molecular biology fifth edition chapter 13 chromatin structure and its effects on transcription...

Molecular BiologyFifth Edition

Chapter 13

Chromatin Structure and Its Effects on

Transcription

Lecture PowerPoint to accompany

Robert F. Weaver

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Chromatin Structure

• Eukaryotic genes do not exist naturally as naked DNA, or even as DNA molecules bound only to transcription factors

• They are complexed with an equal mass of other proteins to form chromatin

• Chromatin is variable and the variations play an enormous role in chromatin structure and in the control of gene expression

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13.1 Histones

• Eukaryotic cells contain 5 kinds of histones– H1– H2A– H2B– H3– H4

• Histone proteins are not homogenous due to:– Gene reiteration– Posttranslational modification

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Properties of Histones

• Abundant proteins whose mass in nuclei nearly equals that of DNA

• Pronounced positive charge at neutral pH

• Most are well-conserved from one species to another

• Not single copy genes, repeated many times– Some copies are identical

– Others are quite different

– H4 has only had 2 variants ever reported

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13.2 Nucleosomes

• Chromosomes are long, thin molecules that will tangle if not carefully folded

• Folding occurs in several ways

• First order of folding is the nucleosome, which have a core of histones, around which DNA winds– X-ray diffraction has shown strong repeats of

structure at 100Å intervals– This spacing approximates the nucleosome

spaced at 110Å intervals

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Histones in the Nucleosome

• Chemical cross-linking in solution:– H3 to H4– H2A to H2B

• H3 and H4 exist as a tetramer (H3-H4)2

• Chromatin is composed of roughly equal masses of DNA and histones– Corresponds to 1 histone octamer per 200 bp

of DNA– Octamer composed of:

• 2 each H2A, H2B, H3, H4• 1 each H1

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H1 and Chromatin

• Treatment of chromatin with trypsin or high salt buffer removes histone H1

• This treatment leaves chromatin looking like “beads-on-a-string”

• The beads named nucleosomes– Core histones form a ball with DNA wrapped

around the outside– DNA on outside minimizes amount of DNA

bending– H1 also lies on the outside of the nucleosome

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Nucleosome Structure

• Central (H3-H4)2 core attached to H2A-H2B dimers

• Grooves on surface define a left-hand helical ramp – a path for DNA winding– DNA winds almost twice around the histone

core condensing DNA length by 6- to 7-X– Core histones contain a histone fold:

• 3 -helices linked by 2 loops• Extended tail of abut 28% of core histone mass• Tails are unstructured

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Crystal Structure of a Nucleosomal Core Particle

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The 30-nm Fiber

• Second order of chromatin folding produces a fiber 30 nm in diameter– The string of nucleosomes condenses to form

the 30-nm fiber in a solution of increasing ionic strength

– This condensation results in another six- to seven-fold condensation of the nucleosome itself

• Four nucleosomes condensing into the 30-nm fiber form a zig-zag structure

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Models for the 30-nm Fiber

• The solenoid and the two-start double helix model each have experimental support

• A technique called single-molecule force spectroscopy was employed to answer the question, ‘which model is correct?’

• Results suggested that most of the chromatin in a cell (presumably inactive) adopts a solenoid shape while a minor fraction (potentially active) forms a 30-nm fiber according to the two-start double helix

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Higher Order Chromatin Folding

• 30-nm fibers account for most of chromatin in a typical interphase nucleus

• Further folding is required in structures such as the mitotic chromosomes

• Model favored for such higher order folding is a series of radial loops Source: Adapted from Marsden, M.P.F. and U.K.

Laemmli, Metaphase chromosome structure: Evidence of a radial loop model. Cell 17:856, 1979.

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Relaxing Supercoiling in Chromatin Loops

• When histones are removed, 30-nm fibers and nucleosomes disappear

• Leaves supercoiled DNA duplex

• Helical turns are superhelices, not ordinary double helix

• DNA is nicked to relax

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13.3 Chromatin Structure and Gene Activity

• Histones, especially H1, have a repressive effect on gene activity in vitro

• Histones play a predominant role as regulators of genetic activity and are not just purely structural

• The regulatory functions of histones have recently been elucidated

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Effects of Histones on Transcription of Class II Genes

• Core histones assemble nucleosome cores on naked DNA

• Transcription of reconstituted chromatin with an average of 1 nucleosome / 200 bp DNA exhibits 75% repression relative to naked DNA

• Remaining 25% is due to promoter sites not covered by nucleosome cores

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Histone H1 and Transcription

• Histone H1 causes further repression of template activity, in addition to that of core histones

• H1 repression can be counteracted by transcription factors

• Sp1 and GAL4 act as both:– Antirepressors preventing histone repressions– Transcription activators

• GAGA factor: – Binds to GA-rich sequences in the Krüppel promoter– An antirepressor – preventing repression by histones

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A Model of Transcriptional Activation

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Nucleosome Positioning

• Model of activation and antirepression asserts that transcription factors can cause antirepression by: – Removing nucleosomes that obscure the

promoter– Preventing initial nucleosome binding to the

promoter

• Both actions are forms of nucleosome positioning – activators force nucleosomes to take up positions around, not within, promoters

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Nucleosome-Free Zones• Nucleosome positioning would result in

nucleosome-free zones in the control regions of active genes

• Assessment in SV40 DNA, a circular minichromosome, was performed to determine the existence of nucleosome-free zones - with the use of restriction sites it was found that the late control region is nucleosome free

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Detecting DNase-Hypersensitive Regions• Active genes tend to have DNase-hypersensitive

control regions• Part of this hypersensitivity is due to absence of

nucleosomes

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Histone Acetylation• Histone acetylation occurs in both cytoplasm

and nucleus• Cytoplasmic acetylation carried out by HAT B

(histone acetyltransferase, HAT) – Prepares histones for incorporation into nucleosomes– Acetyl groups later removed in nucleus

• Nuclear acetylation of core histone N-terminal tails– Catalyzed by HAT A– Correlates with transcription activation– Coactivators of HAT A found which may allow

loosening of association between nucleosomes and gene’s control region

– Attracts bromodomain proteins, essential for transcription

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Histone Deacetylation

• Transcription repressors bind to DNA sites and interact with corepressors which in turn bind to histone deacetylases– Repressors

• Mad-Max

– Corepressors• NCoR/SMRT• SIN3

– Histone deacetylases - HDAC1 and 2

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Model for participation of HDAC in transcription repression

• Assembly of complex brings histone deacetylases close to nucleosomes

• Deacetylation of core histones allows – Histone basic tails to

bind strongly to DNA, histones in neighboring nucleosomes

– This inhibits transcription

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Model for Activation and Repression

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Chromatin Remodeling

• Activation of many eukaryotic genes requires chromatin remodeling

• Several protein complexes carry this out– All have ATPase harvesting energy from ATP

hydrolysis for use in remodeling– Remodeling complexes are distinguished by

ATPase component

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Remodeling Complexes

• SWI/SNF – In mammals, has BRG1 as ATPase– 9-12 BRG1-associated factors (BAFs)

• A highly conserved BAF is called BAF 155 or 170• Has a SANT domain responsible for histone

binding• This helps SWI/SNF bind nucleosomes

• ISWI– Have a SANT domain– Also have SLIDE domain involved in DNA

binding

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Models for SWI/SNF Chromatin Remodeling

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Mechanism of Chromatin Remodeling

• Mechanism of chromatin remodeling involves: – Mobilization of nucleosomes– Loosening of association between DNA and core

histones

• Catalyzed remodeling of nucleosomes involves formation of distinct conformations of nucleosomal DNA/core histones when contrasted with: – Uncatalyzed DNA exposure in nucleosomes– Simple nucleosome sliding along a DNA stretch

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Remodeling in Yeast HO Gene Activation• Chromatin immunoprecipitation (ChIP) can

reveal the order of binding of factors to a gene during activation

• As HO gene is activated:– First factor to bind is Swi5– Followed by SWI/SNF and SAGA containing HAT

Gcn5p– Next general transcription factors and other proteins

bind

• Chromatin remodeling is among the first steps in activation of this gene

• Order could be different in other genes

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Remodeling in the Human IFN- Gene: The Histone Code

The Histone Code: – The combination of histone modifications on a

given nucleosome near a gene’s control region affects efficiency of that gene’s transcription

– This code is epigenetic, not affecting the base sequence of DNA itself

• Activators in the IFN- enhanceosome can recruit a HAT (GCN5) – HAT acetylates some Lys on H3 and H4 in a

nucleosome at the promoter– Protein kinase phosphorylates Ser on H3– This permits acetylation of another Lys on H3

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Remodeling in the Human IFN- Gene: TF Binding

• Remodeling allows TFIID to bind 2 acetylated lysines in the nucleosome through the dual bromodomain in TAF1

• TFIID binding– Bends the DNA– Moves remodeled nucleosome aside– Paves the way for transcription to begin

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Heterochromatin

• Euchromatin: relatively extended and open chromatin that is potentially active

• Heterochromatin: very condensed with its DNA inaccessible– Microscopically appears as clumps in higher

eukaryotes– Repressive character able to silence genes as

much as 3 kb away

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• Formation at the tips of yeast chromosomes (telomeres) with silencing of the genes is the telomere position effect (TPE)

• Depends on binding of proteins– RAP1 to telomeric DNA– Recruitment of proteins in this order:

• SIR3• SIR4• SIR2

Heterochromatin and Silencing

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SIR Proteins

• Heterochromatin at other locations in chromosome also depends on the SIR proteins

• SIR3 and SIR4 interact directly with histones H3 and H4 in nucleosomes– Acetylation of Lys 16 on H4 in nucleosomes

prevents interaction with SIR3– Blocks heterochromatin formation

• Histone acetylation also works in this way to promote gene activity

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Histone Methylation

• Methylation of Lys 9 in N-terminal tail of H3 attracts HP1

• This recruits a histone methyltransferase– Methylates Lys 9 on a neighboring

nucleosome– Propagates the repressed, heterochromatic

state

• Methylation of Lys and Arg side chains in core histones can have either repressive or activating effects

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Histone Methylation

• Methylation of Lys 4 in N-terminal tail of H3 is generally tri-methylated (H3K4Me3) and is usually associated with the 5’-end of an active gene

• This modification appears to be a sign of transcription initiation

• Genome-wide ChIP analysis suggests that this may also play a role in controlling gene expression by controlling the re-starting of paused RNA polymerases

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• Histone modifications can affect gene activity by two mechanisms:

• 1. By altering the way histone tails interact with DNA and with histone tails in neighboring nucleosomes, and thereby altering nucleosome cross-linking

• 2. By attracting proteins that can affect chromatin structure and activity

Summary

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Modification Combinations

• Methylations occur in a given nucleosome in combination with other histone modifications:– Acetylations– Phosphorylations– Ubiquitylations

• Each particular combination can send a different message to the cell about activation or repression of transcription

• One histone modification can also influence other, nearby modifications

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Nucleosomes and Transcription Elongation

• An important transcription elongation facilitator is FACT (facilitates chromatin transcription)

– Composed of 2 subunits:

• Spt16

– Binds to H2A-H2B dimers

– Has acid-rich C-terminus essential for these nucleosome remodeling activities

• SSRP1 binds to H3-H4 tetramers

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Nucleosomes and Transcription Elongation

• FACT facilitates transcription through a nucleosome by promoting loss of at least one H2A-H2B dimer from the nucleosome

• Also acts as a histone chaperone promoting re-addition of H2A-H2B dimer to a nucleosome that has lost such a dimer