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Chapt 13: Chromatin Structure and Its
Effects on Transcription
Student learning outcomes:
• Explain relationship among activators,
chromatin structure and gene activity
• Describe basic structure of the
nucleosome
• Explain how histones interact with
DNA and other proteins to control
transcription
Chromatin in
developing human
spermatid
13-1
• Describe how position of nucleosomes can result in
repression, and how remodeling permits activation.
• Explain how modification of histones can affect
gene expression
• Diagram two new techniques:
DNase hypersensitivity assay
Chromatin immunoprecipitation (ChIP)
• Describe how heterochromatin is condensed,
genetically inactive form
• Important Figures: 1, 2, 3, 7, 9, 12, 13*, 16*, 21, 22, 24, 26*,
27, 29*, 32*, 34*, 35*, 36*; Table 1
• Review problems: 1, 7, 8, 9, 12, 13, 14, 16, 18; Anal Q 2, 313-2
13.1 Histones in Eukaryotic cells
–
–
–
–
–
H1
H2A
H2B
H3
H4
21.5 kD
14.0
13.8
15.4
11.3
• Abundant proteins: mass in
nuclei nearly equals that of DNA
• Pronounced positive charge at
neutral pH: 20% lys and arg
• Each type not homogenous
– Gene reiteration
– Posttranslational
modifications (Ac, Me, PO4-)
Fig. 1 Histones from calf
thymus on SDS-PAGE 13-3
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Not single copy genes: repeated many times
Some copies are identical; Others are different
H4 has only had 2 variants ever reported
** Originally viewed as scaffolds for DNA:
regulatory role for gene expression is more recent
13.2 Nucleosomes
• Each chromosome is 1 long,
thin DNA molecule
• Will tangle if not carefully
folded
• 1st order of folding:
nucleosome:
• beads on string
– X-ray diffraction shows repeats
of structure at 100Å intervals
– Approximates nucleosomes
spaced at 110Å intervals
13-5
Histones in the Nucleosome
• Chemical cross-linking in solution:
– H3 to H4;
H2A to H2B
• H3 and H4 form tetramer (H3-H4)2
• Chromatin: roughly equal masses of DNA, histones:
• 1 histone octamer per 200 bp of DNA
• Octamer composed of:
2 each H2A, H2B, H3, H4
• DNA wrapped on outside
• [1 each of H1 binding to
linker region between
core nucleosomes ->
‘beads on string’ ]
Fig. 3
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Fig. 13.4
Fig. 4 Nucleosome core structure;
DNA on outside;
unstructured histone N-terminal tails;
(H3-H4)2 tetramer
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 histone core,
condensing DNA length 6- to 7-fold
– Core histones contain a histone fold:
• 3 α-helices linked by 2 loops
• Extended tail ~ 28% of core histone mass
• Tails are unstructured
13-8
H1 and Chromatin
• Trypsin or high salt buffer
removes histone H1
• Leaves chromatin looking
like “beads-on-a-string”
• Beads are nucleosomes
– Core histones form ball with
DNA around outside
– DNA on outside minimizes
bending
– H1 also lies on outside of
nucleosome
Fig. 6 H1 and chromatin
13-9
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The 30-nm Fiber
• 2nd order of chromatin folding
produces fiber 30 nm in diameter
– String of nucleosomes condenses
to form 30-nm fiber in solution of
increasing ionic strength
– Condensation results in another 6to 7-fold condensation of
nucleosome itself
• 4 nucleosomes condense into
30-nm fiber, form zig-zag
structure
Fig. 7 tetranucleosome
13-10
Formation of 30-nm Fiber
• Two stacks of nucleosomes
form left-handed helix
– Two helices of polynucleosomes
– Zig-zags of linker DNA
• Role of histone H1?
– 30-nm fiber can’t form without H1
– H1 crosslinks to other H1 more
often than to core histones
Fig. 8 model of
30-nm fiber
13-11
Model: Higher Order Chromatin Folding
• 30-nm fibers are most of
chromatin in typical
interphase nucleus
• Further folding needed in
mitotic chromosomes
• Model for higher order
folding is radial loops
• Can be supercoiling in
loops
Fig. 9 higher order folding
Source: Adapted from Marsden, M.P.F. and U.K. Laemmli,
Metaphase chromosome structure: Evidence of a radial
loop model. Cell 17:856, 1979.
13-12
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13.3 Chromatin Structure and Gene Activity
• Histones, especially H1, repressive effect on gene
activity in vitro
• Two families of 5S rRNA genes studied in Xenopus
laevis (pol III type 1)
– Oocyte genes expressed only in oocytes
• About 20,000 gene copies
– Somatic genes expressed both in oocytes and somatic cells
• About 400 copies
– Somatic genes form more stable complexes with
transcription factors, prevent nucleosomes forming complex
13-13
with the internal control region
Transcription Factors
and Histones Control
5S rRNA expression
• Genes are active when
TFIIIs prevent formation of
nucleosome stable
complexes with internal
control region
• Stable complexes require
histone H1 and exclude
TFIIIs once formed, so
genes are repressed
Fig. 11.39
Fig. 1313-14
Effects of Histones on Transcription of
Class II Genes (pol II)
• Core histones assemble nucleosome cores
on naked DNA
• Transcription of reconstituted chromatin
(average of 1 nucleosome / 200 bp DNA):
• Exhibits 75% repression relative to naked DNA
• Remaining 25% activity is due to promoter sites not
covered by nucleosome cores
13-15
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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 counteracted by transcription factors
• Sp1 and GAL4 act as both:
– Antirepressors prevent histone repression
– Transcription activators
• GAGA factor:
– Binds to GA-rich sequences in Krüppel promoter
– An antirepressor – prevents repression by histones
13-16
Model of Transcriptional Activation:
position of nucleosome is critical
Fig. 16;
Yellow is H1
Fig. 16 Source: Adapted from Laybourn, P.J. and J. T. Kadonaga, Role of nucleosomal cores and histone
H1
13-17
in regulation of transcription by polymerase II. Science 254:243, 1991.
Nucleosome Positioning
• Model of activation and antirepression:
• Transcription factors can cause antirepression by:
– Removing nucleosomes that obscure promoter
– Preventing initial nucleosome binding to promoter
• Both actions are forms of nucleosome positioning:
activators force nucleosomes to positions around,
not within, promoters
• Detect nucleosome-free zones by:
– Electron microsopy after restriction digest
– DNase hypersensitivity assay
13-18
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Detecting nucleosome-free zones
• Nucleosome positioning should give nucleosome-free zones in
control regions of active genes: SV40 virus model system
• Assessment in circular chromosome difficult without some
type of marker – use restriction enzymes
Figs. 17,18, 19
a-c, BamHI,
d-f, BglI
13-19
Detecting DNase-Hypersensitive Regions
• Active genes
tend to have
DNasehypersensitive
control regions
• Part of
hypersensitivity
is absence of
nucleosomes
• Detect as
cleavage
products on
gels with probe
Fig. 22 shows analysis of globin gene expression
13-20
Acetylation of Histone
tails activates gene
expression
• Histone acetyltransferase (HAT) adds acetyl group
• Nuclear acetylation of core histone N-terminal tails:
– Catalyzed by HAT A on specific lysines (HAT B cytoplasm)
• H3 (K9, 13, 18); H4 (K5, 8, 12, 16)
– Correlates with transcription activation (ex. TR/RXR)
– Coactivators of HAT A may loosen association between
nucleosomes and gene’s control region
– Attracts proteins like TAF11250, essential for transcription
• Some coactivators have HAT A activity:
13-21
GCN5, CBP/p300; TAF11250
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Histone Deacetylation represses genes
Transcription repressors bind to DNA sites, interact with
corepressors, which bind histone deacetylases
Deacetylation of histones:
basic histone tails
bind strongly to DNA
Repressors include:
unliganded nuclear receptors
Mad-Max
Corepressors include:
NCoR/SMRT
SIN3
Histone deacetylases:
HDAC 1, 2
Fig. 24
13-22
Chromatin, Activation and Repression
Deacetylation
of core
histones
removes
binding sites
for HAT A
coactivator
proteins that
are essential
for transcription
activation
13-23
**Fig. 26
Chromatin Remodeling
• Activation of many eukaryotic genes requires
chromatin remodeling (loosening, repositioning)
• Several protein complexes do remodeling
– All have ATPase activity: use energy from ATP
hydrolysis to remodel nucleosomes
– Alter structure of nucleosome core to make more
accessible to activators, nucleases
Ex. SWI/SNF from yeast (mating type switch)
also in mammals
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Model SWI/SNF Chromatin Remodeling
Fig. 27
SWI/SNF: In mammals, protein BRG1 is ATPase
9-12 BRG1-associated factors (BAFs);
• a highly conserved BAF is BAF 155 or 170
• its SANT domain binds histones - helps SWI/SNF bind
nucleosomes
13-25
Mechanism of Chromatin Remodeling
– Mobilization of nucleosomes from starting position
– Loosen association between DNA, core histones
– Open up promoters to transcription factors
• Formation of distinct conformations of nucleosomal
DNA/core histones - contrast with:
– Uncatalyzed DNA exposure in nucleosomes
– Simple nucleosome sliding along a DNA stretch
13-26
Testing model of nucleosome remodeling
Movement of nucleosomes opens up different
sequences to restriction enzyme digestion
- Time after addition of ATP and SWI/SNF
Fig. 13.28
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Chromatin Immunoprecipitation technique
Identifies specific sequence
bound by a specific protein
Steps include:
• Crosslink cell proteins to DNA
with formaldehyde in vivo
• Isolate chromatin;
• Carefully shear DNA
• Precipitate desired protein with
antibody and beads
• Reverse crosslink; remove proteins
• Use PCR with specific primers to
see if particular region was bound
Fig. 29
13-28
Ex. Remodeling in Yeast HO Gene Activation
• Chromatin immunoprecipitation (ChIP) reveals order
of factors binding to specific gene during activation
• Yeast have 2 mating types: a and α; can switch
• As HO gene is activated (mating type switch):
– First factor to bind is Swi5
– Followed by SWI/SNF and SAGA (containing HAT GCN5)
– Next general transcription factors and other proteins
• Chromatin remodeling is among first steps in
activation of gene
• Order could be different in other genes
13-29
Timing of histone acetyloation after
activation of Human IFN-β
β Gene
• ChIP analysis:
Infect with virus
to activate IFN
• Antibodies to
precipitate
specific proteins
• Analyze mRNA,
TBP
Fig. 30a
• B. Deplete HAT
activity -> no
acetylation of H4
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Remodeling Human IFN-β
β Gene:
ex. Histone Code
The Histone Code:
– Combination of modifications on
nucleosome near gene’s control
region affects efficiency of
transcription
– Code is epigenetic, not affect
sequence of DNA
1) Activators in IFN-β enhanceosome
recruit GCN5 HAT
– HAT acetylates some Lys on H3 and H4
in nucleosome at promoter
– Protein kinase phosphorylates Ser on H3
– Permits acetylation of another Lys on H3
– Ac-Lys recruits SWI/SNF, remodels
nucleosome
13-31
Remodeling Human IFN-β
β Gene: 2) TF Binding
• Remodeled nucleosome allows TFIID to bind 2 Ac-Lys via
bromodomain in TAFII250 (domain that binds Ac- lys)
• TFIID binding: Bends DNA (TBP), Moves remodeled
nucleosome aside, paves way for transcription to begin
Fig. 32
13-32
Heterochromatin
Euchromatin:
• relatively extended and open
• potentially active
Heterochromatin:
• very condensed,
• DNA inaccessible
– Repressive character
can silence genes 3 kb away
– centromeres
– telomeres
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Heterochromatin
and Silencing at
telomeres
Fig. 34
• Heterochromatin at tips of yeast chromosomes
(telomeres) silences nearby genes:
telomere position effect (TPE)
• Requires binding of specific proteins
– RAP1 to telomeric DNA
– Recruitment of proteins in order: SIR3 SIR4 SIR2
13-34
SIR Proteins
• Heterochromatin at other locations in
chromosome also depends on SIR proteins
(silencing information regulator)
• 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 (HATs) which acetylate
histones promote gene activity
13-35
Histone
Methylation
Fig. 35
• Methylation of Lys 9 in tail of H3 attracts HP1
• Recruits a histone methyltransferase (HMTase)
– Methylates Lys 9 on neighboring nucleosome
– Propagates repressed, heterochromatic state
• Methylation of Lys and Arg side chains in core
histones can have repressive or activating effects
13-36
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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
13-37
Histone tail
modifications
can be
repressive or
activating;
permit fine
level of
control on
gene
expression
Fig. 13.36
Modifications interactions
• Ubiquitination of H2B
K123 by the rad6 protein
is required for methylation
of H3 K79 or K4
• Antibodies specific for
different modifications
• Western blot after
separation of proteins by SDSPAGE
Fig. 13.37
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Modification
Interactions
Fig. 38
• Modifications shown above histone tail activate
– Ser phosphorylation
– Lys acetylation
• Modification below tail (Lys methylation) represses
13-40
Nucleosomes and Transcription Elongation
• An important transcription elongation facilitator is
FACT (facilitates chromatin transcription)
– 2 subunits:
• Spt16 binds to H2A-H2B dimers
– acid-rich C-terminus is essential for these
nucleosome remodeling activities
• SSRP1 binds to H3-H4 tetramers
– Facilitates transcription through nucleosome by
promoting loss of at least one H2A-H2B dimer
• Acts as histone chaperone promoting re-addition of
H2A-H2B dimer to nucleosome that has lost dimer
13-41
Review questions
• 1. Diagram nucleosome, showing rough positions of histones;
on another drawing, show position of the DNA
• 9. Present two models for antirepression by transcription
activators, one in which the gene’s control region is not
blocked by a nucleosome, the other in which it is.
• 12. Diagram technique for detecting DNase hypersensitive
region on DNA
• 18. Describe how you could use Chromatin
immunoprecipitation to detect proteins associated with a
particular gene at various points in cell cycle of yeast.
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