Regulation of Myc-induced cell growth by the histonedemethylase Lid<br />AECC<br />May 5th, 2010<br />
Myc genes are deregulated in human cancers<br />Myc gene deregulated<br />Cancer type<br />c-myc<br />Bladder, breast, col...
Myc target genes<br />- Myc target genes are involved in proliferation, vasculogenesis, cell adhesion <br />RNA POL I<br /...
Myc expression induces cell growth<br />Control<br />dMyc (GMM)<br />Gal4<br />GMR<br />Gal4<br />dMyc<br />Gal4<br />UAS<...
Identifying regulators of dMyc-induced growth<br />Co-activators<br />Negative regulators<br />dMyc<br />dMyc targets<br /...
Identifying regulators of dMyc-induced growth<br />Co-activators<br />Negative regulators<br />dMyc<br />dMyc targets<br /...
Identifying regulators of dMyc-induced growth<br />Co-activators<br />Negative regulators<br />dMyc<br />dMyc targets<br /...
GMM UAS-lid<br />GMM lid +/-<br />WT<br />GMM<br />-dMyc<br />-Histone H3<br />Little ImaginalDiscs (Lid) is limiting fo...
GMM UAS-lid<br />GMM lid +/-<br />WT<br />GMM<br />-dMyc<br />-Histone H3<br />Little Imaginal Discs (Lid) is limiting f...
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 /...
 KDM5b is overexpressed in breast, bladder and prostate cancer
 The C-terminal finger of KDM5a forms an oncogenic fusion with Nup98</li></li></ul><li>Myc<br />What is the mechanism of L...
 Lid co-IPs with dMyc
 Lid is required for dMyc to activate Nop60B  </li></ul>?<br />Lid<br />Nop60B<br />E-box<br />
JmjC is a lysine demethylase domain<br />Nature, 2006<br />- JHDM1A demethylates histone H3 lysine 36<br />- Enzymatic act...
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...
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 h...
Lid demethylates trimethyl H3 lysine 4<br />JmjC<br />PHD<br />PHD<br />PHD<br />ARID<br />JmjN<br />C5HC2<br />NLS<br />h...
Histone H3 lysine 4 methylation correlates with transcriptional activation<br />Correlation with transcription<br />+<br /...
* *<br />dMyc    Lid-JmjC*<br />Lid’s JmjC-encoded demethylase activity is not required for dMyc function<br />JmjC<br />P...
Myc<br />What is the mechanism of Lid function?<br />?<br />Lid<br />Nop60B<br />E-box<br />
Enhanced?<br />dMyc    Lid<br />dMyc<br />dMyc    Lid-JmjC*<br />dMyc    Lid-D<br />Identifying domains of Lid required fo...
Domains of Lid required for dMyc function<br />Enhance dMyc phenotype?<br />JmjN<br />JmjC<br />PHD<br />PHD<br />PHD<br /...
TFIID<br />TAF3<br />H3K4me3<br />Transcriptional initiation<br />PHD fingers read the histone code<br />Nurf<br />BPTF<br...
Lid’s PHD3 binds to H3K4me2/3<br />JmjN<br />ARID<br />PHD<br />PHD<br />PHD<br />JmjC<br />C5HC2<br />ECRAENCHKPTGREVDWVP...
c-Myc binding correlates with high levels of di and trimethylated H3K4<br />- Histone H3 lysine 4 methylation precedes and...
Working model for Lid-dMyc function<br />Me<br />Me<br />Me<br />Me<br />Myc<br />?<br />Activation<br />?<br />Lid<br />M...
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Session 5.1: Secombe

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Julie Secombe

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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 />

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