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Biochemistry 201 Biological Regulatory Mechanisms Lecturer: Geeta Narlikar February 2, 2015 Chromatin Structure and Its Regulation-1 Key Points 1. Packaging of eukaryotic DNA into nucleosomes and higher order folded states provides a means to functionally compartmentalize genes. The DNA in nucleosomes is tightly bound but very dynamic, unpeeling and rebinding on the order of milliseconds. 2. The accessibility of DNA within a nucleosome is very sensitive to its location and can be reduced relative to free DNA from 10-fold to 500,000 fold. 3. The intrinsic preferences of DNA for histone octamers can play a role in positioning nucleosomes in vivo but there is more regulation of nucleosome positions than just sequence 4. There are at least two ways in which arrays of chromatin can fold into the 30 nm fiber. This process is likely regulated by other proteins so that the chromatin fiber may adopt additional packing conformations in vivo. References BOOKS: Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. Molecular Biology of the Cell (5th ed.) Garland Publishing Inc., New York, Chapter 4. ARTICLES:

Nucleosome Structure and Dynamics 1. Luger, K., et al., Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature, 1997. 389(6648): p. 251-60. 2. Li, G., et al., Rapid spontaneous accessibility of nucleosomal DNA. Nat Struct Mol Biol, 2005. 12(1): p. 46-53. 3. Polach, K.J. and J. Widom, Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene

regulation. J Mol Biol, 1995. 254(2): p. 130-49.

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In vivo mapping of nucleosome positions and occupancy 1. Kaplan N et al. The DNA-encoded nucleosome organization of a eukaryotic genome. Nature. 2009 458:362-6. 2. Zhang Y et al. Intrinsic histone-DNA interactions are not the major determinant of nucleosome positions in vivo. Nat Struct Mol Biol.

2009;16:847-52. 3. Albert I et al. Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome. Nature. 2007

446:572-6. 4. Zhang Z, Pugh BF. High-resolution genome-wide mapping of the primary structure of chromatin. Cell. 2011; 144:175-86. 5. Kaplan N et al. Nucleosome sequence preferences influence in vivo nucleosome organization.Nat Struct Mol Biol. 2010; 17:918-20 6. Zhang et al. Evidence against a genomic code for nucleosome positioning, Nat Struct Mol Biol. 2010;17: 920-22 7. Pugh BF. A preoccupied position on nucleosomes. Nat Struct Mol Biol. 2010; 17:923 8. Sasaki S et al. Chromatin-associated periodicity in genetic variation downstream of transcriptional start sites.Science. 2009 323: 401-4. 9. Floer M et al. A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding. Cell. 2010;141:407-18. 10. Zhang Z,et al. A packing mechanism for nucleosome organization reconstituted across a eukaryotic genome. Science. 2011;332:977-80. 11. Vera D L et al. Differential Nuclease Sensitivity Profiling of Chromatin Reveals Biochemical Footprints Coupled to Gene Expression and Functional DNA Elements in Maize. Plant Cell 2014;26:3883-3893 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.

Regulating the DNA Template

Organized  nature  of  eukaryo1c  DNA    observed  more  than  a  100  years  ago  

1882:  Salivary  gland  cell  from    Bloodworm(?)  larvae    observed  by  Walther  Flemming  

Lighter  stains  =  euchroma?n    Darker  stains  =  heterochroma?n    

A  few  decades  ago:  Drosophila  salivary  glands  polytene  chromosomes  stained  with  similar  dyes  

1882:  Salivary  gland  cell  from    Bloodworm(?)  larvae    observed  by  Walther  Flemming  

Lighter  stains  =  euchroma?n    Darker  stains  =  heterochroma?n    

‘puffs”  associated  with  transcrip?on  suggested  major  changes  in    chroma?n  organiza?on  

Outline

Func1ons  of  Chroma1n  

Packing  material   Complex  regulatory  plaCorm  

Replica1on   Transcrip1on   RNA  processing  

coordina1on/coupling  

Outline

Func1ons  of  Chroma1n  

Packing  material   Complex  regulatory  plaCorm  

Replica1on   Transcrip1on   RNA  processing  

coordina1on/coupling  

Intrinsic  proper1es     Regula1on  of  intrinsic  proper1es  

ON  

OFF  

On a simple level packaging into chromatin helps compartmentalize genes !

A  closer  look  at  the  nucleosome  

•  4  histones:  2  copies  of  each  form  an  octamer  that  wraps  147  bp  of  DNA.      •  N-­‐terminal  tails  of  histones  extend  out  (~  1/4th  the  histone  mass).      •  DNA  is  highly  bent.  Histone-­‐DNA  interac?ons  stabilize  this  unfavorable  conforma?on.    •  DNA  becomes  less  accessible  to  DNA  binding  proteins        •  Almost  all  the  histone-­‐DNA  contacts  are  sequence  independent.  Most  interac?ons  are  between  the    -­‐vely  charged  phosphate  backbone  of  DNA  and  +vely  charged  histone  residues.    Yet  some  DNA  sequences  can  bind  histone  octamers  100-­‐fold    more  strongly  than  others.  Why?

DNA  sequence  can  affect  energe?cs  of  DNA  bending  

minor groove compressed

major groove compressed

GC  base-­‐pairs  more  easily  allow    major  groove  compression

5  bp  

5  bp  

Proper  placement  of  AT  and  GC  base-­‐pairs  can  give  DNA  sequences  with  high  histone  octamer  binding  affini?es  rela?ve  to  bulk  genomic  DNA      This  pa_ern  is  seen  in  naturally  occurring  “nucleosome  posi?oning  sequences”    

AT  base-­‐pairs  more  easily  allow    minor  groove  compression  

~  4  s-­‐1  

~  90  s-­‐1  

Nucleosomal  DNA  is  intrinsically  dynamic  

closed   open  

Above  numbers  are  for  a  site  that  is  ~10  bp  in  from  one  end  

Keq=  1/20  =  [open]/[closed]  

closed   open  Keq=  1/20    

+   +  

Kd=  10  nM    

Overall  Kd  for  nucleosomal  site  =  10/(1/20)  =  200  nM  

Keq=  1/20    

Keq=  1/500,000    

The  specific  placement  of  a  histone  octamer  on  the  genome  can  maUer  for    regula1ng  the  access  of  DNA  to  a  transcrip1on  factor  

1.  DNA  accessibility  is  very  sensi?ve  to  its  loca?on  within  a  nucleosome    

minor  groove    binding  protein  

Be_er  binding  worse  binding  

2.  Rota?onal  placement  of  DNA  sequence  on  nucleosome  can  affect  factor  affinity    

Coopera?ve  binding  through  protein-­‐protein  interac?ons  

1.     A   B   A  and  B  directly  interact  

The  architecture  of  the  nucleosome  can  give  coopera?ve  binding  behavior    for  non-­‐interac?ng  proteins    

+   +  

1.    

2.    

A   B  

+  A  B  

A  and  B  directly  interact  

A  and  B  do  not  directly  interact  

Debate  in  the  field:  does  the  genome  encode  its  own  packaging?  

Placement  of  AT  and  GC  base-­‐pairs  every  10  bp  can  increase    histone  octamer  binding  affini?es  rela?ve  to  bulk  genomic  DNA      The  specific  transla?onal  and  rota?onal  posi?on  of  nucleosomes  can  have  a  large  impact  on  accessibility  of  regulatory  sequences  

Do  intrinsic  proper?es  of  DNA  sequences  play  a  significant  role  in  posi?oning  nucleosomes  in  vivo?  

In  vitro      

purified  yeast  DNA  +  purified  histones  

assemble  into  nucleosomes  

map  loca?ons  by  MNase  and  deep  sequencing  or  micro-­‐array  

In  vivo    

compare  to  in  vivo  posi?ons  

Comparison  of  in  vivo  and  in  vitro  maps  in  yeast  suggested    in  vivo  role  for  DNA  sequence  preferences    

Average  occupancy    at  each  base-­‐pair    

number  of  reads  that    cover  the  bp  

average  number  of  reads    per  bp  across  genome  

=  

Kaplan  et  al.  Nature.  2009  458:362-­‐6.      

But,  it  can’t  be  that  simple…  

 

1.  How  can  one  nucleosome  map  explain  mul?ple  transcrip?onal  states?  If  nucleosome  placement  affects  transcrip?on  factor  binding  and  transcrip?on  factor  binding  regulates  expression  of  specific  genes,  then  when  gene  expression  pa_erns  change  upon  changes  in  environment,  differen?a?on  etc.,  the  nucleosome  posi?ons  and  occupancy  should  change  as  well.  

 

2.  What  happens  to  nucleosome  posi?ons  and  occupancy  in  the  coding  region  when  gene  expression  levels  change?        

 

The  devil  is  in  the  details  and  the  differences  may  reveal  more  than  the  similari1es  

1.  Correla?on  between  in  vivo  and  in  vitro  maps  is  greater  in  non-­‐promoter  intergenic  regions  (R=0.83)  and  lower  in  promoter  and  coding  regions  (0.69)    2.  At  coding  regions  deple?on  levels  of  nucleosomes  in  vivo  rela?ve  to  in  vitro  increases  with  the    expression  levels  of  the  associated  genes    3.  Both  maps  show  nucleosome  deple?on  at  transcrip?on  start  sites,  but  level  of  deple?on  in  vivo  is  greater    4.  Level  of  nucleosome  deple?on  around  transcrip?on  factor  binding  sites  generally  correlates  between    the  two  maps  but  there  are  interes?ng  differences.  

Nucleosome  occupancy  near  different  transcrip1on  factor  binding  sites  

Nucleosome  Occupancy  vs.  Posi1oning    

Pugh  BF.    Nat  Struct  Mol  Biol.  2010;  17:923    

(Example:  #  reads)  

MNase Hyper-sensitive and Hyper-resistant Footprints

Vera D L et al. Plant Cell 2014;26:3883-3893

©2014 by American Society of Plant Biologists

MNase  based  nucleosome  mapping  data  from  Maize  

Agarose  gel  run  aqer    deproteinizing  MNase  digested  chroma?n  

Vera D L et al. Plant Cell 2014;26:3883-3893

©2014 by American Society of Plant Biologists

MNase  based  nucleosome  mapping  data  from  Maize  

MNase-Hypersensitive Footprints Correlate with regulatory elements like transcription factor binding sites  

Higher  order  compac1on  of  chroma1n  

One  start   Two  start  

30  nm  Electron  micrographs  

Regula?on  of  higher  order  chroma?n  folding  

1.  Short  inter-­‐nucleosomal  spacing  (<20  bp)  may  favor  two-­‐start  structure  while  longer  spacing  may  favor  one  start  structure  

EM  micrographs  

Is  this  process  regulated  by  non-­‐histone  proteins  in  vivo?  Perhaps  chroma?n  fiber  adopts    addi?onal  packing  conforma?ons  in  vivo.  

 

H2A-­‐H2B  acidic  patch    

viral  LANA  pep?de  bound    to  acidic  patch  

     

H4  N-­‐terminal  tail  bound    to  acidic  patch  

 Histone  tails  mediate  inter-­‐nucleosomal  contacts  through  electrosta1c  interac1ons  

Highly  basic  histone  tails  interact  with  DNA  of  neighboring  nucleosomes  

Histone  H4  tail  interacts  with  an  acidic  patch  formed  by  H2A-­‐H2B  

Kaposi's  sarcoma–associated  herpesvirus  latency-­‐associated  nuclear  an?gen  (LANA)  mediates  viral  genome    a_achment  to  mito?c  chromosomes  by  binding  nucleosomes  through    H2A-­‐H2B  acidic  patch  

Linker  histones  (Histone  H1)  promote  chroma1n  folding