The Role of macroH2A Alternative Splicing in Regulating Chromatin<br />Structure and Function during Cancer and Senescence...
What is macroH2A?<br />Histone<br />1<br />131<br />Canonical H2A<br />N-<br />-C<br />65% identity<br />Macro<br />Linker...
What Factors Lead to the Deposition of macroH2A?<br />?<br />What Effects does macroH2A have on <br />Chromatin Structure ...
MacroH2A1 occupies a large proportion of autosomal chromatin<br />A<br />B<br />C<br />
MacroH2A1 binding defines 3 classes of genes<br />IMR90<br />21567 TSSs<br />+3 kb<br />–7 kb<br />1.00<br />0.67<br />Pro...
What is macroH2A?<br />Histone<br />1<br />131<br />Canonical H2A<br />N-<br />-C<br />65% identity<br />Macro<br />Linker...
mH2A1<br />macro<br />macro<br />PARP<br />mH2A2<br />H2A<br />macro<br />N–<br />–C<br />368<br />H2A<br />macro<br />N–<...
Macro Domains Bind to NAD+-derived Second Messengers<br />NAD+<br />PARP-1<br />NAm<br />PAR<br />SIRT1<br />PARG<br />ADP...
MacroH2A: A histone variant with several variants<br />H2AFY<br />PAR,ADPR and <br />OAADPRBinding<br />1<br />131<br />Ca...
Identification of mH2A1.1 macro domain interacting factors<br />122<br />368<br />Macro<br />160<br />1<br />N-<br />Macro...
PARP-1 interacts specifically with macroH2A1.1 nucleosomes<br />input<br />mH2A1.1<br />mH2A1.2<br />GFP<br />Mononucleoso...
Differential splicing of macroH2A1 in primary and cancer cells<br />140<br />IMR90<br />120<br />100<br />1.4<br />Relativ...
Senescence is an innate tumor suppressive mechanism<br />Hallmarks of senescence<br /><ul><li> “Irreversible” growth arrest
Senescence-associated βgalactosidase activity
Senescence-associated heterochromatic foci
Senescence-associated secretory phenotype</li></ul>Yan and Wajapeyee 2010 Cancer BiolTher<br />Roberts and Der et al. 2007...
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Session 5.2: Gamble

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Matthew Gamble

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Session 5.2: Gamble

  1. 1. The Role of macroH2A Alternative Splicing in Regulating Chromatin<br />Structure and Function during Cancer and Senescence<br />Matthew Gamble<br />AECC Advances Meeting<br />May 5th 2010<br />
  2. 2. What is macroH2A?<br />Histone<br />1<br />131<br />Canonical H2A<br />N-<br />-C<br />65% identity<br />Macro<br />Linker<br />1<br />122<br />368<br />160<br />MacroH2A1<br />-C<br />N-<br />Basic<br />α-satellite DNA (1U35) <br />α-satellite DNA (1AOI)<br />M. musculusH3, H4, H2B (1U35)<br />X. laevisH3, H4, H2B (1AOI)<br />H. sapiensmacroH2A (1U35)<br />X. laevisH2A (1AOI)<br />Chakravarthy (2005) Mol Cell Biol 25:7616<br />Costanzi, Pehrson1998 Nature 393:599<br />
  3. 3. What Factors Lead to the Deposition of macroH2A?<br />?<br />What Effects does macroH2A have on <br />Chromatin Structure and Gene Expression?<br />
  4. 4. MacroH2A1 occupies a large proportion of autosomal chromatin<br />A<br />B<br />C<br />
  5. 5. MacroH2A1 binding defines 3 classes of genes<br />IMR90<br />21567 TSSs<br />+3 kb<br />–7 kb<br />1.00<br />0.67<br />Promoter contains mH2A boundary<br />0.33<br />Log2 ratio (mH2A/input)<br />0.00<br />-<br />0.33<br />-<br />0.67<br />-<br />1.00<br />Expression regulated by macroH2A1<br />10% repressed by macroH2A1<br />33% stimulated by macroH2A1<br />Enriched for genes involved in<br />Cell-cell signaling<br />Development<br />
  6. 6. What is macroH2A?<br />Histone<br />1<br />131<br />Canonical H2A<br />N-<br />-C<br />65% identity<br />Macro<br />Linker<br />1<br />122<br />368<br />160<br />MacroH2A1<br />-C<br />N-<br />Basic<br />α-satellite DNA (1U35) <br />α-satellite DNA (1AOI)<br />M. musculusH3, H4, H2B (1U35)<br />X. laevisH3, H4, H2B (1AOI)<br />H. sapiensmacroH2A (1U35)<br />X. laevisH2A (1AOI)<br />Chakravarthy (2005) Mol Cell Biol 25:7616<br />
  7. 7. mH2A1<br />macro<br />macro<br />PARP<br />mH2A2<br />H2A<br />macro<br />N–<br />–C<br />368<br />H2A<br />macro<br />N–<br />–C<br />371<br />PARP9<br />N–<br />857<br />–C<br />PARP14<br />–C<br />1801<br />801–<br />macro<br />macro<br />PARP<br />macro<br />macro<br />macro<br />WWE<br />PARP<br />PARP15<br />N–<br />–C<br />678<br />macro<br />MACROD1<br />N–<br />–C<br />325<br />macro<br />MACROD2<br />N–<br />–C<br />425<br />SNF2<br />macro<br />CHD1L<br />N–<br />–C<br />897<br />C6orf130<br />N–<br />–C<br />152<br />macro<br />GDAP2<br />N–<br />–C<br />497<br />macro<br />SEC14<br />Vertebrate Macro Domain-Containing Proteins<br />
  8. 8. Macro Domains Bind to NAD+-derived Second Messengers<br />NAD+<br />PARP-1<br />NAm<br />PAR<br />SIRT1<br />PARG<br />ADPR<br />OAADPR<br />Macro domains<br />Altered gene expression?<br />Altered factor recruitment?<br />Altered histone modification?<br />Altered chromatin structure?<br />
  9. 9. MacroH2A: A histone variant with several variants<br />H2AFY<br />PAR,ADPR and <br />OAADPRBinding<br />1<br />131<br />Canonical H2A<br />N-<br />-C<br />Macro<br />Histone<br />Linker<br />1<br />122<br />368<br />160<br />MacroH2A1.1<br />-C<br />N-<br />Basic<br />Alt. Splice<br />1<br />122<br />371<br />160<br />MacroH2A1.2<br />-C<br />N-<br />Basic<br />1<br />122<br />371<br />160<br />MacroH2A2<br />-C<br />N-<br />Basic<br />Kustatscher et al. 2005 Nat Struct Mol Biol 12:624 <br />
  10. 10. Identification of mH2A1.1 macro domain interacting factors<br />122<br />368<br />Macro<br />160<br />1<br />N-<br />MacroH2A1.1<br />Histone<br />-C<br />Basic<br />GST-macro1.1<br />GST<br />-C<br />N-<br />Basic<br />GST<br />GST-macro1.1<br />+<br />+<br />-<br />Nuclear extract<br />MDN1<br />TCOF1<br />-205<br />PELP1<br />-116<br />PARP-1<br />-97<br />Nucleolin<br />-66<br />-45<br />SET<br />nucleophosmin<br />-29<br />
  11. 11. PARP-1 interacts specifically with macroH2A1.1 nucleosomes<br />input<br />mH2A1.1<br />mH2A1.2<br />GFP<br />Mononucleosomes<br />(<br />EtBr<br />)<br />MacroH2A1<br />
  12. 12. Differential splicing of macroH2A1 in primary and cancer cells<br />140<br />IMR90<br />120<br />100<br />1.4<br />Relative expression<br />80<br />1.2<br />60<br />40<br />1<br />20<br />MacroH2A1.1/macroH2A1.2 ratio<br />0.8<br />0<br />mH2A1.1<br />mH2A1.2<br />mH2A2<br />0.6<br />0.4<br />600<br />0.2<br />MCF<br />-<br />7<br />500<br />0<br />400<br />Transcripts per cell<br />300<br />200<br />100<br />0<br />mH2A1.1<br />mH2A1.2<br />mH2A2<br />
  13. 13. Senescence is an innate tumor suppressive mechanism<br />Hallmarks of senescence<br /><ul><li> “Irreversible” growth arrest
  14. 14. Senescence-associated βgalactosidase activity
  15. 15. Senescence-associated heterochromatic foci
  16. 16. Senescence-associated secretory phenotype</li></ul>Yan and Wajapeyee 2010 Cancer BiolTher<br />Roberts and Der et al. 2007 Oncogene 26:3291<br />
  17. 17.
  18. 18. Summary / Working Model<br />*<br />RAS/RAF<br />Cancer<br />Normal<br />MEK<br />PARP-1<br />ERK<br />PARP-1<br />PELP1<br />PELP1<br />1.2<br />1.2<br />1.1<br />1.2<br />Proliferation<br />Growth Suppression<br />Senescence<br />
  19. 19. Is macroH2A1.1 status an additional predictor of PARP inhibitor efficacy?<br />DNA Damage<br />ERK<br />PARP-1<br />PARP-1<br />PELP1<br />Nucleotide<br />Excision<br />Repair<br />Homologous<br />Repair<br />1.1<br />1.2<br />BRCA1<br />BRCA2<br />Growth Suppression<br />Senescence<br />Cancer Cell Survival<br />
  20. 20. Acknowledgements<br />Gamble Lab<br />Einstein<br />Hongshan Chen<br />Susan Horwitz (Mol Pharm)<br /> Hailey McDaid<br /> Laura Klein<br />Charlie Rubin (Mol Pharm)<br />Art Skoultchi (Cell Bio)<br />John Greally (Genetics)<br />Robert Gallagher (Medicine)<br />AmitVerma (Medicine and DMB)<br />Ulrich Steidl (Cell Bio)<br />Joe Sparano (Medicine, Oncology)<br />Leonid Novikov<br />W. Lee Kraus (Cornell, Ithaca)<br />Kristine Frizzell<br />Raga Krishnakumar<br />Christine Yang<br />Eugene Park<br />Tong Zhang<br />Sidney Kimmel Foundation for Cancer Research<br />

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