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