chromatin structure and its regulation–2...3. dorigo b, et al. nucleosome arrays reveal the...
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Biochemistry 201 Biological Regulatory Mechanisms Lecturer: Geeta Narlikar February 21, 2013 Chromatin Structure and Its Regulation–2 Key Points 1. There are at least two ways in which arrays of chromatin can fold into the 30 nm fiber. 2. Histones provide a versatile regulatory platform through their many post-translational modifications. Specific modifications are bound by specialized protein domains that bring about distinct downstream events. 3. Acetylated histones strongly correlate with transcriptional activation and deacetylated histones with transcriptional repression. Acetylation can facilitate transcription initiation directly by disrupting higher order chromatin folding or indirectly by recruiting bromo-domain containing transcription factors. 4. A major pathway of altering chromatin structure is through the action of ATP-utilizing machines or ‘chromatin-remodeling complexes’. These ATPases have homology with DEAD box family DNA helicases. Different classes of chromatin-remodeling complexes generate different biochemical outputs. Not clear how the different biochemical outputs relate to their distinct biological roles. 5. Methylation of histones on lysines has context dependent effects. Trimethylation at Lysine 9 and 27 correlates with gene repression, whereas trimethylation at Lysine 4 often correlates with gene activation. Methylated histones are recognized by specific domains such as chromodomains or PHD fingers. 6. Heterochromatin allows for silencing of large domains of the genome and appears to utilize a combination of mechanisms to do so. 7. The presence of more than one type of binding domain within large regulatory complexes suggests mechanisms for specific recognition of different combinations of histone modifications within one nucleosome.
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nucleosome spacing. Nat Struct Mol Biol, 2006. 13(12): p. 1078-83. 15. Racki LR et al., The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes. Nature. 2009. 462:1016-21. 16. Blosser et al., Dynamics of nucleosome remodelling by individual ACF complexes.
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Science, 2004. 303(5656): p. 343-8. Interplay between SWI/SNF and HATs 1. Hassan, A.H., et al., Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter
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Outline
Func%ons of Chroma%n
Packing material Complex regulatory pla9orm
Replica%on Transcrip%on RNA processing
coordina%on/coupling
Intrinsic proper%es Regula%on of intrinsic proper%es
Higher order compac%on of chroma%n
One start Two start
30 nm Electron micrographs
H2A-‐H2B acidic patch
Histone tails mediate inter-‐nucleosomal contacts through electrosta%c interac%ons
Highly basic histone tails interact with DNA of neighboring nucleosomes
Histone H4 tail interacts with an acidic patch formed by H2A-‐H2B
Linker histones (Histone H1) promote chroma%n folding
Implica%on: more func%ons than just packing DNA
Histones contain many different post-‐transla%onal modifica%ons: concentrated on N-‐terminal tails but also found on internal regions
H2A
H2B
H3
H4
PhosphorylaDon, AcetylaDon MethylaDon UbiquitylaDon
Two Case Studies
Histone H3 and H4 Acetyla%on Histone H3 K9 Methyla%on
EuchromaDn (acDve genes)
HeterochromaDn (repressed genes)
Drosophila salivary glands polytene chromosomes stained to detect the DNA
Lighter stains = euchromaDn Darker stains = heterochromaDn
The role(s) of lysine acetylaDon in histone tails
Hyperacetylation of histones was correlated with active genes over 30 years ago.!!GCN5 was originally identified as a transcriptional co-activator of amino acid biosynthesis genes!!1996: yeast GCN5 was shown to have acetyl transferase activity in vitro!
* acetyl lysine
Lysine acetylaDon by GCN5
CoA S CH C
O
3 + Lys N H
H
H
+
CoA SH 3 + Lys N
H
C
O
CH + H +
GCN5
Results in loss of one posiDve charge
GCN5 is part of SAGA complex
Other funcDons of SAGA: Several genes are SAGA dependent but GCN5 independent
from EM structure
Interact with TBP
Ubp8 Removes monoubiquiDn from H2B (locaDon in complex not known)
Interacts with acDvators
AcetylaDon is reversible
Histone De-‐acetylases (HDACS or KDACs) remove acetyl groups -‐-‐-‐most oZen correlate with gene repression Histone acetyl transferases (HATs or KATs) add acetyl groups -‐-‐-‐most oZen correlate with gene acDvaDon AcetylaDon state is very dynamic -‐-‐-‐turnover within minutes
How does histone acetylaDon enhance transcripDon?
Lys N H H
H
+
How does histone acetylaDon enhance transcripDon?
vs.
(1) Does acetylaDon reduce histone-‐DNA interacDons?
closed open
Keq= [open]/[closed]
HyperacetylaDon of all the histone tails increases Keq by ~2 fold
Lys N H
C
O
CH 3
(2) AcetylaDon has large effects on disrupDng inter-‐nucleosomal contacts and on chromaDn compacDon
H2A-‐H2B acidic patch
N-‐GLGKGGAKRHRKV Ac Ac H4
Single acetylaDon mark on H4 lysine 16 has similar impact on chromaDn compacDon as deleDng H4 tail
12 16
(3) Acetylated lysine provides a recogniDon moDf for an “effector protein”
Lys N H
C
O
CH 3 Bromodomain
Bromodomains specifically recognize acetylated lysines
bromo HAT
GCN5 bromodomain with H4 tail acetylated at K16
Budding yeast has 15 bromodomains
1 bromodomain 8 bromodomains
9 of them are distributed between two ATP-‐dependent chroma%n remodeling complexes
SWI/SNF and RSC can enable many different outcomes
transfer histone octamers
Generate DNA “loops”
exchange and remove histone dimers
move histone octamers
Many open mechanis%c ques%ons
So far four major families of ATP-‐dependent chromaDn remodeling complexes High degree of conserva%on from yeast to humans
gene acDvaDon and some gene repression: mulDple biochemical outcomes
some acDvaDon, mostly gene repression and heterochromaDn formaDon: only movesnucleosomes
gene acDvaDon and repression: only move nucleosomes
gene acDvaDon, DNA replicaDon, DNA damage responses move nucleosomes and exchange variant histones
Not clear (i) if the different families work by similar or disDnct mechanisms and (ii) how their different biochemical outputs relate to their different biological roles
Classified based on their ATPase subunits, which are shown below
A cascade of events at the promoter of the human interferon-‐b gene
Human TFIID contains TAFII250,which is a HAT and also contains a double bromodomain
Sequence Specific binding
Several methyla%on marks with different readouts
Methyl marks are bound by Chromodomains and PHD fingers Methylases put on the mark and De-‐methylases remove the marks
(me)3 (me)3 (me)3 (me)2 OFF OFF
OFF
ON
S
mutually exclusive mutually exclusive
Q
Lysine Arginine
N H H
H
+
N H
C
O
CH 3
N CH + 3 CH 3
CH 3
N C N
NH
H H
2
+
N C N
NH
CH CH
2
+ 3 3
unmodified
acetylated
methylated
InteracDons with residues surrounding the ARKS moDf give posiDon specificity
Hydrophobic cage gives mark specificity
Chromodomain and PHD fingers have independently evolved to use caDon-‐π InteracDons to stabilize methylated lysines
(me)3 (me)3 OFF
OFF
Q
mutually exclusive
A S A
Crystal structure of chromodomain from Drosophila HP1 protein
Position Effect Variegation reveals ability of heterochromatin to spread
heterochromaDn white gene @ normal posDon
chromosome swapping
white gene near heterochromaDn
Drosophila fruit fly
HeterochromaDn can spread over more than 1000 kb of previously euchromaDc chromaDn and heritably silences genes
Effects linked to the HP1 protein and methylaDon of Histone H3 at K9
HP1 binds methyl mark using a chromodomain
1) How does heterochromaDn spread? 2) How is silencing achieved? Is the silencing achieved by heterochromaDn qualitaDvely different than repressors binding at gene promoters? Does chromaDn condensaDon also contribute to gene silencing Any addiDonal mechanisms?
Some open ques%ons
Simple Model for HeterochromaDn iniDaDon and Spread
H3K9Me3
H3K4Ac
Paradoxically, in some well-‐studied model systems like fission low levels of transcripDon are needed to enable heterochromaDn formaDon and funcDon…….. RNAi based mechanisms provide a second path to recruiDng methylase
“Histone Code” Hypothesis
“Histone modificaDons, on one or more tails, act sequenDally or in combinaDon to form a 'histone code' that is, read by other proteins to bring about disDnct downstream events”
Turner, B.M.. Bioessays, 2000. 22(9): p. 836-‐45. Strahl, B.D. and C.D. Allis, Nature, 2000. 403(6765): p. 41-‐5.
PHD fingers and chromo-‐ and bromodomains are present in large complexes
ATP-‐dependent chromaDn remodeling complex -‐opens up chromaDn
H3-‐K4(me3) ??
Nucleosome as a template to integrate signals
A new role for histone modificaDons: allosteric regulaDon of enzyme acDvity
EED-‐C-‐term domain
Binding of H3K27 methyl mark by EED C-‐term domain sDmulates methylase acDvity of EZH2, the H3K27methylase
Histone Variants -more Diversity and more Regulation Variants are deposited by specific histone chaperones or ATP-dependent remodeling enzymes