Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Session 5.1 Secombe

328 views

Published on

Julie Secombe

Published in: Health & Medicine, Technology
  • Be the first to comment

  • Be the first to like this

Session 5.1 Secombe

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

×