Transcript Inquiry into Life Twelfth Edition

Molecular Biology
Lecture 20
Chapter 13
Chromatin Structure
and Its Effects on
Transcription
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Histones
• Eukaryotic cells
contain 5 kinds of
histones
–
–
–
–
–
H1
H2A
H2B
H3
H4
Source: Panyim and Chalkley, Arch. Biochem.
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& Biophys. 130, 1969, f. 6A, p.343.
Properties of Histones
• 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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Nucleosomes
• 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
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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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Fig. 13.4
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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
– H1 also lies on the outside of the nucleosome
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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 about 28% of core histone mass
• Tails are unstructured
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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 30nm fiber form a zig-zag structure
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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
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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,
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1979.
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
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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
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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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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.
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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
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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
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Detecting DNaseHypersensitive 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
• Unliganded nuclear receptors
• Mad-Max
– Corepressors
• NCoR/SMRT
• SIN3
– Histone deacetylases - HDAC1 and 2
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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
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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
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