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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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References Higher Order Chromatin folding *1. Tremethick DJ. Higher-order structures of chromatin: the elusive 30 nm fiber.

Cell. 2007. 128:651-4.

*2. Bassett A, et al. The folding and unfolding of eukaryotic chromatin. Curr Opin Genet Dev. 2009 19:159-65. 3. Dorigo B, et al. Nucleosome arrays reveal the two-start organization of the chromatin fiber.

Science. 2004. 306:1571-3. 4. Schalch, T., et al., X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature, 2005. 436(7047):

p. 138-41. 5. Routh A, Sandin S and Rhodes D, Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure. Proc Natl Acad Sci. 2008 105:8872-7. 6. Horn, P.J., et al., The SIN domain of the histone octamer is essential for intramolecular folding of nucleosomal arrays. Nat

Struct Biol, 2002. 9(3): p. 167-71. Reviews on Histone Modifications *1. Turner, B.M., Histone acetylation and an epigenetic code. Bioessays, 2000. 22(9): p. 836-45. *2. Strahl, B.D. and C.D. Allis, The language of covalent histone modifications. Nature, 2000. 403(6765): p. 41-5. 3. Peterson, C.L. and M.A. Laniel, Histones and histone modifications. Curr Biol, 2004. 14(14): p. R546-51. 4. Taverna, S.D., et al., How chromatin-binding modules interpret histone modifications: lessons from professional pocket

pickers. Nat Struct Mol Biol, 2007. 14(11): p. 1025-40.

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*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, 2007 8:p. 983-94

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, 1997. 11(13): p. 1640-50. 2. Wu, P.Y., et al., Molecular architecture of the S. cerevisiae SAGA complex. Mol Cell, 2004. 15(2): p. 199-208. *3. Brownell, J.E., et al., Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene

activation. Cell, 1996. 84(6): p. 843-51. 4. Daniel, J.A., et al., Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription. J Biol Chem,

2004. 279(3): p. 1867-71. 5. Jacobson, R.H., et al., Structure and function of a human TAFII250 double bromodomain module. Science, 2000. 288(5470):

p. 1422-5. 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, 2000. 19(22): p. 6141-9. 7. Shogren-Knaak, M., et al., Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science, 2006.

311(5762): p. 844-7. ATP-dependent Chromatin Remodeling Enzymes 1. Clapier CR and Cairns BR.The biology of chromatin remodeling complexes. Annu. Rev. Biochem. 2009. 78:273-304 2. Cairns, B.R., Chromatin remodeling: insights and intrigue from single-molecule

studies. Nat Struct Mol Biol, 2007. 14: 989-996.

3. Racki L.R. and Narlikar GJ. ATP-dependent chromatin remodeling enzymes:

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two heads are not better, just different. Curr Opin Genet Dev. 2008. 18:137-44. 4. Peterson, C.L. and I. Herskowitz, Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global

activator of transcription. Cell, 1992. 68(3): p. 573-83. 5. Cote, J., et al., Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science, 1994.

265(5168): p. 53-60. 6. Lorch, Y., M. Zhang, and R.D. Kornberg, Histone octamer transfer by a chromatin-remodeling complex. Cell, 1999. 96(3): p.

389-92. 7. 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, 2001. 8(6): p. 1219-30. 8. Bruno, M., et al., Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities. Mol Cell, 2003. 12(6):

p. 1599-606. 10. Zhang Y. et al., DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC. Mol Cell.

2006. 24:559-68 11. Lia G., et al., Direct observation of DNA distortion by the RSC complex. Mol Cell, 2006. 21: 417-25 12. Langst, G., et al., Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone

octamer. Cell, 1999. 97(7): p. 843-52. *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, 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.

Nature. 2009. 462:1022-7.

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17. Fan HY, Trotter KW, Archer TK, Kingston RE. Swapping function of two chromatin remodeling complexes. Mol Cell. 2005. 17:805-15. 18. *Mizuguchi, G., et al., ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex.

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

nucleosomes. Cell, 2002. 111(3): p. 369-79. 2. Hassan, A.H., K.E. Neely, and J.L. Workman, Histone acetyltransferase complexes stabilize swi/snf binding to promoter

nucleosomes. Cell, 2001. 104(6): p. 817-27. *3. Agalioti, T., G. Chen, and D. Thanos, Deciphering the transcriptional histone acetylation code for a human gene. Cell, 2002.

111(3): p. 381-92. 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, 2003. 17(15): p. 1870-81. 2. Jacobs, S.A. and S. Khorasanizadeh, Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail.

Science, 2002. 295(5562): p. 2080-3. 3. Kirmizis, A., et al., Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation. Nature, 2007. 449(7164):

p. 928-32. *4. Li, H., et al., Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF. Nature, 2006.

442(7098): p. 91-5.

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*5. Lan, F., et al., Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression. Nature,

2007. 448(7154): p. 718-22. *6. Pena, P.V., et al., Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2. Nature, 2006.

442(7098): p. 100-3. 7. Shi, X., et al., ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature, 2006. 442(7098): p.

96-9. *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, 316:1050-4. 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):4552-64.

2. Hines KA et al., Domains of heterochromatin protein 1 required for Drosophila melanogaster heterochromatin spreading.Genetics. 2009;182(4):967-77.

3. Grewal SI, Elgin SC.Transcription and RNA interference in the formation of heterochromatin.Nature. 2007;447(7143):399-406.

4. Wallrath LL, Elgin SC. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 1995; 9(10):1263-77.

5. Margueron R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature. 2009;461(7265):762-7.

6. 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. 2012 Jan 11.

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