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CHARACTERIZATION OF THE MURINE ANGELMAN SYNDROME IMPRINTING CENTER

By

EMILY YVONNE SMITH

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010

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© 2010 Emily Y. Smith

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To my mom and dad

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ACKNOWLEDGMENTS

I would like to thank all past and present members of the Resnick lab with whom I have

had the pleasure to work with: Chris Futtner, Danielle Maatouk, Lori Kellam, Jessica Walrath,

Karen Johnstone, Edwin Peery, Mike Lewis, and Amanda DuBose. Special thanks goes to our

undergraduate assistants, Ryan Hallett and Chelsea Batten, who have spent countless hours

helping with my research and completing many less than desirable lab duties.

In addition, I would like to thank my mentor, Dr. Jim Resnick. He has provided me with

incredible guidance in the lab, in my career, and has also given me invaluable life advice. I am

eternally grateful for his extraordinary friendship and for always going out of his way to help, no

matter the circumstances. The support he has given me and his other graduate students has been

phenomenal and I could not have asked for a better mentor.

Special thanks goes to my sister, brother-in-law, boyfriend, and friends for always

providing great distractions from my research and reminding me of the most important things in

life.

Most importantly, I would like to thank my parents for their unconditional love and

immeasurable support throughout my life. Words could never express my gratitude for

everything they have done for me and for the great role models they have been in every aspect of

life.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS.................................................................................................................... 4

LIST OF TABLES................................................................................................................................ 8

LIST OF FIGURES .............................................................................................................................. 9

ABSTRACT ........................................................................................................................................ 11

CHAPTER

1 INTRODUCTION....................................................................................................................... 13

Genomic Imprinting .................................................................................................................... 13 The Discovery of Imprinted Genes .................................................................................... 14 The Epigenetics of Imprinting ............................................................................................ 15

Prader-Willi and Angelman Syndromes .................................................................................... 17 Molecular Classes of PWS and AS .................................................................................... 18 The PWS/AS Locus ............................................................................................................. 19

2 MATERIALS AND METHODS ............................................................................................... 29

Mouse Husbandry ....................................................................................................................... 29 Transgenic Lines .................................................................................................................. 29 Genotyping ........................................................................................................................... 29

RNA Isolation .............................................................................................................................. 30 Northern Blot ............................................................................................................................... 31 SnoRNA Northern Blot .............................................................................................................. 31 RT-PCR ....................................................................................................................................... 32 Southern Blot ............................................................................................................................... 33 Primordial Germ Cell Purification ............................................................................................. 34 High Molecular Weight Genomic DNA Preparation ................................................................ 35 Bisulfite Sequence Analysis ....................................................................................................... 36

Bisulfite Conversion ............................................................................................................ 36 Bisulfite-Treated DNA Purification ................................................................................... 36 Bisulfite PCR ....................................................................................................................... 36 Cloning PCR Products......................................................................................................... 37 Plasmid Sequencing ............................................................................................................. 38

3 THE IMPRINTED BAC TRANSGENE ................................................................................... 39

Introduction ................................................................................................................................. 39 Attempts to Identify the Murine AS-IC ............................................................................. 39 Founding the BAC Transgenic Model System .................................................................. 41

Results .......................................................................................................................................... 44

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The 425Δ5-7 BAC Possesses a Functional AS-IC ............................................................ 44 Imprinted expression of the 425Δ5-7 transgene......................................................... 44 Epigenetic imprinting marks on the 425Δ5-7 transgene............................................ 45

Snrpn Upstream Exon Expression Patterns are Conserved on the 425Δ5-7A Transgene ......................................................................................................................... 47

Upstream exon expression in the brain ....................................................................... 48 Upstream exon expression in the ovary ...................................................................... 48 Snrpn upstream exon expression is not restricted to the brain and ovary ................ 49

Snrpn Upstream Exons are Silent in the Germline Before the Establishment of Imprints ............................................................................................................................. 50

Discussion .................................................................................................................................... 51

4 IDENTIFICATION OF THE AS-IC ......................................................................................... 62

Introduction ................................................................................................................................. 62 Results .......................................................................................................................................... 63

Three Snrpn Upstream Exons Constitute the AS-IC on the 425D18 BAC...................... 63 Expression of the 425ΔU1-U3 transgene is not imprinted ........................................ 63 Epigenetic imprinting marks are absent on the 425ΔU1-U3 transgene .................... 64

Discussion .................................................................................................................................... 65

5 A SINGLE UPSTREAM EXON IS SUFFICIENT TO IMPRINT THE BAC TRANSGENE ............................................................................................................................. 72

Introduction ................................................................................................................................. 72 Results .......................................................................................................................................... 73

The 425ΔU2/U3 BAC Displays AS-IC Activity ............................................................... 73 The 425ΔU2/U3 BAC exhibits imprinted expression patterns ................................. 73 The 425ΔU2/U3 BAC is epigenetically imprinted .................................................... 74

U1 Usage on the Imprinted 425ΔU2/U3 BAC Transgene ................................................ 75 U1 is not transcribed from the 425ΔU2/U3E transgene in newborn brain............... 76 The 425ΔU2/U3E transgene does not express U1 in the ovary ................................ 76

Discussion .................................................................................................................................... 76

6 SILENCING OF THE ENDOGENOUS LOCUS BY A PATERNALLY TRANSMITTED TRANSGENE ............................................................................................... 83

Introduction ................................................................................................................................. 83 Results .......................................................................................................................................... 83

Imprint Analysis of the 425ΔU2/U3A Transgene: Uncoupling of the Epigenetic and Expression Imprints ......................................................................................................... 83

The 425ΔU2/U3A transgene does not display imprinted expression patterns ......... 83 The DNA methylation imprint is present on the 425ΔU2/U3A transgene ............... 84

U1 Usage From the 425ΔU2/U3A BAC Transgene.......................................................... 84 Endogenous Snrpn is Silenced Upon Paternal Transmission of the 425ΔU2/U3A

Transgene ......................................................................................................................... 85

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The 425ΔU2/U3A transgene cannot silence Snrpn from another BAC transgene ................................................................................................................... 86

Silencing by the 425ΔU2/U3A transgene is independent of imprinting .................. 87 Regulation of Other Genes at the Endogenous PWS/AS Locus by the 425ΔU2/U3A

Transgene ......................................................................................................................... 88 The upstream cluster genes are not repressed by the 425ΔU2/U3A transgene ........ 88 The 425ΔU2/U3A transgene exerts varying effects on the downstream cluster

genes .......................................................................................................................... 89 Discussion .................................................................................................................................... 92

7 CONCLUSIONS AND FUTURE DIRECTIONS .................................................................. 107

APPENDIX: PCR PRIMER SET SEQUENCES .......................................................................... 115

LIST OF REFERENCEs .................................................................................................................. 117

BIOGRAPHICAL SKETCH ........................................................................................................... 126

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LIST OF TABLES

Table page 6-1 Gene regulation at the PWS/AS locus by the 425ΔU2/U3A transgene............................ 106

A-1 List of primers used in PCR and RT-PCR experiments .................................................... 115

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LIST OF FIGURES

Figure page 1-1 The imprint lifecycle. ............................................................................................................. 23

1-2 Molecular classes of PWS and AS. ....................................................................................... 24

1-3 The PWS/AS domain.. ........................................................................................................... 25

1-4 Model for IC function at the PWS/AS locus.. ...................................................................... 26

1-5 Identifying the boundaries of the PWS-IC and AS-IC. ....................................................... 27

1-6 Organization of the SNPRN upstream exons. ....................................................................... 28

3-1 Schematic representation of the Snrpn containing BAC transgenes.. ................................ 55

3-2 Snrpn RT-PCR analysis of the 425Δ5-7 transgenic lines. ................................................... 56

3-3 Schematic diagram of the Snrpn DMR. ................................................................................ 57

3-4 Bisulfite sequence analysis of the 425Δ5-7H transgene. ..................................................... 58

3-5 Snrpn upstream exon expression from the 425Δ5-7A transgene. ....................................... 59

3-6 Snrpn upstream exon expression in various tissues. ............................................................ 60

3-7 Snrpn upstream exon expression in the developing germline.. ........................................... 61

4-1 The modified 425D18 BAC transgenes.. .............................................................................. 69

4-2 Snrpn expression analysis of the 425ΔU1-U3D transgene.................................................. 70

4-3 Bisulfite sequence analysis of the 425ΔU1-U3D transgene. ............................................... 71

5-1 Comparisons of the three modified 425D18 BAC transgenes. ........................................... 79

5-2 Snrpn expression analysis of the 425ΔU2/U3E transgene.. ................................................ 80

5-3 Bisulfite sequence analysis of the 425ΔU2/U3E transgene. ............................................... 81

5-4 Snrpn upstream exon usage from the 425ΔU2/U3E transgene.. ......................................... 82

6-1 Snrpn expression from the 425ΔU2/U3A transgene............................................................ 97

6-2 Bisulfite sequence analysis of the 425ΔU2/U3A transgene. ............................................... 98

6-3 Snrpn upstream exon usage from the 425ΔU2/U3A transgene.. ........................................ 99

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6-4 Snrpn expression analysis with the 425U2/U3A and 380A paternally transmitted transgenes. ............................................................................................................................ 100

6-5 Endogenous Snprn repression in fetal gonads upon paternal transmission of the 425ΔU2/U3A transgene.. ..................................................................................................... 101

6-6 Upstream cluster gene expression in the 425ΔU2/U3A transgenic line. .......................... 102

6-7 Effects on Ube3a-ats and Ube3a expression in the 425ΔU2/U3A transgenic line. ........ 103

6-8 SnoRNA expression in the 425ΔU2/U3A transgenic line. ................................................ 104

6-9 SnoRNA expression analysis with the 425ΔU2/U3A and 380A maternally transmitted transgenes. ......................................................................................................... 105

7-1 A working model for imprint establishment at the PWS-IC ............................................. 112

7-2 Structure of the GDF9flox380 BAC transgene.. .................................................................. 113

7-3 Schematic diagram of the transcriptional terminator allele.. ............................................. 114

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

CHARACTERIZATION OF THE MURINE ANGELMAN SYNDROME IMPRINTING

CENTER

By

Emily Yvonne Smith

May 2010 Chair: James Resnick Major: Medical Sciences− Genetics

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurological

disorders resulting from improper gene expression from the imprinted domain on chromosome

15q11-q13, the PWS/AS locus. This locus is controlled by a bipartite imprinting center

consisting of the PWS-IC and the AS-IC. Evidence suggests that the PWS-IC acts as a positive

element to promote gene expression from the paternal allele. The AS-IC acts in the oocyte to

inactivate the PWS-IC on the future maternal allele thus silencing the paternally expressed genes.

The PWS-IC is located just 5’ to and including exon one of SNRPN whereas the AS-IC is 35 kb

upstream of SNRPN. Importantly, the AS-IC includes two of several SNRPN alternative

upstream exons.

The PWS/AS locus is well conserved in the mouse but a murine AS-IC remains

uncharacterized. As in humans, the mouse Snrpn locus includes several upstream exons

postulated to function in silencing the maternal allele. We have taken a transgenic approach to

study the potential regulatory role of these alternative exons. To do so, we utilized the bacterial

artificial chromosome (BAC) 425D18, which contains Snrpn and approximately 120 kb of

5’sequence in which three alternative upstream exons reside. We first confirmed that this BAC

transgene displayed proper imprinted expression in multiple transgenic lines thus demonstrating

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the presence of a functional AS-IC. Imprinting was further examined by analysis of the

epigenetic status of the Snrpn differentially methylated region (DMR), which lies within the

PWS-IC.

To determine whether the upstream exons on the 425D18 BAC confer silencing upon

maternal transmission, we used recombineering techniques to create targeted deletions of these

exons. Deletion of the three upstream exons resulted in robust Snrpn expression upon both

maternal and paternal transmission of the transgene as well as a loss of the epigenetic imprint at

the Snrpn DMR. These results indicate that the three upstream exons comprise the AS-IC on the

425D18 BAC. Our data support a model in which transcription arising from the AS-IC and

continuing through the PWS-IC results in epigenetic modification of the PWS-IC. Further

experiments utilized this BAC transgenic system to investigate mechanisms of AS-IC action.

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CHAPTER 1 INTRODUCTION

Genomic Imprinting

Sexual reproduction is a fundamental feature of life that generates genetic diversity

through the production of offspring possessing two unique sets of chromosomes, one inherited

maternally and one paternally inherited. Typically, homologous genes on these chromosomes

are expressed at similar levels; however, there exists a small number of genes that do not display

biallelic expression patterns, genes which are subject to a phenomenon termed genomic

imprinting. Genomic imprinting is an epigenetic regulatory mechanism that acts at a subset of

chromosomal regions and results in parent-of-origin specific monoallelic gene expression. The

epigenetic imprint is a heritable mark that is established in gametes and maintained in somatic

cells. Imprinted expression patterns are frequently both tissue and developmental-stage specific

and appropriate control of these expression patterns is vital as imprinted genes regulate many

aspects of growth and development.

To date, 112 imprinted genes have been identified in mammals, 96 in the mouse and 53 in

humans, including 37 which overlap (Morison et al., 2005). These imprinted genes are

frequently found clustered together in specific regions of the genome, coordinately regulated by

imprinting control centers (ICs) that direct allele specific differences in transcription, DNA

methylation and histone modifications (Kitsberg et al., 1993; Lewis and Reik, 2006; Margueron

et al., 2005; Razin and Cedar, 1994). The IC is a cis-acting DNA element that is capable of

long-range regulation, however, the mechanisms of this regulation are still not fully understood

(Ferguson-Smith and Surani, 2001). Furthermore, studies have shown that different imprinted

regions are not necessarily subject to the same regulatory mechanisms. A major focus of current

research in the field of genomic imprinting is on IC mechanisms as they act upon imprinted loci.

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The Discovery of Imprinted Genes

Evidence for genomic imprinting was first discovered in the mid-1980s through murine

embryological and classical genetic experiments (Barton et al., 1984; McGrath and Solter, 1984).

In these studies, diploid mouse embryos possessing either two female pronuclei (gynogenotes) or

two male pronuclei (androgenotes) were created from one-cell-stage embryos via nuclear

transplantation. The authors found that both androgenetic and gynogenetic embryos failed to

complete normal embryogenesis, demonstrating that the maternal and paternal genetic

contribution is not functionally equivalent. Both types of embryos were capable of developing to

the blastocyst stage but died shortly after implantation. Embryonic growth, albeit delayed, was

evident in the gynogenotes; however, these embryos lacked significant extraembryonic tissue

and thus failed to survive to term. Conversely, androgenotes developed substantial

extraembryonic tissue but exhibited little, if any, development of the embryo proper. Murine

androgenetic development mimics that of the naturally occurring hydatidiform mole in humans,

a paternally diploid conceptus that contains ample extraembryonic tissues but lacks a fetus

(Bagshawe and Lawler, 1982).

Additional genetic studies analyzing mice bearing uniparental disomies provided further

evidence for the existence of genomic imprinting in mammals. The combination of results from

a number of studies proved that for proper embryonic development, contributions from both

parents are only necessary for certain chromosomal regions of the genome. Mice possessing

uniparental disomies displayed normal survival rates and were phenotypically wild type for a

majority of the chromosomes (Lyon et al., 1975). In contrast, embryos inheriting maternal or

paternal disomies of certain chromosomes, those that are now known to contain imprinted loci,

either failed to survive to term or survived but displayed developmental abnormalities (Cattanach

and Kirk, 1985; Searle and Beechey, 1978). In addition, the developmental aberrations observed

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in these mice varied markedly depending on the parental origin of the disomy. The failure of a

duplication of one parental chromosome to complement the deficiency of the other suggested a

differential functioning of alleles located within these chromosomes. These findings further

demonstrated the existence of an imprinting mark at certain genomic loci, a heritable mark

affecting gene activity in a parent-of-origin specific manner.

The Epigenetics of Imprinting

Since the existence of genomic imprinting was first revealed, intensive efforts have gone

into discovering a mechanism for this phenomenon. Given that both parental alleles may be

identical in DNA sequence, it follows that the differential expression of imprinted genes is

controlled by an epigenetic regulatory mechanism. Epigenetic marks, including DNA

methylation and histone modifications, provide a means for cells to generate diverse patterns of

gene expression from identical DNA sequences. The majority of imprinted domains are

coordinately regulated by cis-acting ICs that direct epigenetic modifications and expression

patterns throughout the locus, oftentimes acting over regions of several megabases (Lewis and

Reik, 2006).

Although the exact mechanisms of IC regulation are not entirely clear, DNA methylation is

recognized as playing a vital role in the process. Methylation of DNA is carried out by DNA

methyltransferases (Dnmts) at the 5’ position of cytosine residues in CpG dinucleotides. One

function of this repressive epigenetic modification is to inhibit transcription at promoter regions

(Bird, 2002). At imprinted loci, allele-specific differential DNA methylation is frequently

observed at distinct sequences termed differentially methylated regions (DMRs). ICs typically

contain DMRs as well as other allele-specific differences in chromatin structure such as DNase I

hypersensitivity sites and covalent modifications of histone tails (Lewis and Reik, 2006). A

recent study demonstrated a mechanistic link between DNA methylation and histone methylation

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at several murine ICs, providing support for a model in which DNA methylation controls histone

modifications at these loci (Henckel et al., 2009). Typically, repressive histone modifications

including methylated lysines 9 and 27 on histone H3 (H3K9 and H3K27), reside on the

methylated allele. The unmethylated allele generally displays activating histone modifications

such as methylation of lysine 4 on histone H3 (H3K4) and acetylation of histones H3 and H4.

The epigenetic marks that define the imprint are established at ICs in the germline,

allowing for differential gene expression between parental alleles over the course of

development. In order to be propagated throughout the generations, imprinting marks must go

through a life cycle consisting of three stages: erasure, establishment and maintenance (Figure 1-

1). It is essential for imprints to be erased and re-established in a parent-specific manner in the

germline so that upon fertilization, each zygote contains one maternally imprinted set of

chromosomes and one paternally imprinted set of chromosomes. Erasure of imprinting marks

occurs in the primordial germ cells (PGCs) as they colonize the developing gonad between 10.5-

12.5 days post coitus (dpc) (Hajkova et al., 2002; Lee et al., 2002). Imprint erasure is associated

with the demethylation of IC DMRs and well as with the biallelic expression or biallelic

silencing of imprinted genes in PGCs by 12.5 dpc (Szabo and Mann, 1995). DNA demethylation

during this time in development is part of a germ cell-specific global epigenetic reprogramming;

DMRs are maintained in the soma at this stage of embryogenesis.

The timing of imprint erasure is critical as it ensures both the maternally inherited and the

paternally inherited alleles are at an equivalent epigenetic state before sexual differentiation of

PGCs occurs at 13.5 dpc. It is not until after sexual differentiation that imprints are re-

established in a sex-specific manner in the germline. In the male embryo, paternally methylated

DMRs are established in the mitotically arrested fetal prospermatogonia between 15.5-18.5 dpc

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(Davis et al., 2000). In the oocyte, maternally methylated DMRs are established after birth

during the oocyte growth phase, corresponding with meiotic prophase I (Lucifero et al., 2004;

Lucifero et al., 2002). Dnmt3a and Dnmt3l, two members of the de novo family of

methyltransferases, are required for the acquisition of methylation imprints (Bourc'his and

Bestor, 2004; Bourc'his et al., 2001; Hata et al., 2002; Kaneda et al., 2004). The one known

exception is the Rasgrf1 DMR, which also requires Dnmt3b for establishment of DNA

methylation (Kato et al., 2007).

After genomic imprints are established in the germline, it is imperative that they are

maintained following fertilization and stably inherited in the somatic cells throughout the course

of multiple cell divisions. During pre-implantation development, the maternal and paternal

genomes undergo extensive epigenetic reprogramming and the majority of DNA methylation is

lost; however, imprinted genes are protected from this global demethylation (Tremblay et al.,

1995). The maintenance methyltransferase, Dnmt1, acts on hemimethylated DNA after

replication to methylate CpG dinucleotides on the newly synthesized strand. Dnmt1 is required

to maintain methylation imprints in both pre- as well as post-implantation development

(Hirasawa et al., 2008; Li et al., 1993). How Dnmt1 activity is specified to imprinted regions in

pre-implantation embryos is still under investigation.

Prader-Willi and Angelman Syndromes

Due to their monoallelic expression patterns, imprinted genes are functionally hemizygous,

resulting in a genetic vulnerability that contributes to developmental disorders and disease,

carcinogenesis, and embryonic lethality. My research is focused on two distinct neurogenetic

imprinting disorders, PWS and AS. The prevalence of each disorder is approximately one in

every 15,000 live births. Patients with either syndrome exhibit significant developmental and

behavioral problems. PWS is characterized by an initial failure to thrive in infancy which

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evolves into severe hyperphagia and obesity within the first few years of life (Holm et al., 1993).

Other traits include hypogonadism, small hands and feet, short stature, mild to moderate mental

retardation and obsessive-compulsive disorder. AS clinical manifestations include severe mental

retardation, profound speech impairment, sleep disorders, microcephaly, gait ataxia, and seizures

(Williams et al., 2001). AS patients also display behavioral abnormalities including a happy

disposition with frequent laughter and smiling, uplifted hand-flapping, and hyperactivity

(Williams et al., 2006).

Both PWS and AS are the result of improper gene expression from the imprinted locus on

chromosome 15q11-q13, a region known as the PWS/AS locus. This locus contains the

paternally expressed genes MKRN3, MAGEL2, NDN, SNURF/SNRPN (referred to henceforth as

SNRPN), a UBE3A-antisense transcript (UBE3A-ATS), and multiple small nucleolar RNA

(snoRNA) encoding genes. UBE3A, an E6-associated-protein ubiquitin-protein ligase, is also

located within the PWS/AS domain. This gene is biallelically expressed in non-neuronal tissues

but preferentially expressed from the maternal allele in portions of the brain (Albrecht et al.,

1997; Rougeulle et al., 1997; Vu and Hoffman, 1997). Mutations leading to a loss of UBE3A

function from the maternal chromosome result in AS (Kishino et al., 1997; Matsuura et al.,

1997). PWS has classically been thought to be the result of a loss of multiple paternal gene

products from the PWS/AS locus; however, two recently described individuals, one diagnosed

with PWS and one with several PWS-associated characteristics, both bear microdeletions limited

to the HBII-85 snoRNA C/D cluster thereby challenging this notion (de Smith et al., 2009;

Sahoo et al., 2008).

Molecular Classes of PWS and AS

There are several genetic mechanisms that lead to AS and PWS, each resulting in a loss of

gene expression from the normally active allele (Figure 1-2). In PWS, it is a loss of paternal

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gene expression from the PWS/AS locus and in AS it is a loss of maternal gene expression from

this region. Approximately 70% of PWS cases are a result of a large interstitial deletion on

paternal chromosome 15 while roughly 29% of PWS patients exhibit maternal uniparental

disomy (UPD) of chromosome 15. A third molecular class of PWS, representing approximately

1% of patients, is defined as an imprinting defect (ID) in which the paternal chromosome

possesses a maternal imprint at the PWS/AS locus. Rare patients display balanced chromosomal

translocations where HBII-85 snoRNA expression is absent (Wirth et al., 2001). These

translocation patients, in addition to the HBII-85 snoRNA-containing microdeletion patients

mentioned previously, provide evidence that this snoRNA cluster plays a key role in the

pathology of PWS.

There are five distinct molecular classes of AS with varying phenotypic severities

depending on the origin of the defect. The first class, representing about 70% of patients, is a

large de novo deletion of maternal chromosome 15. Five percent of patients fall into the second

molecular class of AS, bearing a UPD of paternal chromosome 15. The third class of AS

represents the ID patients, those possessing a paternal imprint at the PWS/AS locus on the

maternal chromosome (Reis et al., 1994). This class constitutes less than 5% of all AS cases.

Class four is caused by a mutation of UBE3A, comprising 10% of cases, and the final 10% of

cases (class five) are of an unknown origin. Patients in classes one and five display the most

severe phenotypes while those in classes two and three exhibit less severe clinical

manifestations.

The PWS/AS Locus

The PWS/AS imprinted domain is located within a 2 Mb region on the long arm of

chromosome 15 (Figure 1-3A). This locus contains two imprinted gene clusters commonly

referred to as the downstream and the upstream cluster based on their position relative to the IC.

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The upstream cluster contains the paternally expressed MKRN3, MAGEL2, and NDN genes. The

downstream cluster is comprised of the paternally expressed SNRPN, several snoRNAs including

HBII-85, HBII-52, and HBII-13, as well as UBE3A-ATS. The paternally expressed downstream

gene products are thought to be processed from one single large transcript of over 460 kb that

originates at SNRPN exon one (Runte et al., 2001). Imprinting of the paternally expressed genes

in both the upstream as well as the downstream cluster appears to be ubiquitous throughout the

different tissues of the body. Contrasting to these paternal expression patterns are the expression

patterns of two other genes, UBE3A and ATP10A, which are also located within the downstream

cluster. ATP10A displays variable imprinted expression in the brain with some individuals

demonstrating biallelic expression and others exhibiting monoallelic expression in this tissue

(Hogart et al., 2008). As mentioned previously, UBE3A shows preferential expression from the

maternal allele exclusively in brain tissues.

Located between the upstream and downstream imprinted gene clusters is a cis-acting

bipartite IC, comprised of the PWS-IC and the AS-IC, that regulates both epigenetic

reprogramming as well as gene expression at this locus (Figure 1-4) (Buiting et al., 1995b;

Dittrich et al., 1996). Prevailing models of IC function suggest that the PWS-IC serves as a

positive element to activate gene expression from the paternal allele. The AS-IC is posited to act

as a negative element that directs inhibitory epigenetic modifications at the PWS-IC during

oogenesis, thereby silencing the paternally expressed genes on the future maternal allele

(Brannan and Bartolomei, 1999; Dittrich et al., 1996; Shemer et al., 2000).

Rare PWS and AS patients display IDs at the PWS/AS domain due to mutations or

microdeletions of the IC (Buiting et al., 1995a). By analyzing the shortest region of overlap of

these microdeletions, the boundaries of the PWS-IC and AS-IC have been defined. The PWS-IC

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is located within a region of less than 4.3 kb 5’ to and including exon one of SNRPN (Figure 1-

5A) (Ohta et al., 1999b; Sutcliffe et al., 1994). The SNPRN DMR is a CpG island contained

within the PWS-IC that is methylated specifically on the maternal allele, representing an

essential epigenetic imprint at this locus. The paternal SNRPN DMR is unmethylated and

displays additional epigenetic marks indicative of open chromatin structure including high levels

of histone H3 and H4 acetylation as well as histone H3 lysine 4 methylation (Fulmer-Smentek

and Francke, 2001; Saitoh and Wada, 2000). In PWS ID cases without a PWS-IC deletion, the

SNPRN DMR is hypermethylated on both alleles, indicating a maternal imprint on the paternal

chromosome, and expression of the paternal genes is absent (Ohta et al., 1999b).

Mapping of microdeletions in AS ID patients has localized the AS-IC to a sequence of

0.88 kb approximately 35 kb upstream of the PWS-IC (Figure 1-5B) (Buiting et al., 1999; Ohta

et al., 1999a; Saitoh et al., 1996). These patients display hypomethylation of the maternal PWS-

IC along with biallelic expression of the paternal genes. Notably, within the 0.88 kb of AS-IC

sequence are two of several alternative upstream exons of SNRPN, u5 and u6 (Figure 1-6A)

(Farber et al., 1999; Wawrzik et al., 2009). These SNRPN alternative exons are the result of

multiple duplication events of the IC region. Several alternatively spliced transcripts, termed

IC/SNRPN transcripts, originate from these upstream exons and are postulated to have a role in

silencing the PWS-IC on the maternal allele (Dittrich et al., 1996).

The PWS/AS locus is highly conserved in both gene order and expression patterns at the

orthologous region on central mouse chromosome 7, thereby providing an excellent model for

studying imprinting mechanisms within this domain (Figure 1-3B). The PWS-IC location and

function are also conserved, allowing for in-depth investigation into the role of this element in

imprinting regulation. Hindering these studies is the absence of an identifiable murine AS-IC

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element. No conserved sequence from the human AS-IC exists in the mouse however, the

murine Snprn does possess a number of alternative upstream exons similar to the human locus.

Nine have been identified to date, termed U1-U9, spanning over 450 kb upstream of Snrpn exon

one (Figure 1-6B) (Bressler et al., 2001; Landers et al., 2004). These exons generate multiple

transcripts, most frequently splicing into exon two of Snrpn but also splicing downstream of the

Snrpn gene as well. Notably, while Snrpn is widely expressed in adult tissues, published reports

have shown that the upstream exons are transcribed specifically from the paternal allele in the

brain as well as in the oocyte and granulosa cells (Bressler et al., 2001; Le Meur et al., 2005;

Mapendano et al., 2006). Significantly, transcription in the oocyte corresponds with the

establishment of the maternal imprint at the PWS-IC.

The focus of my research is to identify and characterize the murine AS-IC. I have

investigated the role of the Snrpn alternative upstream exons in establishing the maternal imprint

in the oocyte. Mainly, I explored the function of transcription from the upstream exons in the

epigenetic modification of the PWS-IC on the maternal allele. A recent study from Chotalia et

al. demonstrated that transcription through the Gnas DMR in growing oocytes is required to

establish maternal germline methylation imprints at this domain (Chotalia et al., 2009). This

study supports a role for transcription in establishing imprints in the female germline. My

experimental approach utilizes bacterial artificial chromosome (BAC) transgenes containing

Snrpn along with a significant amount of upstream sequence to study a potential regulatory role

of the upstream exons in genomic imprinting mechanisms.

23

Figure 1-1. The imprint lifecycle. Genomic imprints go through three stages during mammalian development: establishment, maintenance and erasure. Imprints are first established in a sex-specific manner in the germline during gametogenesis. Upon fertilization, imprints must be maintained in the soma of the developing embryo. Contrastingly, in the primordial germ cells imprints are erased between 10.5-12.5 dpc during a germline-specific epigenetic reprogramming event. After erasure, female embryos re-establish maternal imprinting marks in the oocytes and male embryos re-establish paternal imprinting marks in the sperm.

24

Figure 1-2. Molecular classes of PWS and AS. PWS and AS result from improper gene expression from chromosome 15q11-q13, the PWS/AS locus (grey bar). PWS is caused by a loss of paternal gene products while AS is caused by a lack of UBE3A (green bar) expression from the maternal chromosome. A large interstitial deletion including the PWS/AS locus on the paternal chromosome (blue bar) or a maternal uniparental disomy of chromsome 15 cause PWS. Additionally, a microdeletion or mutation of the PWS-IC on the paternal chromosome so that it behaves as if inherited maternally also leads to PWS. Large deletions on maternal chromsome 15 (red bar) including the q11-q13 region, a paternal uniparental disomy, or maternal UBE3A defects result in AS. Also, a microdeletion or mutation of the maternal AS-IC leads to paternal imprints on the maternal chromosome, causing AS.

25

Figure 1-3. The PWS/AS domain. This locus is highly conserved in gene order and expression patterns from human chromosome 15q11-13 to central mouse chromosome 7. A) The human PWS/AS locus. B) The murine PWS/AS locus. This domain consists of two gene clusters, an upstream and a downstream cluster (in relation to the IC). The downstream cluster of paternally expressed genes is thought to be transcribed as a single long transcript from which multiple gene products are processed. Arrows represent the direction of transcription.

26

Figure 1-4. Model for IC function at the PWS/AS locus. The bipartite IC consists of the PWS-IC and the AS-IC. The PWS-IC functions as a positive element to activate expression of both the upstream cluster genes and the paternally expressed downstream cluster genes. On the maternal allele, the AS-IC acts as a negative element to inhibit the PWS-IC and thereby silence the paternally expressed genes.

27

Figure 1-5. Identifying the boundaries of the PWS-IC and AS-IC. A) Microdeletions were mapped from PWS patients displaying imprinting defects. The boundaries of the PWS-IC were defined by the shortest region of overlap (SRO) of these deletions. The PWS-IC is located within 4.3 kb of sequence just 5’ to and including SNRPN exon one. B) Microdeletions from AS patients exhibiting imprinting defects were mapped to identify the AS-IC. The SRO was mapped to a region of 0.88 kb approximately 35 kb upstream of the PWS-IC.

28

Figure 1-6. Organization of the SNPRN upstream exons. Schematic diagrams of the A) human SNRPN locus and the B) murine Snrpn locus. Verified transcription start sites are indicated by arrows.

29

CHAPTER 2 MATERIALS AND METHODS

Mouse Husbandry

Transgenic Lines

The 425D18 BAC was obtained from the Roswell Park Cancer Institute RPCI-23 murine

BAC library, which was derived from the C57BL/6J strain. BAC recombineering as well as

preliminary BAC injections were performed by Chris Futtner at the University of Florida

(Futtner, 2007). Additional injections were performed by the UF Mouse Models Core Facility.

All BACs were injected as supercoils into the male pronucleus of fertilized oocytes, which were

obtained from superovulated FVB/N female mice. The resulting pups were screened for the

transgene at three weeks of age by PCR. At eight weeks of age, transgenic founders were mated

with wild-type partners to establish the lines and ensure germline transmission of the transgene.

Lines were initially maintained on the FVB background however, for bisulfite sequence analysis,

the lines were crossed for two generations to the B6.cast.c7 strain. This is a congenic strain that

has a Mus musculus domesticus C57BL/6J background but contains a region of chromosome 7

from the species Mus musculus castaneous (cast) (Wakeland et al., 1997). By performing this

cross, we were able to obtain transgenic mice on a homozygous cast c7 background, allowing us

to distinguish the endogenous alleles from the transgene in our bisulfite sequence analysis.

Genotyping

At three weeks of age, pups were weaned. During this process they were sex separated,

ear punched for identification and tail clipped for genotyping. Genomic DNA was isolated from

tail biopsies by overnight incubation at 55°C in 0.4 mL tail lysis buffer (100 mM Tris pH 8.5, 5

mM EDTA, 0.2% SDS, 200 mM NaCl) supplemented with 100 ug/mL proteinase K.

Phenol:chloroform:isoamyl alcohol (25:24:1) DNA extraction was performed followed by

30

ethanol precipitation. Purified genomic DNA was subject to PCR with the appropriate primer set

for the desired genotyping. For the 425 BAC transgene, the x5-7del-F1/R2 primer set (Appendix

A) was developed. These primers were designed to allow for differentiation between the

modified BAC Snrpn, which possesses a deletion between exons five to seven, and the

endogenous locus. The primer set flanks the deletion so that the transgene product is

approximately 0.8 kb while the endogenous product is about 2.3 kb.

For detection of the cast allele, a PCR amplification of the Ndn gene was performed

followed by a restriction endonuclease digest. The Ndnpoly-F/R primer set was used (Appendix

A) to amplify a region of the Ndn gene containing a polymorphic AvaII site between the cast

allele and the domesticus allele. The AvaII site exists on the domesticus allele, at position 117 of

the Ndn transcript, but not on the cast allele. Thus, after AvaII digestion of the PCR reactions at

37°C for three hours, the digests were electrophoresed on an agarose gel and the two alleles

differentiated based on band size.

RNA Isolation

RNA was extracted from murine neonatal whole brains as well as various adult tissues

using RNA-Bee reagent (Tel-Test, Inc.) per manufacturer’s instructions. For each tissue sample,

approximately 4 mL of RNA-Bee was added followed by homogenization with a Polytron

homogenizer. Next 0.4 mL chloroform was added, samples were shaken vigorously, placed on

ice for five minutes and centrifuged at 12,000g for 15 minutes. After centrifugation, the RNA

was contained in the upper aqueous phase, separated from the DNA and proteins in the inter and

organic phases. The aqueous phase was thus transferred to a new tube, combined with an equal

volume of isopropanol, placed on ice for 15 minutes, and centrifuged at 12,000g for 15 minutes

31

to pellet the RNA precipitate. Each sample was then washed in 75% ethanol, dried briefly under

vacuum, and resuspended in 100 ul diethylpyrocarbonate (DEPC)-treated water.

Northern Blot

Purified RNA from whole neonatal mouse brains was used for Northern blot analyses to

investigate gene expression patterns. Four volumes of formaldehyde sample buffer were added

to 10 ug of total RNA. Samples were heat denatured for five minutes at 65°C, placed on ice, and

run on 1% agarose formaldehyde gels for several hours at 70 volts. The gels were soaked in 20X

SSC for 30 minutes and RNA transferred onto Hybond N+ nylon membranes in 10X SSC

overnight. The following day, the membranes were washed in 2X SSC and baked at 80°C for

two hours in a vacuum oven. Prehybridization was carried out in 15 mL Church and Gilbert

hybridization buffer (1% BSA, 1 mM EDTA pH 8.0, 0.25 M sodium phosphate buffer pH 7.2,

7% SDS) at 65°C for two hours (Church and Gilbert, 1984). DNA probes were prepared with

the Prime-It II random primer labeling kit (Stratagene) and α32P-dCTP (Perkin-Elmer), purified

with the QIAquick nucleotide removal kit (Qiagen), and subsequently denatured by boiling for

five minutes. After prehybridization, the membranes were hybridized overnight at 65°C in 10

mL Church and Gilbert hybridization buffer combined with denatured probe. The following day,

membranes were washed two times for 20 minutes each at 65°C in 0.2X SSCP/0.1% SDS and

exposed to film.

SnoRNA Northern Blot

Northern blot analyses were also performed to investigate snoRNA expression. For

detection of these small RNA molecules, total RNA was separated on 8% denaturing

polyacrylamide gels (7M urea, 1X TBE buffer). Gels were pre-run in 1X TBE buffer for 30

minutes at 250 volts. For each sample, 10 ug of RNA was combined with formaldehyde sample

buffer, heated to 95°C for five minutes, placed on ice and then run on the gel at 250 volts for

32

approximately two hours. The gels were equilibrated on ice in 0.5X TBE for 20 minutes and

subsequently transferred to Hybond N+ nylon membranes using a semi-dry blotting apparatus

(Trans-blot SD, BioRad). Electroblotting was carried out at 20 volts for one hour and

membranes were then baked at 80°C overnight in a vacuum oven. Membranes were

prehybridized in Church and Gilbert hybridization buffer for two hours at 58°C. Oligonucleotide

probes were made by end-labeling reactions using T4 polynucleotide kinase (Invitrogen) and

γ32P-ATP (Perkin-Elmer). Oligonucleotides, complementary to snoRNA sequences, used in

probe synthesis were as follows: MBII-85 5’-TTCCGATGAGAGTGGCGGTACAGA-3’, MBII-

52 5’-CCTCAGCGTAATCCTATTGAGCATGAA-3’, and 5.8S rRNA 5’-

TCCTGCAATTCACATTAATTCTCGCAGCTAGC-3’. Probes were purified using the

QIAquick nucleotide removal kit (Qiagen). Membranes were hybridized with purified probe

overnight at 58°C in 10 mL Church and Gilbert hybridization buffer. The following day,

membranes were washed three times for 15 minutes each at room temperature in 0.2X

SSCP/0.1%SDS and exposed to film.

RT-PCR

RNA isolated from several tissue types was subject to gene expression analysis by RT-

PCR. RNA samples were DNase treated prior to reverse transcription to eliminate genomic

DNA contamination. For each sample, 10 ug of RNA was treated with DNase I (Invitrogen) for

15 minutes at room temperature. The reaction was stopped by the addition of EDTA and ten

minutes incubation at 65°C. Next, the samples were divided into two aliquots of 5 ug each. One

aliquot was subject to reverse transcription while the other, generated for use as a control, was

treated in parallel but in the absence of reverse transcriptase to ensure any PCR product observed

was amplified from the cDNA. First strand cDNA was synthesized by adding 1 ul of 500 ug/mL

random primers (Invitrogen) to 5 ug RNA and bringing up to 26.4 ul total volume with sterile

33

ddH2O. Samples were heated to 68°C for three minutes and placed on ice. Then, 1.6 ul of a

mixture of 2.5mM dNTPs, 8 ul reverse transcriptase buffer, 2 ul of 100 mM DTT, 1ul RNase

OUT, and 1 ul Superscript II reverse transcriptase (Invitrogen) were added to each sample.

Reactions were carried out at 37°C for 60 minutes. One microliter of cDNA was used as

template in PCR reactions under the following conditions: 10 mM Tris-HCl, 50 mM KCl, 1.5

mM MgCl2 , four dNTPs at 0.125 mM each, 10% betaine, 1.5 units Taq DNA polymerase

(NEB), and the appropriate primers (Appendix A) at 10 uM each. Reactions were subject to an

initial denaturation at 95°C for five minutes followed by 34 cycles of 94°C for 30 s, 55°C for 30

s and 72°C for 60 s. An extension step of five minutes at 72°C completed the reaction.

Southern Blot

RT-PCR products were subject to Southern blotting procedures as described (Sambrook et

al., 1989). Samples were run on 1.2% agarose/0.5X TBE gels at 95 volts for approximately one

hour. The DNA was denatured by placing the gel in alkali solution (1.5 M NaCl and 0.5 N

NaOH) for 45 minutes and then in neutralizing solution (1.5 M NaCl and 1 M Tris pH 7.4) for 90

minutes. The DNA was subsequently transferred onto Hybond N+ nylon membranes in 10X

SSC overnight. The following day, membranes were washed in 2X SSC and baked at 80°C for

two hours in a vacuum oven. Prehybridization was carried out in 15 mL Church and Gilbert

hybridization buffer (1% BSA, 1 mM EDTA pH 8.0, 0.25 M sodium phosphate buffer pH 7.2,

7% SDS) at 65°C for two hours (Church and Gilbert, 1984). DNA probes were prepared with

the Prime-It II random primer labeling kit (Stratagene) and α32P-dCTP (Perkin-Elmer), purified

with the QIAquick nucleotide removal kit (Qiagen), and subsequently denatured by boiling for

five minutes. After prehybridization, the membranes were hybridized overnight at 65°C in 10

mL Church and Gilbert hybridization buffer combined with denatured probe. The following day,

34

membranes were washed two times for 20 minutes each at 65°C in 0.2X SSCP/0.1% SDS and

exposed to film.

Primordial Germ Cell Purification

PGCs were immunomagnetically purified from embryos at 13.5 dpc for gene expression

analysis. Initially, timed matings were set up and females examined for copulatory plugs. Noon

on the day a plug was first visible was reasoned to be 0.5 dpc in embryonic development. At

13.5 dpc embryos were removed from the mothers’ uteri and placed into 1X PBS. The fetal

gonads were then dissected out, sex segregated, and each sex pooled together for purification.

Around eight sets of gonads were desired per sex per purification. Once collected, 500 ul of

trypsin-EDTA was added to each sample and the tissue incubated for five minutes at 37°C.

Following incubation, the samples were triturated to break up the tissue and then centrifuged at

2,000 rpm for two minutes. After centrifugation the trypisn-EDTA was removed and 1 mL of

PBS-DNase (1X PBS, 5 mM EDTA, 0.5% BSA, and 20 ug/mL DNase) was added. Samples

were mixed, centrifuged, and then the PBS-DNase solution was removed. Subsequently, 160 ul

of fresh PBS-DNase was added and the tissue was again triturated in order to obtain a single cell

suspension. Samples were placed on ice and 40 ul of the TG-1 antibody, a primary mouse IgM

antibody that binds to the cell surface of PGCs, was added followed by a 30 minute incubation.

After this incubation the cells were washed three times with PBS-DNase and incubated for 30

minutes on ice in a rat anti-mouse IgM secondary antibody (Miltenyi Biotech #130-047-302).

This secondary antibody has an iron bead conjugated to it for purification of PGCs through

MACs columns on the magnetic MACs Separator (Miltenyi Biotech).

For PGC purification, the MACs columns were placed on a Miltenyi Mini-MACs magnet

and prewashed with 1X PBS/3% DNase equilibration buffer. Each cell suspension was passed

35

through the column three times to ensure that the majority of the PGCs had adhered to the

column. The immunodepleted fraction, which contained the somatic cells of the fetal gonad, was

collected for use as a control. Columns were then washed four times with PBS-DNase. The

PGCs were subsequently eluted from the columns in PBS-DNase after removal from the magnet.

PGC purity was assayed by alkaline phosphatase staining.

High Molecular Weight Genomic DNA Preparation

Genomic DNA was extracted from mouse neonatal whole brains for Southern blot analysis

and bisulfite sequence analysis. Frozen brains were homogenized with a dounce homogenizer in

2.5 mL of 1X SSC, 1% SDS, 0.25 mg/mL Pronase E (Sigma). The homogenates were poured

into 15 mL round bottom Falcon tubes and the homogenizer washed with an additional 2.5 mL

homogenizing solution, which was then combined with the homogenate. Samples were vortexed

and placed at 37°C for one hour. After incubation, 5 mL of phenol:chloroform:isoamyl alcohol

(25:24:1) was added and samples were vortexed and spun for five minutes at 2500 rpm. The

DNA-containing aqueous layer was collected and the extraction repeated two more times, once

as above and once with chloroform alone. After the chloroform extraction, the DNA was ethanol

precipitated and resuspended in 400 ul ddH2O. Next, 25 ul of 2 mg/mL RNase A was added and

the samples were incubated for 30 minutes at 37°C. Then 25 ul of 5 mg/mL Pronase E was

added and samples again incubated at 37°C for 30 minutes. The phenol:chloroform:isoamyl

alcohol extractions and ethanol precipitation was repeated as described above. The purified

genomic DNA was subsequently resuspended in 200 ul ddH2O, incubated at 55°C for an hour

and then allowed to sit at room temperature overnight to ensure resuspension.

36

Bisulfite Sequence Analysis

Bisulfite Conversion

Murine genomic DNA was subject to bisulfite sequence analysis in order to determine the

methylation status of a region within the Snrpn DMR (Clark et al., 1994). For each sample, 5 ng

of genomic DNA was denatured in a solution of 0.3 M NaOH for 30 minutes at 37°C. The

denatured DNA was then combined with 2.0 M sodium metabisulfite/10 mM hydroquinone

solution, for a final concentration of 1.5 M bisulfite/0.5 mM hydroquinone, and incubated at

55°C for 16-20 hours.

Bisulfite-Treated DNA Purification

After bisulfite conversion, free bisulfite was removed with the Promega Wizard DNA

clean-up system per manufacturer’s instructions. DNA was eluted in 45 ul ddH2O and 5 ul 3 M

NaOH was added for a final concentration of 0.3 M. After a 15 minute incubation at 37°C, the

DNA was ethanol precipitated overnight at -80°C. The next day, the DNA was pelleted by

centrifugation, washed in 70% ethanol and resuspended in 100 ul ddH2O.

Bisulfite PCR

Following purification, the bisulfite-converted genomic DNA was subject to PCR

amplification. Primers were designed to anneal to the bisulfite-converted DNA sequence. A 364

bp region within the Snrpn DMR, which spans 14 CpG dinucleotides, was amplified with

primers W18 and W19 (Appendix A). PCR conditions were as follows: 10 mM Tris-HCl, 50

mM KCl, 1.5 mM MgCl2 , four dNTPs at 0.125 mM each, 1.5 units Jumpstart Taq DNA

polymerase (Sigma), and the appropriate primers at 10 uM each. To each 25 ul reaction, 1ul of

template DNA was added. The PCR reactions were subject to an initial denaturation step at

95°C for 15 minutes followed by 38 cycles of: 94°C for 45 s, 54°C for 60 s and 72°C for 90 s. A

37

10 minute final extension at 72°C completed the reactions. PCR products were run on 1.2% low

melt agarose gels containing ethidium bromide. The appropriate sized bands were excised and

purified with the Wizard DNA clean-up system. Three microliters of the 40 ul purifications were

run on 1.2% gels to verify DNA quality before cloning.

Cloning PCR Products

The purified bisulfite PCR products were ligated into the pGEM-T Easy Vector System

(Promega) per manufacturer’s protocol. Briefly, 5 ul of 2X rapid ligation buffer, 1 ul pGEM-T

Easy vector, 1 ul purified DNA, 2 ul ddH2O, and 1 ul of T4 ligase were combined and incubated

at 4°C overnight.

The following day, the ligations were transformed into XL1-Blue subcloning-grade

competent cells (Stratagene). For each sample, 6 ul of the ligation reaction was added to a 50 ul

aliquot of bacteria and placed on ice for 20 minutes. The bacteria were then heat shocked at

42°C for 45 seconds, placed on ice for two minutes and subsequently shaken for 30 minutes at

37°C in 0.9 mL of SOC medium. The transfomations were plated onto Luria-Bertani (LB) plates

(1% tryptone, 0.5% yeast extract, 1% sodium chloride and 1.5% agar) containing 50 ug/mL

ampicillin. In addition, the plates were supplemented with 100 ul of 100 mM IPTG and 25 ul of

40 mg/mL X-Gal for blue-white colony selection (Sambrook et al., 1989). Plates were incubated

overnight at 37°C. The next day, white colonies were picked and cultured overnight at 37°C in 3

mL LB broth, consisting of 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 50 ug/mL

ampicillin. Plasmid DNA was purified from the cultures using the QIAprep Spin Miniprep Kit

(Qiagen) as directed.

38

Plasmid Sequencing

Purified plasmids were sequenced using ABI Prism BigDye Terminator reagent (Applied

Biosystems) and run on an ABI Prism 377XL Automated DNA Sequencer at the Center for

Epigenetics DNA Sequence Core (University of Florida). Sequencing reactions were set up as

follows: 1 ul of 3.2 pmol/ul SP6 primer, 2 ul of 5X sequencing buffer, 2 ul DNA, 2 ul BigDye

Terminator, and 3 ul ddH2O. Reactions were subject to PCR for 24 cycles of: 96°C for 30 s,

50°C for 15 s and 60°C for 4 minutes. After PCR, the reactions were purified by passage

through Performa DTR Gel Filtration Columns (Edge Biosystems) and taken to the Center for

Epigenetics for sequencing. Sequence files were analyzed using Sequencher 4.2 (Gene Codes

Corporation).

39

CHAPTER 3 THE IMPRINTED BAC TRANSGENE

Introduction

Attempts to Identify the Murine AS-IC

Imprinted genes characteristically reside in clusters that are coordinately regulated in cis by

IC elements. These imprinted domains typically span over one megabase of DNA and thus the

ICs must be capable of exerting their control across extensive amounts of sequence. A bipartite

IC consisting of the PWS-IC and the AS- IC exerts control over the entire PWS/AS locus, a

domain encompassing approximately two megabases of sequence. Although the human AS-IC

has been localized to a region about 35 kb upstream of the PWS-IC, the location of the murine

AS-IC remains unknown. Several targeted deletion approaches to knock out AS-IC function

have proven unsuccessful, including two deletions made by our lab that removed sequences at

the same location as the human AS-IC (relative to the PWS-IC). These knockouts consisted of

an 8.2 kb deletion, from -29 to -37 relative to Snrpn exon one, and a 12.8 kb deletion, from -37

to -24 relative to Snrpn exon one (Peery et al., 2007). Both knockout models were viable and

fertile with no discernable phenotype. Notably, these deletions left all of the currently identified

Snrpn alternative upstream exons intact.

The Beaudet lab also made mutations in the region upstream of Snrpn in an attempt to

knockout AS-IC function (Wu et al., 2006). One of these mutations consisted of a large deletion

encompassing 80 kb of sequence from -13 to -93 relative to Snrpn exon one, a region containing

two of the most proximal Snrpn upstream exons but not U2 or U4-U9. This deletion generated

mice displaying an imprinting defect with incomplete penetrance. Upon maternal inheritance of

this deletion, the DNA methylation imprint at the maternal PWS-IC was lost or partially lost in

over half of the pups analyzed. There was no detectable imprinting defect when the paternal

40

chromosome carried this deletion. The other mutation generated in this study was described as

an “insertion/duplication mutation” 13 kb upstream of Snrpn exon one, designated the ICan allele.

This insertion included Hprt exons three to nine, a loxP site, a puromycin resistance (PurR)

cassette transcribed in the opposite orientation of Snrpn, and a six kb duplication of target site

sequence. Paternal transmission of the ICan allele had no consequence. In contrast, maternal

inheritance of the ICan allele resulted in biallelic expression of Snrpn, a loss of DNA methylation

at the maternal PWS-IC, and a dramatic reduction in Ube3a expression in the brain. Thus, the

maternally inherited ICan mutant displays several phenotypes associated with loss of AS-IC

function. The mechanism of interference with AS-IC activity created by the ICan

insertion/duplication remains unclear. One possibility is that transcription from the PurR cassette

interferes with production of the Snrpn upstream exon transcripts.

In additional efforts to identify the AS-IC and characterize the mechanism utilized in

imprinting regulation at the PWS/AS locus, several transgenic mouse models have been

generated. In these models, an imprinted transgene is defined as displaying 1) Snrpn expression

upon paternal but not maternal transmission and 2) DNA methylation at the transgene PWS-IC

exclusively after maternal transmission. Previously, our lab produced transgenic lines from a P1

phage clone containing Snrpn as well as 33 kb of 5’ flanking sequence, sequence lacking any of

the identified alternative upstream exons. Imprinting of this transgene varied based on copy

number with a single copy line expressing Snrpn upon both maternal and paternal inheritance but

with a two-copy line displaying maternally imprinted Snrpn expression (Blaydes et al., 1999).

This model indicated that all the elements required for AS-IC function are not present on the

transgene but that in multi-copy this deficiency is overcome and imprinting is observed. One

41

explanation for this result is that the extra copy of the transgene adds back genomic sequence

necessary for imprinting to occur.

Another transgenic study, published by Shemer et al., also utilized a Snrpn P1 phage clone.

In contrast to our transgene, this transgene contained approximately 50 kb of sequence 5’ to

Snrpn, including the most proximal upstream exon, U1. Two high copy number lines were

examined: one line containing ten copies and the other containing 20 copies of the transgene.

DNA methylation analysis using methylation sensitive restriction enzyme digests showed that

this transgene was methylated at two sites within the PWS-IC upon maternal but not paternal

inheritance (Shemer et al., 2000). Analysis of transgene expression in this study was

complicated by the presence of endogenous Snrpn background. The transgene was reported as

being imprinted, however, close inspection of figure 2C in this paper suggests biparental Snrpn

expression albeit at a much higher level after paternal transmission relative to the expression

level after maternal transmission.

Overall, these mouse models for identifying the AS-IC and investigating mechanisms of

regulation have proven unsuccessful and have yielded perplexing results. Several deletion

models have failed to completely knockout IC function while traditional transgenic models have

provided inconclusive results. Therefore, we have taken a different transgenic approach, one

utilizing BAC transgenes, to identify the murine AS-IC.

Founding the BAC Transgenic Model System

BAC transgenes have many advantages over conventional plasmid transgenes when

studying imprinted loci, mainly due to their large size. Up to 300 kb of genomic sequence can be

contained in one BAC thus providing a great vector for examining interactions between ICs and

the genes they regulate, which can be located hundreds of kilobases away. The large amount of

genomic sequence that can be incorporated into one BAC increases the probability that any other

42

existing elements necessary for proper imprinting will be included in the transgene. Another

advantage of BAC transgenes is that their large size typically results in integration at low copy

number, which is important as previous studies have shown that transgene copy number affects

imprinting at the PWS/AS locus. Furthermore, smaller plasmid based transgenes are frequently

influenced by the chromatin state at the integration site as well as by the flanking endogenous

sequence. These position effects cause differences in expression patterns among multiple

transgenic lines, producing conflicting results and making for cumbersome analyses. The

substantial amount of sequence contained in the BAC vectors can act as a buffer against these

effects, allowing for accurate and consistent expression patterns. One additional advantage of

BAC constructs is that they are easily modified via BAC recombination techniques. This simple

procedure allows for manipulation of the transgene, which can be useful for distinguishing it

from the endogenous locus or for examining the effects of modifications to the transgene such as

a deletion of regulatory sequences.

To study imprinting mechanisms at the PWS/AS locus and identify the murine AS-IC, we

created a BAC transgenic model system. Our system is based on the discovery of an imprinted

BAC transgene, a discovery indicating that all the genetic information necessary for imprinting

at this locus is contained within the transgene. Once an imprinted transgene was discovered,

implying that a functional AS-IC is located within this transgene sequence, modifications could

be made to knockout IC function. Loss of imprinting on a modified transgene would thereby

identify the AS-IC and provide a model to investigate IC mechanisms.

To create our model system, we screened the Roswell Park Cancer Institute (RPCI) RPCI-

23 murine BAC library, which was derived from the C57BL/6J mouse strain, for clones

containing the entire Snrpn gene (Chamberlain, 2003). In addition to Snrpn, we looked for

43

clones with varying amounts of upstream sequence as we hypothesized that the AS-IC is

contained within the Snrpn upstream exons. Twenty BACs were identified and of those, three

chosen for further study: 380J10, 425D18, and 215A9. The 380J10 BAC contains Snrpn as well

as 16 kb of sequence upstream and 140 kb of sequence downstream. The 425D18 BAC spans

from 120 kb 5’ to 65 kb 3’ relative to Snrpn while the 215A9 BAC extends 150 kb 5’ and 20 kb

3’ to Snrpn. Importantly, the 380J10 BAC possesses no upstream exons whereas the 425D18

BAC includes U1, U2, and U3 and the 215A9 BAC includes U1, U2, U3 and U4 (Figure 3-1).

Multiple lines of transgenics were generated from these three BACs and each line was examined

for Snrpn expression patterns.

In order to analyze imprinting in these transgenics, we had to be able to distinguish

expression of the transgene from endogenous Snrpn expression. Therefore, we made use of a

Snrpn mutant previously constructed by our lab, the ΔSmN mouse line. This mutant carries a

deletion between exons five to seven of the Snrpn gene, resulting in the absence of standard

Snrpn transcript and instead producing low levels of a larger fusion transcript that includes Snrpn

exons one to four and the neomycin resistance gene, which was inserted into the deletion during

targeting (Yang et al., 1998). Paternal transmission of the ΔSmN allele abolishes endogenous

Snrpn expression allowing for analysis of transgene expression without endogenous background.

Results from these experiments showed that the 380J10 BAC, the BAC lacking Snrpn upstream

exons, expresses Snrpn upon both maternal and paternal transmission, demonstrating that the

transgene does not contain sufficient sequence to confer imprinting. Conversely, both the 215A9

BAC and the 425D18 BAC displayed imprinted expression patterns, indicating that there is a

functional AS-IC located within these transgenes (Futtner, 2007).

44

As these transgenic studies showed that the 120 kb of sequence upstream of Snrpn on the

425D18 BAC is sufficient to establish imprinting, we chose to make this BAC the basis of our

model system to identify the AS-IC. To simplify expression analysis of the transgene and

eliminate the need for complex breeding schemes with lines bearing deletions in Snrpn or the

PWS-IC, we used BAC recombineering techniques to modify the transgene. We made a deletion

of approximately 242 bps of coding sequence between Snrpn exons five to seven. We chose this

deletion because it is the same deletion that was made previously in the lab at the endogenous

locus (the ΔSmN mutation) and it proved to have no effect on imprinting at the locus. We

termed this modified transgene 425Δ5-7. After thorough analysis of this transgene, further

modifications were made to identify the AS-IC as will be discussed in the upcoming chapters.

Results

The 425Δ5-7 BAC Possesses a Functional AS-IC

Imprinted expression of the 425Δ5-7 transgene

To begin our study, it was imperative to first examine the 425Δ5-7 BAC to ensure that it

was properly imprinted, displaying appropriate epigenetic imprinting marks as well as imprinted

expression patterns. The BAC was injected the into fertilized FVB/N oocytes and the oocytes

subsequently implanted into pseudopregnant females in an attempt to obtain multiple founders

(Futtner, 2007). Injections were performed by Chris Futtner, a previous graduate student in the

lab. From these injections, we were able to establish three transgenic lines suitable for analysis:

line A, H, and I. Because copy number influenced the imprinted status of previously analyzed

transgenic lines, we deemed it necessary to obtain at least one single copy line for our studies.

Copy number was analyzed via Southern blot on high molecular weight genomic DNA isolated

from postnatal day 1 (P1) whole brains. Line A was determined to be a single copy line while

lines H and I were found to bear multiple copies of the transgene (data not shown).

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After determining the copy number of each line, we investigated expression patterns to

verify the imprinted status of the 425Δ5-7 BAC. We expected to find imprinted expression such

that offspring inheriting the transgene maternally would silence Snrpn but offspring inheriting

the transgene paternally would express Snrpn. As this transgene bears a deletion in the body of

Snrpn, a simple RT-PCR was performed to determine expression of the transgene while at the

same time distinguishing it from the background endogenous expression. To distinguish

between the two transcripts, we designed primers in regions flanking the deletion (N2.1-F/N6.2-

R) (Appendix A). Our forward primer sequence is located within Snrpn exon four and our

reverse primer sequence is located within exon eight, generating amplicons of 513 bps from the

endogenous transcript and 350 bps from the transgenic transcript (Figure 3-2A). We set up

matings to obtain offspring with either a maternally or paternally inherited transgene. On the day

of birth, litters were collected and brains harvested from the pups. Total RNA from P1 whole

brains was extracted and subject to DNase treatment followed by reverse transcription to produce

cDNA. The cDNAs were then used as templates in RT-PCR reactions to analyze Snprn

expression. Our results showed that the transgene was expressed upon paternal transmission but

silenced upon maternal transmission in each of the three lines (Figure 3-2B-D). Given that

multiple lines demonstrated the same expression patterns, we concluded that the 425Δ5-7 BAC

was imprinted, as was the unmodified 425D18 BAC transgene. For the majority of our further

studies with this transgene, we used the 425Δ5-7A line since a single copy line was desirable for

analysis.

Epigenetic imprinting marks on the 425Δ5-7 transgene

A common and functionally important feature of imprinted loci is the presence of

differential epigenetic marks at imprinting control regions. These epigenetic marks are essential

for the establishment and maintenance of imprinting. Notably, within the PWS-IC resides the

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Snrpn DMR, which is hypermethylated on the maternal allele and unmethylated on the paternal

allele in somatic cells (Gabriel et al., 1998; Shemer et al., 1997). This is a germline DMR,

meaning the methylation imprint is erased in fetal germ cells and reapplied in a sex-specific

manner during gametogenesis. The Snrpn DMR obtains its maternal-specific methylation

imprint postnatally during the oocyte growth phase (Lucifero, Mann et al. 2004).

To investigate whether the 425Δ5-7 BAC undergoes appropriate epigenetic

reprogramming in the germline, we examined methylation at the Snrpn DMR in newborn brain

DNA using genomic bisulfite sequence analysis (Clark et al., 1994). Bisulfite treatment of DNA

results in the deamination of unmethylated cytosine residues, converting them to uracil

nucleotides while methylated cytosines remain unaffected by the treatment. After bisulfite

treatment, DNA is amplified by PCR and the unmethylated cytosines are represented as thymines

in the amplicon. By cloning the PCR product and sequencing individual clones, the methylation

status of a region of interest can be determined. For our analysis, we amplified a fragment of

364 bps that included the 5’ flanking region of Snrpn as well as exon one (Figure 3-3). This

sequence is located within the Snrpn DMR and spans 14 CpG dinucleotides. For this experiment

to be informative, we had to have the ability to distinguish the transgene sequences from the

endogenous sequences; therefore, we transferred the 425Δ5-7 transgene onto a C57BL/6J line

congenic for the PWS/AS domain of Mus musculus castaneus chromosome 7 (B6.cast.c7)

(Wakeland et al., 1997). We then utilized sequence polymorphisms between the C57BL/6J

derived transgene and the endogenous cast alleles, one of which was located within our 364 bp

amplicon. Our initial analysis was performed on the 425Δ5-7H line, which we crossed to the

B6.cast.c7 line. Subsequently, we mated the F1 offspring to generate pups for collection of

newborn brains. Whole brain genomic DNA was isolated and bisulfite sequence analysis

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performed on samples that were positive for the transgene and that also exhibited cast c7

homozygosity as indicated by PCR genotyping experiments. Sequences were examined for the

presence of a single nucleotide polymorphism (SNP) at base number 69 of the Snrpn DMR

amplicon. The C57BL/6J transgene was identified by a guanine nucleotide while the

endogenous cast alleles were identified by a thymine at this position. We compared data

between paternally and maternally inherited transgenic brains after performing experiments on at

least two brains per mode of inheritance. Our results from the sequence analysis were as

expected, the paternally transmitted 425Δ5-7H transgene displayed hypomethylation of the

Snrpn DMR while the maternally transmitted transgene possessed a hypermethylated DMR

(Figure 3-4A). The endogenous alleles displayed a mixture of methylated and unmethylated

clones as both the maternal and paternal alleles are represented in these sequences (Figure 3-4B).

Taken together, this methylation data along with the expression analysis confirm that BAC

425Δ5-7 harbors AS-IC activity.

We are in the process of repeating the genomic bisulfite sequencing experiment with the

425Δ5-7A line as we want to verify our methylation data with this single copy transgenic line.

We expect the results to be the same as those obtained with the 425Δ5-7H line; the transgene

will exhibit hypomethylation of the Snrpn DMR upon paternal transmission but display

hypermethylation of the DMR upon maternal transmission. These results, combined with the

425Δ5-7H methylation data, will provide compelling evidence indicating that the appropriate

epigenetic imprinting marks are established on the 425Δ5-7 transgene at the Snrpn DMR, a

DMR that is located within the PWS-IC.

Snrpn Upstream Exon Expression Patterns are Conserved on the 425Δ5-7A Transgene

In our proposed model for AS-IC function, transcription of the Snrpn alternative upstream

exons is vital for epigenetically modifying the PWS-IC on the maternal allele and thereby

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silencing the paternally expressed genes. As the 425Δ5-7 transgene displays appropriate

imprinted expression and epigenetic imprinting marks, we hypothesized that upstream exon

expression from this transgene would mimic the endogenous upstream exon expression patterns.

Specifically, we expected to find upstream exon containing transcripts from the transgene

exclusively in oocytes and in postnatal brain upon paternal transmission as published reports

have shown (Bressler et al., 2001; Le Meur et al., 2005; Mapendano et al., 2006). As stated

earlier, we chose the 425Δ5-7A line for our analysis as this was a single copy line. To examine

upstream exon expression patterns, we needed a way to distinguish the transgene-generated

transcript from the endogenous alleles. This was made possible by a LoxP site that remained in

the coding region of Snrpn after recombineering the deletion in exons five through seven. We

designed an RT-PCR experiment with a primer set (SnrpnU1-F2/Lox Tg-R2) that included a

forward primer in U1 and a reverse primer in the LoxP sequence (Appendix A), generating

amplicons exclusively from the transgene (Figure 3-5A). RT-PCRs were Southern blotted with a

probe for Snrpn U1 to exon three sequence to intensify signal strength.

Upstream exon expression in the brain

Initially, we performed RT-PCR on newborn whole brain cDNAs from pups inheriting the

transgene either maternally or paternally. As expected, transcripts derived from the transgene

that included U1 were detected in brain tissue after paternal transmission. Lower levels of

expression were detected after maternal transmission (Figure 3-5B). This leaky expression is

expected, as imprinting is never 100% efficient. The observed expression patterns mimic

upstream exon expression from the endogenous locus in the brain.

Upstream exon expression in the ovary

Next, we analyzed upstream exon expression from the transgene in the ovary. Detecting

expression in this tissue is important as the epigenetic imprint is established at the PWS-IC in

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growing oocytes. Thus, transcription of the Snrpn upstream exons, originating from the

transgene, in growing oocytes would provide supporting evidence for our hypothesis on the AS-

IC identity and function. In a study from the Trasler lab investigating the timing of DNA

methylation imprint establishment in the maternal germline, the Snrpn DMR was found to

acquire methylation in growing oocytes between 5-25 days postpartum (dpp) (Lucifero et al.,

2004). Therefore, we examined ovaries from three-week-old female mice, the age at which

imprints are being established in the oocytes, for upstream exon expression. We harvested

ovaries from four transgenic females (maternal transmission of the transgene) and two non-

transgenic littermates for negative control tissue. As all alleles in the oocytes are reset as

maternal alleles, the mode of parental inheritance of the transgene was irrelevant. We performed

RT-PCR using the U1-LoxP primer set on cDNAs generated from ovary RNA. We then

Southern blotted the gel with a probe consisting of Snrpn U1-exon three sequence to intensify

the signal. Significantly, we detected upstream exon containing transcripts derived from the

transgene in the ovary (Figure 3-5C).

Snrpn upstream exon expression is not restricted to the brain and ovary

To complete our Snprn upstream exon expression comparisons between the transgene and

the endogenous locus, we performed RT-PCR on a panel of tissues from an adult 425Δ5-7A

paternally transmitted transgenic male. We made cDNAs from brain, testis, kidney, heart, and

liver tissue and used 425Δ5-7A transgenic ovary cDNA as a positive control. We first

performed RT-PCR looking specifically at upstream exon expression from the transgene by

using the U1-LoxP primer set and subsequently Southern blotting the gel with the Snrpn U1-

exon three probe. Surprisingly, we discovered that upstream exon-containing transcripts were

present in every tissue analyzed with low levels of expression in testis, kidney, and liver and high

levels of expression in ovary, brain, and heart (Figure 3-6). We next analyzed upstream exon

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expression from the endogenous allele by RT-PCR with the same forward U1primer (U1-F2) but

a reverse primer in Snrpn exon five (5’RACE Ex5-R), which consisted of sequence lying within

the exon five to seven deletion region (Appendix A). Again, we Southern blotted this gel with

the Snrpn U1-exon three probe. In contrast to published reports, we found expression of the

endogenous upstream exons in all tissues analyzed (Figure