Matthew Gamble

1. The Role of macroH2A Alternative Splicing in Regulating Chromatin Structure and Function during Cancer and Senescence Matthew Gamble AECC Advances Meeting May 5th 2010

2. What is macroH2A? Histone 1 131 Canonical H2A N- -C 65% identity Macro Linker 1 122 368 160 MacroH2A1 -C N- Basic α-satellite DNA (1U35) α-satellite DNA (1AOI) M. musculusH3, H4, H2B (1U35) X. laevisH3, H4, H2B (1AOI) H. sapiensmacroH2A (1U35) X. laevisH2A (1AOI) Chakravarthy (2005) Mol Cell Biol 25:7616 Costanzi, Pehrson1998 Nature 393:599

3. What Factors Lead to the Deposition of macroH2A? ? What Effects does macroH2A have on Chromatin Structure and Gene Expression?

4. MacroH2A1 occupies a large proportion of autosomal chromatin A B C

5. MacroH2A1 binding defines 3 classes of genes IMR90 21567 TSSs +3 kb –7 kb 1.00 0.67 Promoter contains mH2A boundary 0.33 Log2 ratio (mH2A/input) 0.00 - 0.33 - 0.67 - 1.00 Expression regulated by macroH2A1 10% repressed by macroH2A1 33% stimulated by macroH2A1 Enriched for genes involved in Cell-cell signaling Development

6. What is macroH2A? Histone 1 131 Canonical H2A N- -C 65% identity Macro Linker 1 122 368 160 MacroH2A1 -C N- Basic α-satellite DNA (1U35) α-satellite DNA (1AOI) M. musculusH3, H4, H2B (1U35) X. laevisH3, H4, H2B (1AOI) H. sapiensmacroH2A (1U35) X. laevisH2A (1AOI) Chakravarthy (2005) Mol Cell Biol 25:7616

7. mH2A1 macro macro PARP mH2A2 H2A macro N– –C 368 H2A macro N– –C 371 PARP9 N– 857 –C PARP14 –C 1801 801– macro macro PARP macro macro macro WWE PARP PARP15 N– –C 678 macro MACROD1 N– –C 325 macro MACROD2 N– –C 425 SNF2 macro CHD1L N– –C 897 C6orf130 N– –C 152 macro GDAP2 N– –C 497 macro SEC14 Vertebrate Macro Domain-Containing Proteins

8. Macro Domains Bind to NAD+-derived Second Messengers NAD+ PARP-1 NAm PAR SIRT1 PARG ADPR OAADPR Macro domains Altered gene expression? Altered factor recruitment? Altered histone modification? Altered chromatin structure?

9. MacroH2A: A histone variant with several variants H2AFY PAR,ADPR and OAADPRBinding 1 131 Canonical H2A N- -C Macro Histone Linker 1 122 368 160 MacroH2A1.1 -C N- Basic Alt. Splice 1 122 371 160 MacroH2A1.2 -C N- Basic 1 122 371 160 MacroH2A2 -C N- Basic Kustatscher et al. 2005 Nat Struct Mol Biol 12:624

10. Identification of mH2A1.1 macro domain interacting factors 122 368 Macro 160 1 N- MacroH2A1.1 Histone -C Basic GST-macro1.1 GST -C N- Basic GST GST-macro1.1 + + - Nuclear extract MDN1 TCOF1 -205 PELP1 -116 PARP-1 -97 Nucleolin -66 -45 SET nucleophosmin -29

11. PARP-1 interacts specifically with macroH2A1.1 nucleosomes input mH2A1.1 mH2A1.2 GFP Mononucleosomes ( EtBr ) MacroH2A1

12. Differential splicing of macroH2A1 in primary and cancer cells 140 IMR90 120 100 1.4 Relative expression 80 1.2 60 40 1 20 MacroH2A1.1/macroH2A1.2 ratio 0.8 0 mH2A1.1 mH2A1.2 mH2A2 0.6 0.4 600 0.2 MCF - 7 500 0 400 Transcripts per cell 300 200 100 0 mH2A1.1 mH2A1.2 mH2A2

14. Senescence-associated βgalactosidase activity

15. Senescence-associated heterochromatic foci

16. Senescence-associated secretory phenotypeYan and Wajapeyee 2010 Cancer BiolTher Roberts and Der et al. 2007 Oncogene 26:3291

18. Summary / Working Model * RAS/RAF Cancer Normal MEK PARP-1 ERK PARP-1 PELP1 PELP1 1.2 1.2 1.1 1.2 Proliferation Growth Suppression Senescence

19. Is macroH2A1.1 status an additional predictor of PARP inhibitor efficacy? DNA Damage ERK PARP-1 PARP-1 PELP1 Nucleotide Excision Repair Homologous Repair 1.1 1.2 BRCA1 BRCA2 Growth Suppression Senescence Cancer Cell Survival

20. Acknowledgements Gamble Lab Einstein Hongshan Chen Susan Horwitz (Mol Pharm) Hailey McDaid Laura Klein Charlie Rubin (Mol Pharm) Art Skoultchi (Cell Bio) John Greally (Genetics) Robert Gallagher (Medicine) AmitVerma (Medicine and DMB) Ulrich Steidl (Cell Bio) Joe Sparano (Medicine, Oncology) Leonid Novikov W. Lee Kraus (Cornell, Ithaca) Kristine Frizzell Raga Krishnakumar Christine Yang Eugene Park Tong Zhang Sidney Kimmel Foundation for Cancer Research