5. Heterochromatin allows for silencing of large domains of the genome and utilizes a combination of mechanisms to do so.

Transcription

1 Biochemistry 201 Biological Regulatory Mechanisms Lecturer: Geeta Narlikar February 1, 2016 Chromatin Structure and Its Regulation 2 Key Points 1. 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. 2. 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 bromodomain containing transcription factors. 3. 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. 4. 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. 5. Heterochromatin allows for silencing of large domains of the genome and utilizes 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. 8. There seem to be both regular and more liquid like ways to fold chromatin into compact states. 1

2 References Reviews on Histone Modifications *1. Turner, B.M., Histone acetylation and an epigenetic code. Bioessays, (9): p *2. Strahl, B.D. and C.D. Allis, The language of covalent histone modifications. Nature, (6765): p Peterson, C.L. and M.A. Laniel, Histones and histone modifications. Curr Biol, (14): p. R Taverna, S.D., et al., How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol, (11): p *5. Ruthenburg A.J., Li H., Patel D.J., Allis C.D., Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol, :p Histone Acetylation 1. Grant, P.A., et al., Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev, (13): p Wu, P.Y., et al., Molecular architecture of the S. cerevisiae SAGA complex. Mol Cell, (2): p *3. Brownell, J.E., et al., Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell, (6): p Daniel, J.A., et al., Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription. J Biol Chem, (3): p Jacobson, R.H., et al., Structure and function of a human TAFII250 double bromodomain module. Science, (5470): p

3 6. Owen, D.J., et al., The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p. Embo J, (22): p Shogren-Knaak, M., et al., Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science, (5762): p ATP-dependent Chromatin Remodeling Enzymes 1. Clapier CR and Cairns BR.The biology of chromatin remodeling complexes. Annu. Rev. Biochem : Cairns, B.R., Chromatin remodeling: insights and intrigue from single-molecule studies. Nat Struct Mol Biol, : Racki L.R. and Narlikar GJ. ATP-dependent chromatin remodeling enzymes: two heads are not better, just different. Curr Opin Genet Dev : Peterson, C.L. and I. Herskowitz, Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription. Cell, (3): p Cote, J., et al., Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science, (5168): p Lorch, Y., M. Zhang, and R.D. Kornberg, Histone octamer transfer by a chromatin-remodeling complex. Cell, (3): p Narlikar, G.J., M.L. Phelan, and R.E. Kingston, Generation and interconversion of multiple distinct nucleosomal states as a mechanism for catalyzing chromatin fluidity. Mol Cell, (6): p Bruno, M., et al., Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities. Mol Cell, (6): p Zhang Y. et al., DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC. Mol Cell :

4 11. Lia G., et al., Direct observation of DNA distortion by the RSC complex. Mol Cell, : Langst, G., et al., Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone octamer. Cell, (7): p *14. Yang, J.G., et al., The chromatin-remodeling enzyme ACF is an ATP-dependent DNA length sensor that regulates nucleosome spacing. Nat Struct Mol Biol, (12): p Racki LR et al., The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes. Nature : Blosser et al., Dynamics of nucleosome remodelling by individual ACF complexes. Nature : Fan HY, Trotter KW, Archer TK, Kingston RE. Swapping function of two chromatin remodeling complexes. Mol Cell : *Mizuguchi, G., et al., ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science, (5656): p Interplay between SWI/SNF and HATs 1. Hassan, A.H., et al., Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes. Cell, (3): p Hassan, A.H., K.E. Neely, and J.L. Workman, Histone acetyltransferase complexes stabilize swi/snf binding to promoter nucleosomes. Cell, (6): p *3. Agalioti, T., G. Chen, and D. Thanos, Deciphering the transcriptional histone acetylation code for a human gene. Cell, (3): p

5 Chromodomains and PHD fingers 1. Fischle, W., et al., Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev, (15): p Jacobs, S.A. and S. Khorasanizadeh, Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science, (5562): p Kirmizis, A., et al., Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation. Nature, (7164): p *4. Li, H., et al., Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF. Nature, (7098): p *5. Lan, F., et al., Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression. Nature, (7154): p *6. Pena, P.V., et al., Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2. Nature, (7098): p Shi, X., et al., ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature, (7098): p *8. Li B, Gogol M, Carey M, Lee D, Seidel C, Workman JL. Combined action of PHD and chromo domains directs the Rpd3S HDAC to transcribed chromatin. Science. 2007, 316: Heterochromatin formation and regulation 1. Verschure PJ et al., In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol Cell Biol. 2005; 25(11):

6 2. Hines KA et al., Domains of heterochromatin protein 1 required for Drosophila melanogaster heterochromatin spreading.genetics. 2009;182(4): Wallrath LL, Elgin SC. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 1995; 9(10): Reyes-Turcu FE, Grewal SI., Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast. Curr Opin Genet Dev Jan 11. Higher Order Chromatin folding 1. Maeshima K et al. Liquid-like behavior of chromatin. Curr Opin Genet Dev Jan 27;37: Grigoryev SA, et al. Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes.proc Natl Acad Sci Jan 19. [Epub ahead of print] 3. Bassett A, et al. The folding and unfolding of eukaryotic chromatin. Curr Opin Genet Dev : Schalch, T., et al., X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature, (7047): p

7 Outline Func%ons of Chroma%n Intrinsic proper%es Packing material Regula%on of intrinsic proper%es Complex regulatory pla9orm coordina%on/coupling Replica%on Transcrip%on RNA processing 1

8 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 H2A- H2B acidic patch Linker histones (Histone H1) promote chroma%n folding 2

9 Histones contain many different post- transla%onal modifica%ons: concentrated on N- terminal tails but also found on internal regions H2A H2B H3 H4 PhosphorylaAon, AcetylaAon MethylaAon UbiquitylaAon Implica%on: more func%ons than just packing DNA 3

10 Two Case Studies Histone H3 and H4 Acetyla%on Histone H3 K9 Methyla%on EuchromaAn (acave genes) HeterochromaAn (repressed genes) Drosophila salivary glands polytene chromosomes stained to detect the DNA Lighter stains = euchromaan Darker stains = heterochromaan 4

11 The role(s) of lysine acetylaaon in histone tails * acetyl lysine 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! 5

12 Lysine acetylaaon by GCN5 CoA S O C CH 3 H + + Lys N H H GCN5 O CoA SH + Lys N H C CH 3 + H + Results in loss of one posiave charge 6

13 GCN5 is part of SAGA complex Interact with TBP from EM structure Ubp8 Removes monoubiquian from H2B (locaaon in complex not known) Interacts with acavators Other funcaons of SAGA: Several genes are SAGA dependent but GCN5 independent 7

14 AcetylaAon is reversible Histone De- acetylases (HDACS or KDACs) remove acetyl groups most o\en correlate with gene repression Histone acetyl transferases (HATs or KATs) add acetyl groups most o\en correlate with gene acavaaon AcetylaAon state is very dynamic turnover within minutes How does histone acetylaaon enhance transcripaon? 8

15 How does histone acetylaaon enhance transcripaon? Lys H + N H H vs. Lys N H O C CH 3 (1) Does acetylaaon reduce histone- DNA interacaons? closed open K eq = [open]/[closed] HyperacetylaAon of all the histone tails increases K eq by ~2 fold 9

16 (2) AcetylaAon has larger effects (greater than 10- fold) on disrupang inter- nucleosomal contacts and on chromaan compacaon Ac Ac N- GLGKGGAKRHRKV H4 H2A- H2B acidic patch Single acetylaaon mark on H4 lysine 16 has similar impact on chromaan compacaon as deleang H4 tail 10

17 (3) Acetylated lysine provides a recogniaon moaf for an effector protein Lys N H O C CH 3 Bromodomain Bromodomains specifically recognize acetylated lysines 11

18 GCN5 bromodomain with H4 tail acetylated at K16 HAT bromo 12

19 Budding yeast has 15 bromodomains 1 bromodomain 8 bromodomains 9 of them are distributed between two ATP- dependent chroma%n remodeling complexes 13

20 SWI/SNF and RSC can enable many different outcomes transfer histone octamers move histone octamers Generate DNA loops exchange and remove histone dimers Many open mechanis%c ques%ons 14

21 So far four major families of ATP- dependent chromaan remodeling complexes High degree of conserva%on from yeast to humans Classified based on their ATPase subunits, which are shown below gene acavaaon and some gene repression: mulaple biochemical outcomes some acavaaon, mostly gene repression and heterochromaan formaaon: only movesnucleosomes gene acavaaon and repression: only move nucleosomes gene acavaaon, DNA replicaaon, DNA damage responses move nucleosomes and exchange variant histones Not clear (i) if the different families work by similar or disanct mechanisms and (ii) how their different biochemical outputs relate to their different biological roles 15

22 A cascade of events at the promoter of the human interferon- b gene Sequence Specific binding Human TFIID contains TAFII250,which is a HAT and also contains a double bromodomain 16

23 Several methyla%on marks with different readouts Q S (me) 2 (me) 3 (me) 3 (me) 3 OFF ON OFF OFF mutually exclusive mutually exclusive Methyl marks are bound by Chromodomains and PHD fingers Methylases put on the mark and De- methylases remove the marks 17

24 Lysine Arginine H + N H H unmodified H N N C H + NH 2 O N H C CH 3 acetylated CH N + 3 CH 3 CH 3 methylated CH 3 N N C CH 3 + NH 2 18

25 Chromodomain and PHD fingers have independently evolved to use caaon- π InteracAons to stabilize methylated lysines Hydrophobic cage gives mark specificity Crystal structure of chromodomain from Drosophila HP1 protein Q (me) 3 OFF A S A (me) 3 OFF mutually exclusive InteracAons with residues surrounding the ARKS moaf give posiaon specificity 19

26 Position Effect Variegation reveals ability of heterochromatin to spread white normal posaon heterochromaan chromosome swapping white gene near heterochromaan Drosophila fruit fly HeterochromaAn can spread over more than 1000 kb of previously euchromaac chromaan and heritably silences genes Effects linked to the HP1 protein and methylaaon of Histone H3 at K9 HP1 binds methyl mark using a chromodomain 20

27 1) How does heterochromaan spread? 2) How is silencing achieved? Is the silencing achieved by heterochromaan qualitaavely different than repressors binding at gene promoters? Does chromaan condensaaon also contribute to gene silencing Any addiaonal mechanisms? Some open ques%ons 21

28 Simple Model for HeterochromaAn iniaaaon and Spread H3K9Me3 H3K4Ac Paradoxically, in some well- studied model systems like fission low levels of transcripaon are needed to enable heterochromaan formaaon and funcaon.. RNAi based mechanisms provide a second path to recruiang methylase 22

29 Histone Code Hypothesis Histone modificaaons, on one or more tails, act sequenaally or in combinaaon to form a 'histone code' that is, read by other proteins to bring about disanct downstream events Turner, B.M.. Bioessays, (9): p Strahl, B.D. and C.D. Allis, Nature, (6765): p

30 PHD fingers and chromo- and bromodomains are present in large complexes H3- K4(me3)?? ATP- dependent chromaan remodeling complex - opens up chromaan Nucleosome as a template to integrate signals 24

31 Histone Variants -more Diversity and more Regulation Variants are deposited by specific histone chaperones or ATP-dependent remodeling enzymes 25

32 What do we know about higher- order chroma%n folding? 30 nm fibers -seen in some terminally differentiated cells -seen with reconstituted chromatin -not seen in proliferating cells -not seen in mitotic chromosomes 26

33 Some newer models for packing of Interphase chroma%n TAD: topologically associated domain Current Opinion in Genetics & Development, Volume 37, 2016,

34 Methods to look for long- range DNA interac%ons 28

35 Chromosomes seem to occupy distinct territories in three-dimensional (3D) space. Job Dekker, and Tom Misteli Cold Spring Harb Perspect Biol 2015;7:a by Cold Spring Harbor Laboratory Press