sequences. The

formation of nucleoprotein complexes at these sequences were originally thought to form

boundaries that limit the regulatory signals for hsp70 from exerting influence on neighboring

genes (UDVARDY et al. 1985). The BEAF proteins bind to the scs’ sequence and other genomic

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sequences containing clusters of CGATA motifs (CUVIER et al. 1998; ZHAO et al. 1995). In

addition, BEAF also binds to many genomic regions without these motifs (pers. communication,

N. Jiang).

32A and 32B have been shown to interact in vivo, as protein-specific antibodies for 32A

or 32B will immunprecipitate complexes from nuclear extracts consisting of both proteins. The

scs’ sequence described above is comprised of two sets of CGATA motifs. Termed the “B” and

“D” sites, they are located on opposite sides of a positioned nucleosome (see Chapter 3). Band

shift experiments show that 32A binds strongly to the D site, but will not bind to the B site. 32B

will bind to both sites, but its affinity for the B site is much lower than for the D site. Both 32B

and heterocomplexes of 32A and 32B from nuclear extracts will cooperatively bind to the B site

in the presence of the D site in vitro (HART et al. 1997). Thus BEAF complexes can

cooperatively interact even when their binding sites are separated by 200 bp of DNA.

Despite these previous characterizations - the different DNA-binding domains, the in vivo

interaction of 32A and 32B, the cooperative binding properties observed at scs’, and the

characterization of scs’, we still do not have a comprehensive description of how BEAF mediates

it’s insulator properties. It is also unclear if BEAF’s role as an insulator protein is its only role in

the nucleus. While the presence of a BEAF-binding site may confer insulator properties in

transgene assays, this could be an indirect consequence of BEAF’s primary role.

Here, in an attempt to better characterize 32A and 32B individually, we have

fluorescently-tagged 32A with Green Fluorescent Protein (32A-GFP) and 32B with Red

Fluorescent Protein (32B-RFP). This study will report on the preliminary work of utilizing these

fluorescently-tagged proteins to characterize BEAF protein dynamics and behavior in vivo.

Our results presented here will demonstrate that 32A and 32B distribution on polytene

chromosomes are different and they exhibit different behaviors during mitosis. Finally,

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preliminary FRAP experiments indicate that the proteins have different dynamics during

interphase.

Materials and Methods

Plasmid Construction and Microinjection: 32A-GFP and 32B-RFP plasmid constructs

were made using standard cloning techniques. Briefly, a plasmid containing a previously

characterized full length genomic BEAF-EGFP construct underwent PCR mutagenesis to delete

~200 base pairs spanning that ATG start codons and most of the coding sequences for the ~80

amino acid unique amino-terminal regions of 32A or 32B (ROY et al. 2007). The sequences 1kb

upstream of the 32A-specific and 32B-specific transcribed regions were kept intact in case

unknown regulatory regions are present, and the shared exon was also left intact. Coding

sequences for monomeric RFP were PCR amplified from pTRW (CAMPBELL et al. 2002) and

substituted for EGFP coding sequences to result in plasmids with BEAF-32A-GFP or BEAF-

32B-RFP. Both BEAF-32A-GFP and BEAF-32B-RFP full length constructs were inserted into a

pCaSpeR4 vector containing the mini-white gene for easy selection, P-element insertion

sequences and the boundary elements scs and scs’ to protect the transgene from chromosomal

position effects. Microinjections were done with the p399 helper plasmid on a Nikon Diaphot

inverted phase contrast microscope with Narishige Injectors in the lab of P. DiMario and with the

technical expertise of R. Rosby.

Microscopy: Mitotic brain cells and interphase salivary gland nuclei were examine

following established protocols with minor modifications (DIMARIO et al. 2006). Briefly,

wandering 3rd instar larvae were dissected in Brower’s solution (0.15M PIPES, 3mM MgSO4,

1.5mM EGTA, 1.5% NP40) with 2% formaldehyde. Glands or brains where then transferred to

PBS/1%TTX-100 and 3.7% formaldehyde with a 1:100 dilution of 1 mg/ml DAPI for 20

minutes. After rinsing in 50% glycerol the glands were gently squashed between a coverslip and

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microscope slide. For brains the tissue was smeared into a monolayer by rotating the coverslip

around the slide. Slides were immediately viewed with a 100X oil immersion lens with a Ziess

Axioskop microscope equipped with a Spot RT Slider CCD camera. Images were cropped and

labeled using Adobe Photoshop.  

Fluorescence Recovery After Photobleaching: Salivary glands of wandering 3rd instar

larvae were selected, rinsed and dissected in Nutritive Media (Shields and Sang M3 Insect

Medium, 5% Fetal Calf Serum, 2.5% Fly Extract, 10 ug/ml porcine insulin) (CHERBAS 2007).

The nuclei of cells with similar brightness were chosen for FRAP experiments. A region of

interest (ROI) in these nuclei was photobleached with a laser beam set to 100%. Fluorescence

recovery in this ROI was monitored over time using the FRAP software provided by Leica. As

controls, fluorescence intensities were monitored within and outside the tested nucleus. Recovery

times for 3 replicates were evaluated by analysis of the recovery plots determined by the Leica

software, and the data is presented as the mean ± standard deviation.

Results

BEAF-32A and BEAF-32B Demonstrate Different Patterns of Localization: Since

early models predicted that 32A and 32B interact to form functional complexes, it was of interest

to determine their relative localization on polytene chromosomes. Salivary glands from 3rd instar

larvae expressing 32A-GFP or 32B-RFP, or both, were fixed and squashed. Polytene

chromosomes are divided into regions of condensed and uncondensed chromatin, and are

distinguishable by the intensity of DNA staining that appears with DAPI. Condensed chromatin

stains darkly and uncondensed chromatin stains lightly. The analysis of BEAF banding patterns

indicates that 32A-GFP localizes broadly and is uniformly associated with the DNA. However

there are a small number of loci where distinct and exclusive 32A-GFP bands appear. In contrast,

32B-RFP exhibits a very localized and banded pattern, more often found in decondensed regions.

96  

Curiously, 32A-GFP and 32B-RFP sometimes appear to border each other in a manner that

suggests that one excludes the other, although this does not agree with biochemical evidence.

Figure 4-1 BEAF 32A-GFP and BEAF-32B-RFP exhibit distinct banding patterns on polytenechromosomes from 3rd instar larvae. A) The 32A-GFP genotype is +/+; BEAF AB-KO/CyO; 32A-GFP and the32B-RFP fluorescence is from larvae with the genotype 32B-RFP; BEAF AB-KO; +/+. Scale bar = 10 µm. B)All images are from genotype 32B-RFP/+;BEAF AB-KO/CyO;32A-GFP/+. Banding patterns of 32A-GFP and32B-RFP exhibit distinct banding patterns, with 32A localizing with DNA and 32B forming discreet bandsin primarily euchromatic regions. Scale bar = 5 µm.

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The pattern suggests that 32A binds with very little sequence specificity, while 32B is targeted to

many fewer sites.

BEAF-32A Remains on Chromosomes Throughout Mitosis and BEAF-32B

Disassociates During Prophase: Brains from 3rd instar larvae of 32B-RFP/+; BEAF AB-KO/CyO;

32A-GFP/+ were fixed, stained with DAPI and squashed to form a monolayer of cells on a slide.

The squash could then be scanned for cells undergoing mitosis identifiable by DAPI

staining. Multiple cells undergoing mitosis displayed the observations seen in Figure 4-2. At

prophase, metaphase, and anaphase 32A-GFP remains associated with condensed chromosomes.

Alternatively, 32B-RFP begins to disassociate during prophase, and is seen diffuse throughout

Figure 4-2 Brain cells from 3rd instar larvae undergoing mitosis demonstrate that BEAF-32Aremains associated with chromatin throughout mitosis whereas BEAF-32B disassociates duringprophase. The genotype of the individuals above is 32B-RFP/+;BEAFAB-KO/CyO;32A-GFP/+.Scale bar = 5 µm.

98  

the cell and unassociated with the chromatin in metaphase and anaphase. This is similar many

other proteins that disassociate from condensed mitotic chromosomes, such as RNA polymerase

II (CHEN et al. 2005).

Fluorescence Recovery After Photobleaching (FRAP) Experiments in Polytene

Nuclei Demonstrate that BEAF-32A and BEAF-32B Have Different but Inter-Dependent

Protein Dynamics: Salivary glands from wandering, third instar larvae were dissected in a

nutritive media supplemented with fetal calf serum, fly extract, and insulin. The larvae were

either homozygous for the BEAF-32A-GFP transgene on the 3rd chromosome (32A-GFP),

homozygous for the BEAF-32B-RFP transgene on the X chromosome (32B-RFP), or

homozygous for a null BEAF mutation on the second chromosome and homozygous for BEAF-

32B-RFP on the X chromsosome (KO/KO; 32B-RFP). Our attempts to make a fly line

homozygous for endogenous BEAF mutations and the 32A-GFP transgene were unsuccessful,

indicating that 32A alone is not sufficient to rescue the lethality observed in the BEAFAB-KO line,

however 32B is necessary for viability (data not shown). This is in agreement with our previous

finding that a fly line with a BEAF-32A null mutation demonstrates normal viability (ROY et al.

2007). The dissected glands were then placed in an examination chamber prepared for inverted

confocal microscopy with the media described above. After locating an intact, fluorescing nuclei

the region of interest (ROI) was selected and photobleached. A time course of images was taken

and the fluorescence recovery time was determined using software provided by Leica. The

average of three replicates is displayed in Figure 4-3.

When both 32A and 32B are present, the results indicate that 32A recovers about 3 times

faster than 32B. However, if 32A is not present then the recovery time of 32B appears to

increase, although the data suggests the increase is not statistically significant. The faster

recovery times suggests that 32A has faster dynamics, perhaps due to lower affinity binding to

 

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100  

transgene. The experiments described here highlight additional differences between 32A and

32B.

We have shown here that the proteins demonstrate very different localization on polytene

chromosomes. It appears as though 32A binds with little specificity, coating the DNA in both

bands and interbands. It also has faster recovery kinetics in FRAP experiments, suggesting it

binds with a lower affinity then 32B, yet it remains associated with mitotic chromosomes.

Previous experiments have demonstrated that polytene chromosome structure from larvae with

null mutations in 32A appears slightly perturbed (ROY et al. 2007). Combined, this data suggests

that 32A may play a very general role in chromatin structure that is particularly important during

mitosis.

The 32B protein demonstrates what appears to be a much higher specificity for many

discrete genomic loci. The highly localized banding pattern is reminiscent of transcription factors

such as DREF (data not shown) or active chromatin complexes such as the dosage compensation

protein MOF, suggesting that 32B may be more actively involved in specific gene regulation

processes (SMITH et al. 2000). Also in support of this hypothesis, many transcription-related

proteins such as the components of RNA polymerase II are known to disassociate from

chromatin during mitosis similar to what is observed with 32B (CHEN et al. 2005). The fact that

32B has a slower recovery time in FRAP experiments when 32A is not present indicates that

while their binding patterns may be different, there is a functional relationship between the two

proteins. This evidence would suggest that 32A causes or influences 32B to be a more dynamic,

mobile protein. This could be because 32A destabilizes 32B on the DNA, or the presence of 32A

has indirect influences via other proteins on 32B.

This work succeeded in creating the transgenic lines and documenting our initial

observations regarding 32A and 32B in vivo. Despite many attempts we were unsuccessful in

101  

documenting live images of the proteins during mitosis, or in confocal microscopy of diploid

cells. Such experiments would help address questions such as whether BEAF, like Su(Hw),

forms “insulator bodies” at the nuclear periphery (BLANTON et al. 2003; BYRD and CORCES

2003). Furthermore, the FRAP experiments presented here will require additional replicates and

more technical control and normalization procedures for verification. However, the implication

that 32A and 32B have different behaviors yet influence each other is certainly worthy of further

investigation.

References ARAVIND, L., 2000 The BED finger, a novel DNA-binding domain in chromatin-boundary-

element-binding proteins and transposases. Trends Biochem Sci 25: 421-423. BLANTON, J., M. GASZNER and P. SCHEDL, 2003 Protein:protein interactions and the pairing of

boundary elements in vivo. Genes Dev 17: 664-675. BYRD, K., and V. G. CORCES, 2003 Visualization of chromatin domains created by the gypsy

insulator of Drosophila. J Cell Biol 162: 565-574. CAMPBELL, R. E., O. TOUR, A. E. PALMER, P. A. STEINBACH, G. S. BAIRD et al., 2002 A

monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99: 7877-7882. CHEN, D., M. DUNDR, C. WANG, A. LEUNG, A. LAMOND et al., 2005 Condensed mitotic

chromatin is accessible to transcription factors and chromatin structural proteins. J Cell Biol 168: 41-54.

CHERBAS, P., 2007 Additions to tissue culture medium, pp. 1-3 in Drosophila Genomics

Resource Center. University of Indiana. CUVIER, O., C. M. HART and U. K. LAEMMLI, 1998 Identification of a class of chromatin

boundary elements. Mol Cell Biol 18: 7478-7486. DIMARIO, P., R. ROSBY and Z. CUI, 2006 Direct Visualization of GFP-Fusion Proteins on

Polytene Chromosomes. Drosophila Information Service 89: 115-118. EMBERLY, E., R. BLATTES, B. SCHUETTENGRUBER, M. HENNION, N. JIANG et al., 2008 BEAF

regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dual-core binding sites. PLoS Biol 6: 2896-2910.

GILBERT, M. K., Y. Y. TAN and C. M. HART, 2006 The Drosophila boundary element-associated

factors BEAF-32A and BEAF-32B affect chromatin structure. Genetics 173: 1365-1375.

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HART, C. M., K. ZHAO and U. K. LAEMMLI, 1997 The scs' boundary element: characterization of

boundary element-associated factors. Mol Cell Biol 17: 999-1009. KELLUM, R., and P. SCHEDL, 1991 A position-effect assay for boundaries of higher order

chromosomal domains. Cell 64: 941-950. KELLUM, R., and P. SCHEDL, 1992 A group of scs elements function as domain boundaries in an

enhancer-blocking assay. Mol Cell Biol 12: 2424-2431. ROY, S., M. K. GILBERT and C. M. HART, 2007 Characterization of BEAF mutations isolated by

homologous recombination in Drosophila. Genetics 176: 801-813. SMITH, E. R., A. PANNUTI, W. GU, A. STEURNAGEL, R. G. COOK et al., 2000 The drosophila MSL

complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol Cell Biol 20: 312-318.

UDVARDY, A., E. MAINE and P. SCHEDL, 1985 The 87A7 chromomere. Identification of novel

chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. J Mol Biol 185: 341-358.

ZHAO, K., C. M. HART and U. K. LAEMMLI, 1995 Visualization of chromosomal domains with

boundary element-associated factor BEAF-32. Cell 81: 879-889. 

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Chapter 5: Conclusion

Most previous work regarding BEAF has focused on the functional consequences of

BEAF on gene expression and on characterizing BEAF-binding sequences. The early enhancer

blocking and position-independent expression assays demonstrated that BEAF-binding

sequences can function as an insulator sequence by blocking promoter activation of an enhancer

when placed between them and by protecting a transgene from chromosomal position effects.

These experiments were generally interpreted in the context of a model that proposed BEAF’s

primary role is to divide the genome into independently regulated domains, sequestering the

promiscuous activity of enhancers from activating genes in other “domains”.

Subsequent models used for elucidating a mechanism for how these functions are

accomplished are similar to models for another protein, Su(Hw). This insulator protein has been

proposed to be a component of insulator bodies, dynamic protein and RNA complexes that bind

to DNA and form loops of chromatin. More recent research has suggested that BEAF functions

by regulating chromatin dynamics by way of competing with histone methyl transferases.

When the work described in Chapter 2 was initiated, the evidence regarding BEAF

function was limited to in vitro analysis and the use of engineered transgenes. This allowed

researchers to elucidate many essential properties of BEAF-binding sequences and to determine

basic properties of the BEAF proteins, such as the interaction of 32A and 32B in vivo. These

experiments were limited, however, because they could not demonstrate that the insulator

properties observed were BEAF-dependent functions. This could however be accomplished by

eliminating or reducing BEAF expression and demonstrating an effect on insulator function.

A peptide comprising the BEAF-interfering domain, or BID was expressed in flies using

the tissue-specific GAL4 system. The relevant components of this peptide interact with the C-

terminal, self-interaction domain of endogenous BEAF, thereby forming complexes lacking

104  

DNA binding domains and reducing DNA binding. Upon BID expression a loss of BEAF-

binding was observed on polytene chromosomes. We demonstrated that the expression of BID in

flies will prevent a BEAF-dependent insulator from blocking activation of a reporter gene, and

that multiple lines with inserts of a transgene that is bracketed by a BEAF-dependent insulator

and scs show an increase in reporter gene expression upon BID expression, indicating a loss of

protection against chromosomal position effects. Finally, as evidence that BEAF affects

chromatin we demonstrated that genes placed near centromeric heterochromatin are more

susceptible to silencing in the presence of BID, indicating that heterochromatin spreads into

regions of active transcription. These assays were the first to demonstrate that a reduction in

BEAF affected the function of a BEAF-binding sequence as an insulator. Also notable after

expression of BID is the perturbation to the structure of polytene chromosomes, suggesting that

BEAF functions by affecting chromatin structure. Subsequent work in our lab demonstrated that

a BEAF knockout also had effects on polytene chromosome structure, although it was more

pronounced on the male X-chromosome.

While the BID protein has already been used as a research tool in subsequent

publications, it has the potential for future use. It is likely that BEAF interacts with other, as yet

unknown proteins. Since the BID protein only consists of the C-terminal domain it would be

useful to use this fact to determine the nature of any such interaction. For example, if a protein of

interest can co-precipitate with BEAF, but not with BID, then one can deduce that the interaction

is either dependent on an interaction with BEAF’s N-terminal, or, that the protein-protein

interaction is DNA dependent interaction. Furthermore, BID-BEAF complexes are not found on

DNA but they ostensibly remain in the nucleus. It would be of interest to determine if this

complex is associated with the biochemically defined nuclear matrix, as has been suggested, or if

this matrix interaction is a DNA-dependent interaction. Finally, the GAL4 expression system

105  

used to drive BID expression can interfere with BEAF in a tissue-specific manner, thus

additional genetic screens for proteins that interact with BEAF could help better elucidate a

mechanism for BEAF function.

Subsequent work with the BEAFAB-KO line was aimed at determining if the effects

observed on chromosome structure caused by a lack of BEAF were due to perturbations in

nucleosome positioning or caused by other effects to chromatin structure. Micrococcal nuclease

digestion and Southern blot analysis revealed that genome-wide nucleosome positioning

appeared normal. However, when looking at BEAF-binding sequences located near promoters,

we observed subtle alterations in hypersensitive sites. By indirect-end labeling and a

nucleosome-scanning assay we demonstrate what appears to be a subtle shifting of nucleosomes

near promoter regions in the BEAFAB-KO line. This process of subtle shifting has been

demonstrated as a regulatory mechanism at such loci as the PHO5 gene where nucleosome

remodeling at gene regulatory regions to expose additional binding sites for regulatory proteins

to bind. A shifting of nucleosomes at promoters has also been demonstrated as a genome-wide

mechanism for gene regulation in human cells.

Finally, analyzing fluorescently-tagged 32A and 32B peptides revealed further insights

into BEAF function. The fact that 32B comes off of DNA during mitosis and has banding

patterns reminiscent of transcription factors is consistent with this protein being related to

transcription, whereas 32A seems to be generally localized with chromatin and not gene-specific.

Furthermore FRAP experiments are consistent with 32A being less tightly bound to DNA as its

recovery after photobleaching is faster than 32B.

Taken together, the data presented here tells us that BEAF functions as an insulator and

that BEAF may accomplish this by affecting nucleosome positioning or dynamics. While more

data is needed for a comprehensive model, the effect on nucleosome positioning may occur

106  

because BEAF binds to the DNA in two locations that bracket a nucleosome, then BEAF

proteins may interact with each other via their C-terminus to both stabilize each other and

“clamp” the nucleosome in place. This is supported by several lines of evidence. First, earliest

mapping of hypersensitive sites near the BEAF binding sites at BE28, BE76, and scs’ in Kc cells

always consist of two hypersensitive sites with an intervening region of ~ 200 base pairs –

precisely the distance needed to bracket a nucleosome. In the case of scs’ both hypersensitive

sites are BEAF binding sites. In BE76 and scs’, regions that are in gene regulatory regions, the

CGATA motifs are located at the edge of a positioned nucleosome. Furthermore, in scs’ and

BE76 the hypersensitive sites at the edges of the nucleosome become fuzzy in the absence of

BEAF, perhaps because if BEAF is not present to act as a “clamp” then the nucleosome may be

displaced.

Another possibility is that the BEAF proteins have distinct roles, where 32A binds in a

general fashion, perhaps by regulating many nucleosomes, and 32B is involved in gene-specific

nucleosome regulation. This is supported by BEAF fluorescence data showing 32A binding in a

general and diffuse pattern, while 32B binds in a more punctate pattern reminiscent of many

transcription factors. In this case either 32A or 32B may aid in the recruitment of gene-specific

transcription factors or other components necessary for transcription. The alternative is also a

possibility – that the presence of BEAF may exclude regulatory components. Based on this

model, and the fact that expression of most BEAF-associated genes decrease with a lack of

BEAF, we might expect that BEAF recruits activating regulatory components or inhibits

repressive components at most loci. Evidence for this more dynamic model is that BEAF appears

to affect histone modifications in a different way at different promoters, and that gene expression

in the BEAFAB-KO line may be increased or decreased depending on the gene. Furthermore,

analysis of CGATA motifs in the BEAF binding regions on chromosome 2L (Chapter 3, figure

 

3-1), reve

between

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107 

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108

Appendix: Permission to Include Copyrighted Material

109

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112

 

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114

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Vita Matthew Gilbert was born in Bowling Green, Ohio. When he was 11 years old his

parents, Philip and Marilyn Gilbert, moved their family of six to Kihei, Maui, Hawaii. Matthew

started college at the University of Hawaii at Manoa, and after two years transferred to Louisiana

State University where he obtained his bachelor’s degree in zoology in 2000. After working for

two years in New York City at The Rockefeller University he returned to Louisiana State

University and enrolled in the graduate program.

Matthew is married to Katherine Latapie Gilbert from Chalmette, Louisiana, and their

first child, Marley Patricia Gilbert, is due five days after the submission of this dissertation.