Download Chromatin Structure and Its Effects on Transcription

Chromatin Structure and Its Effects
on Transcription
Chapter 13
Histones
• Eukaryotic cells contain 5
kinds of histones
–
–
–
–
–
H1
H2A
H2B
H3
H4
• Each histone type - not
homogenous
– Gene reiteration
– Posttranslational
modification
Features of Histones
• Abundant proteins
• 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 - 2 variants
Nucleosomes
• Chromosomes are long, thin molecules –tangle - if
not carefully folded
• Folding occurs in several ways
- First order of folding is the nucleosome
– Maurice Wilkins - X-ray diffraction has shown strong
repeats of structure at 100Å intervals
– 110Å spacing
Histones in the Nucleosome
• Chemical cross-linking in solution: (Korenberg)
– 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
– 1 histone octamer/200 bp of DNA
– Octamer composed of:
• 2 each of H2A, H2B, H3, H4
• 1 each of H1
H1 and Nucleosomes
• Treatment of chromatin with trypsin or high salt
buffer removes histone H1
• Left 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
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 by deacetylases
Histone Acetylation
• Nuclear acetylation of core histone N-terminal tails
– Catalyzed by HAT A
– Attracts bromodomain proteins - essential for
transcription
– Correlates with transcription activation
– Coactivators of HAT A found which may allow loosening
of association between nucleosomes and gene’s control
region
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
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
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
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
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
SWI/SNF Chromatin Remodeling
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
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
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
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
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
Modification Interactions
• The modifications
shown above the tail are
activating
– Ser phosphorylation
– Lys acetylation
• Modification below the
tail (Lys methylations) is
repressive
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
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
has lost such a dimer
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