Julie Secombe

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

11. KDM5b is overexpressed in breast, bladder and prostate cancer

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.

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

31. Lid co-IPs with dMyc