Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth 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.
13.1 Histones
• Eukaryotic cells
contain 5 kinds of
histones
–
–
–
–
–
H1
H2A
H2B
H3
H4
• Each histone type
isn’t homogenous
– Gene reiteration
– Posttranslational
modification
Source: Panyim and Chalkley, Arch. Biochem.
13-2
& Biophys. 130, 1969, f. 6A, p.343.
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
13-3
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
– X-ray diffraction has shown strong repeats of
structure at 100Å intervals
– This spacing approximates the nucleosome
spaced at 110Å intervals
13-4
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
13-5
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
13-6
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
13-7
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
13-8
Formation of the 30-nm Fiber
• Two stacks of nucleosomes form a lefthanded 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
13-9
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,
13-10
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
13-11
13.3 Chromatin Structure and
Gene Activity
• Histones, especially H1, have a repressive
effect on gene activity in vitro
• Two families of 5S rRNA genes studied are
oocyte and somatic genes
– Oocyte genes are expressed only in oocytes
– Somatic genes are expressed both in oocytes
and somatic cells
– Somatic genes form more stable complexes
with transcription factors
13-12
Transcription Factors and
Histones Control the 5S rRNA
• Genes active by
TFIIIs preventing
formation of
nucleosome stable
complexes with
internal control region
• Stable complexes
require histone H1
and exclude TFIIIs
once formed so that
genes are repressed
13-13
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
13-14
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
13-15
Model of Transcriptional
Activation
Source: Adapted from Laybourn, P.J. and J. T. Kadonaga, Role of nucleosomal cores and histone H1 in
regulation of transcription by polymerase II. Science 254:243, 1991.
13-16
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
13-17
Nucleosome-Free Zones
• Nucleosome positioning would result in
nucleosome-free zones in the control regions of
active genes
• Assessment in a circular chromosome can be
difficult without some type of marker
13-18
Detecting DNaseHypersensitive Regions
• Active genes tend to have DNase-hypersensitive
control regions
• Part of this hypersensitivity is due to absence of
13-19
nucleosomes
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
13-20
Histone Deacetylation
• Transcription repressors bind to DNA sites
and interact with corepressors which in
turn bind to histone deacetylases
– Repressors
• Unliganded nuclear receptors
• Mad-Max
– Corepressors
• NCoR/SMRT
• SIN3
– Histone deacetylases - HDAC1 and 2
13-21
Ternary Protein Complexes
• 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
13-22
Activation and Repression
Source: Adapted from Wolfe, A.P., 1997. Sinful repression. Nature 387:16-17.
Deacetylation of core histones removes binding
sites for bromodomain proteins that are essential
for transcription activation
13-23
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
13-24
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
13-25
SWI/SNF Chromatin
Remodeling
13-26
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
13-27
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
13-28
Chromatin Immunoprecipitation
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 Lys in the nucleosomes through
the dual bromodomain in TAFII250
• 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 of at 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
Modification Interactions
• The modifications
shown above the tail
are activating
– Ser phosphorylation
– Lys acetylation
• Modification below
the tail (Lys
methylations) is
repressive
13-36
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
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
– 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
13-38
has lost such a dimer