molecular biology fifth edition chapter 13 chromatin structure and its effects on transcription...
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Molecular BiologyFifth Edition
Chapter 13
Chromatin Structure and Its Effects on
Transcription
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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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