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Session 5.1: Secombe

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Julie Secombe

Julie Secombe

Published in: Health & Medicine, Technology

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  • 1. Regulation of Myc-induced cell growth by the histonedemethylase Lid
    AECC
    May 5th, 2010
  • 2. Myc genes are deregulated in human cancers
    Myc gene deregulated
    Cancer type
    c-myc
    Bladder, breast, colon, gastric, melanoma, myeloma, ovary, prostate, lung
    N-myc
    Neuroblastoma, breast, lung
    L-myc
    Breast, lung
  • 3. Myc target genes
    - Myc target genes are involved in proliferation, vasculogenesis, cell adhesion
    RNA POL I
    ribosomal RNA
    Myc
    RNA POL II
    Growth
    metabolism, translation,
    ribosomal proteins
    RNA POL III
    tRNAs
  • 4. Myc expression induces cell growth
    Control
    dMyc (GMM)
    Gal4
    GMR
    Gal4
    dMyc
    Gal4
    UAS
    dMyc
    dMyc
    dMyc
    Expression of dMyc in post-mitotic cells during eye development
    GMR-Gal4, UAS-dMyc
  • 5. Identifying regulators of dMyc-induced growth
    Co-activators
    Negative regulators
    dMyc
    dMyc targets
    Cell growth
    Large, rough eye
  • 6. Identifying regulators of dMyc-induced growth
    Co-activators
    Negative regulators
    dMyc
    dMyc targets
    Cell growth
    Large, rough eye
    Less rough (suppressed)
  • 7. Identifying regulators of dMyc-induced growth
    Co-activators
    Negative regulators
    dMyc
    dMyc targets
    Cell growth + apoptosis
    Large, rough eye
    More rough (enhanced)
  • 8. GMM UAS-lid
    GMM lid +/-
    WT
    GMM
    -dMyc
    -Histone H3
    Little ImaginalDiscs (Lid) is limiting for dMyc-induced cell growth
    GMM, lid/+
    Control
    GMM
  • 9. GMM UAS-lid
    GMM lid +/-
    WT
    GMM
    -dMyc
    -Histone H3
    Little Imaginal Discs (Lid) is limiting for dMyc-induced cell growth
    GMM, lid/+
    GMM, UAS-Lid
    Control
    GMM
  • 10. Lid and its orthologs
    JmjC
    PHD
    PHD
    PHD
    ARID
    JmjN
    C5HC2
    Lid
    A. gambie Lid
    C. elegans rbr-2
    H. sapiens KDM5a
    H. sapiens KDM5b
    H. sapiens KDM5c
    H. sapiens KDM5d
    D. rerio Lid
    • KDM5a, b and care regulators of transcription
    • 11. KDM5b is overexpressed in breast, bladder and prostate cancer
    • 12. The C-terminal finger of KDM5a forms an oncogenic fusion with Nup98
  • Myc
    What is the mechanism of Lid function?
    • Lid has domains that implicate it as a regulator of transcription
    • 13. Lid co-IPs with dMyc
    • 14. Lid is required for dMyc to activate Nop60B
    ?
    Lid
    Nop60B
    E-box
  • 15. JmjC is a lysine demethylase domain
    Nature, 2006
    - JHDM1A demethylates histone H3 lysine 36
    - Enzymatic activity requires the JmjC domain, Fe2+ and a-ketoglutarate
  • 16. M
    M
    M
    M
    M
    M
    CH3
    CH3
    CH3
    H3C
    CH3
    CH3
    trimethyl
    dimethyl
    monomethyl
    unmodified
    N+
    NH+
    NH2+
    NH3+
    JmjC-dependent demethylase activities
    H3
    ARTKYTARKSTGGKAPRKQLATKAARKSAPATGGVK..K..K
    H4
    SGRGKGGKGLGKGGAKRHRKVLR
  • 17. What is the mechanism of Lid function?
    JmjC
    PHD
    PHD
    PHD
    ARID
    JmjN
    C5HC2
    Is Lid a histone demethylase?
    Is this activity required for dMyc-induced cell growth?
  • 18. Lid demethylates trimethyl H3 lysine 4
    JmjC
    PHD
    PHD
    PHD
    ARID
    JmjN
    C5HC2
    NLS
    hs-FLP ; UAS-Lid
    actin>CD2>Gal4, UAS-GFP
    GFP
    a-Lid
    a-H3K4me3
    GFP
    Larval Fat Body Cells
  • 19. Histone H3 lysine 4 methylation correlates with transcriptional activation
    Correlation with transcription
    +
    K4me3
    +
    K4me2
    +/-
    K4me1
    Adapted from Li and Workman, 2007
    - Lid’s demethylase activity implicates it as a transcriptional repressor
  • 20. * *
    dMyc Lid-JmjC*
    Lid’s JmjC-encoded demethylase activity is not required for dMyc function
    JmjC
    PHD
    PHD
    PHD
    ARID
    JmjN
    C5HC2
    NLS
    dMyc Lid
    dMyc
    - Expression of either wildtype or JmjC* Lid alone has no effect on eye development
  • 21. Myc
    What is the mechanism of Lid function?
    ?
    Lid
    Nop60B
    E-box
  • 22. Enhanced?
    dMyc Lid
    dMyc
    dMyc Lid-JmjC*
    dMyc Lid-D
    Identifying domains of Lid required for Myc-induced cell growth?
    Lid domain
    UAS
  • 23. Domains of Lid required for dMyc function
    Enhance dMyc phenotype?
    JmjN
    JmjC
    PHD
    PHD
    PHD
    ARID
    C5HC2
    NLS
    * *
    X
    (
    )
    (
    )
    (
    )
    X
    (
    )
    (
    )
    nd
    X
  • 24. TFIID
    TAF3
    H3K4me3
    Transcriptional initiation
    PHD fingers read the histone code
    Nurf
    BPTF
    Pygo
    BHC80
    H3K4me0
    H3K4me2
    H3K4me2/3
    Wnt-induced transcriptional activation
    Tethers the LSD1 repressor to chromatin
    Nucleosome remodeling
  • 25. Lid’s PHD3 binds to H3K4me2/3
    JmjN
    ARID
    PHD
    PHD
    PHD
    JmjC
    C5HC2
    ECRAENCHKPTGREVDWVPCDGGCNEWFHMYCVGLNRSQIKPDDDYICIRCT
    H3 21-40
    H3K4
    H3K9
    H3K27
    5% input
    H3 1-21
    Beads
    H4
    me1
    me2
    me3
    me1
    me2
    me3
    me1
    me2
    me3
    49
    PHD3
    38
  • 26. c-Myc binding correlates with high levels of di and trimethylated H3K4
    - Histone H3 lysine 4 methylation precedes and is independent of Myc binding.
  • 27. Working model for Lid-dMyc function
    Me
    Me
    Me
    Me
    Myc
    ?
    Activation
    ?
    Lid
    Me
    Me
    K4
    K4
    E-box
    Nop60B
    • E boxes (CACGTG) occur on average every 4kb
    • 28. One of the functions on Lid might be to ‘recruit’ Myc to sites that are
    enriched for H3K4me2/3
  • 29. Thank you
    Einstein
    Christina Greer
    FHCRC
    Ling Li
    Bob Eisenman
    Salk institute
    Satchin Panda
    Hiep Le
  • 30. Lid
    1838 AA
    JmjC
    PHD
    PHD
    PHD
    ARID
    JmjN
    C5HC2
    NLS
    • Lid has domains that implicate it as a regulator of transcription
    • 31. Lid co-IPs with dMyc
    • 32. Lid is required for dMyc to activate Nop60B
  • What is the mechanism of Lid function?
    JmjC
    PHD
    PHD
    PHD
    ARID
    JmjN
    C5HC2
    Is Lid a histonedemethylase? YES. H3K4me3 specific
    Is this activity required for dMyc-induced cell growth? NO