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Session 5.2: Gamble
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Matthew Gamble

Matthew Gamble

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Session 5.2: Gamble Presentation Transcript

  • 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
  • 13. Senescence is an innate tumor suppressive mechanism
    Hallmarks of senescence
    • “Irreversible” growth arrest
    • 14. Senescence-associated βgalactosidase activity
    • 15. Senescence-associated heterochromatic foci
    • 16. Senescence-associated secretory phenotype
    Yan and Wajapeyee 2010 Cancer BiolTher
    Roberts and Der et al. 2007 Oncogene 26:3291
  • 17.
  • 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