6. Heterochromatin allows for silencing of large domains of the genome and appears to utilize a combination of mechanisms to do so.

Transcription

1 Biochemistry 201 Biological Regulatory Mechanisms Lecturer: Geeta Narlikar February 18, 2014 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 bromodomain 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. 1

2 References Higher Order Chromatin folding *1. Tremethick DJ. Higher-order structures of chromatin: the elusive 30 nm fiber. Cell : *2. Bassett A, et al. The folding and unfolding of eukaryotic chromatin. Curr Opin Genet Dev : Dorigo B, et al. Nucleosome arrays reveal the two-start organization of the chromatin fiber. Science : Schalch, T., et al., X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature, (7047): p Routh A, Sandin S and Rhodes D, Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure. Proc Natl Acad Sci : Horn, P.J., et al., The SIN domain of the histone octamer is essential for intramolecular folding of nucleosomal arrays. Nat Struct Biol, (3): p 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

3 *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 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: 3

4 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 : 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 :

5 17. 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 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

6 *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): Hines KA et al., Domains of heterochromatin protein 1 required for Drosophila melanogaster heterochromatin spreading.genetics. 2009;182(4): Grewal SI, Elgin SC.Transcription and RNA interference in the formation of heterochromatin.nature. 2007;447(7143): Wallrath LL, Elgin SC. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 1995; 9(10): Margueron R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature. 2009;461(7265): 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. 6

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 Higher order compac%on of chroma%n Electron micrographs 30 nm One start Two start 2

9 Regula:on of higher order chroma:n folding Short inter- nucleosomal spacing (<20 bp) may favor two- start structure while longer spacing may favor one start structure EM micrographs In vivo folding may be regulated by other proteins. Perhaps the chroma:n fiber adopts addi:onal packing conforma:ons in vivo?

10 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 4

11 Histones contain many different post- transla%onal modifica%ons: concentrated on N- terminal tails but also found on internal regions H2A H2B H3 H4 Phosphoryla:on, Acetyla:on Methyla:on Ubiquityla:on Implica%on: more func%ons than just packing DNA 5

12 Two Case Studies Histone H3 and H4 Acetyla%on Histone H3 K9 Methyla%on Euchroma:n (ac:ve genes) Heterochroma:n (repressed genes) Drosophila salivary glands polytene chromosomes stained to detect the DNA Lighter stains = euchroma:n Darker stains = heterochroma:n 6

13 The role(s) of lysine acetyla:on 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! 7

14 Lysine acetyla:on 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 posi:ve charge 8

15 GCN5 is part of SAGA complex Interact with TBP from EM structure Ubp8 Removes monoubiqui:n from H2B (loca:on in complex not known) Interacts with ac:vators Other func:ons of SAGA: Several genes are SAGA dependent but GCN5 independent 9

16 Acetyla:on is reversible Histone De- acetylases (HDACS or KDACs) remove acetyl groups most oben correlate with gene repression Histone acetyl transferases (HATs or KATs) add acetyl groups most oben correlate with gene ac:va:on Acetyla:on state is very dynamic turnover within minutes How does histone acetyla:on enhance transcrip:on? 10

17 How does histone acetyla:on enhance transcrip:on? Lys H + N H H vs. Lys N H O C CH 3 (1) Does acetyla:on reduce histone- DNA interac:ons? closed open K eq = [open]/[closed] Hyperacetyla:on of all the histone tails increases K eq by ~2 fold 11

18 (2) Acetyla:on has larger effects (greater than 10- fold) on disrup:ng inter- nucleosomal contacts and on chroma:n compac:on Ac Ac N- GLGKGGAKRHRKV H4 H2A- H2B acidic patch Single acetyla:on mark on H4 lysine 16 has similar impact on chroma:n compac:on as dele:ng H4 tail 12

19 (3) Acetylated lysine provides a recogni:on mo:f for an effector protein Lys N H O C CH 3 Bromodomain Bromodomains specifically recognize acetylated lysines 13

20 GCN5 bromodomain with H4 tail acetylated at K16 HAT bromo 14

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

22 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 16

23 So far four major families of ATP- dependent chroma:n remodeling complexes High degree of conserva%on from yeast to humans Classified based on their ATPase subunits, which are shown below gene ac:va:on and some gene repression: mul:ple biochemical outcomes some ac:va:on, mostly gene repression and heterochroma:n forma:on: only movesnucleosomes gene ac:va:on and repression: only move nucleosomes gene ac:va:on, DNA replica:on, DNA damage responses move nucleosomes and exchange variant histones Not clear (i) if the different families work by similar or dis:nct mechanisms and (ii) how their different biochemical outputs relate to their different biological roles 17

24 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 18

25 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 19

26 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 20

27 Chromodomain and PHD fingers have independently evolved to use ca:on- π Interac:ons 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 Interac:ons with residues surrounding the ARKS mo:f give posi:on specificity 21

28 Position Effect Variegation reveals ability of heterochromatin to spread white normal pos:on heterochroma:n chromosome swapping white gene near heterochroma:n Drosophila fruit fly Heterochroma:n can spread over more than 1000 kb of previously euchroma:c chroma:n and heritably silences genes Effects linked to the HP1 protein and methyla:on of Histone H3 at K9 HP1 binds methyl mark using a chromodomain 22

29 1) How does heterochroma:n spread? 2) How is silencing achieved? Is the silencing achieved by heterochroma:n qualita:vely different than repressors binding at gene promoters? Does chroma:n condensa:on also contribute to gene silencing Any addi:onal mechanisms? Some open ques%ons 23

30 Simple Model for Heterochroma:n ini:a:on and Spread H3K9Me3 H3K4Ac Paradoxically, in some well- studied model systems like fission low levels of transcrip:on are needed to enable heterochroma:n forma:on and func:on.. RNAi based mechanisms provide a second path to recrui:ng methylase 24

31 Histone Code Hypothesis Histone modifica:ons, on one or more tails, act sequen:ally or in combina:on to form a 'histone code' that is, read by other proteins to bring about dis:nct downstream events Turner, B.M.. Bioessays, (9): p Strahl, B.D. and C.D. Allis, Nature, (6765): p

32 PHD fingers and chromo- and bromodomains are present in large complexes H3- K4(me3)?? ATP- dependent chroma:n remodeling complex - opens up chroma:n Nucleosome as a template to integrate signals 26

33 A new role for histone modifica:ons: allosteric regula:on of enzyme ac:vity EED- C- term domain Binding of H3K27 methyl mark by EED C- term domain s:mulates methylase ac:vity of EZH2, the H3K27methylase 27

34 Histone Variants -more Diversity and more Regulation Variants are deposited by specific histone chaperones or ATP-dependent remodeling enzymes 28