Transcript Chapter 13 Lecture PowerPoint - McGraw Hill Higher Education

Lecture PowerPoint to accompany
Molecular Biology
Fifth Edition
Robert F. Weaver
Chapter 13
Chromatin Structure
and Its Effects on
Transcription
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
13-2
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
13-3
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
13-4
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
13-5
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
13-6
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
13-7
Nucleosome Structure
• Central (H3-H4)2 core attached to H2AH2B 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 a-helices linked by 2 loops
• Extended tail of abut 28% of core histone mass
• Tails are unstructured
13-8
Crystal Structure of a Nucleosomal
Core Particle
13-9
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 30nm fiber form a zig-zag structure
13-10
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
13-11
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,
13-12
1979.
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
13-13
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
13-14
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
13-15
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
13-16
A Model of Transcriptional Activation
13-17
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
13-18
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
13-19
Detecting DNase-Hypersensitive Regions
• Active genes tend to have DNase-hypersensitive
control regions
• Part of this hypersensitivity is due to absence of
nucleosomes
13-20
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
13-21
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
13-22
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
13-23
Model for Activation and Repression
13-24
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
13-25
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
13-26
Models for SWI/SNF Chromatin
Remodeling
13-27
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
13-28
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
13-29
Remodeling in the Human IFN-b 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-b 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
13-30
Remodeling in the Human IFN-b 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
13-31
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
13-32
Heterochromatin and Silencing
• 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
13-33
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
13-34
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
13-35
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
13-36
Summary
• 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
13-37
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
13-38
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
13-39
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 readdition of H2A-H2B dimer to a nucleosome that
has lost such a dimer
13-40