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Epigenomics

Epigenomics. Rob Mitra 4 /25/11. Outline . Epigenetic phenomena What is epigenetics ? Overview of the types of epigenetic marks Technologies for Surveying the marks What are we learning?. Ting Wang. Agouti slide.

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Epigenomics

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  1. Epigenomics Rob Mitra 4/25/11

  2. Outline • Epigenetic phenomena • What is epigenetics? • Overview of the types of epigenetic marks • Technologies for Surveying the marks • What are we learning?

  3. Ting Wang

  4. Agouti slide • IAP retrotransposon in the Agouti gene (encodes a signaling molecule typically expressed only in hair follicles that induces a yellow color) • Yellow mice – ectopic expression from a promoter in the Retrotransposon • CpG methylation silences this promoter variably

  5. Variable expression 45% of your genome contains retrotransposon Folate and other methyl-donors affect variagation

  6. Outline • Epigenetic phenomena • What is epigenetics? • Overview of the types of epigenetic marks • Technologies for Surveying the marks • What are we learning?

  7. Ting Wang

  8. Epigenetic Marks • DNA methylation • Histone Modification

  9. Ting Wang

  10. DNA methylation SAM = S-adenosylmethionine

  11. Ting Wang

  12. Ting Wang

  13. Ting Wang

  14. Ting Wang

  15. How is methylation established? Ting Wang

  16. How is methylation maintained? Ting Wang

  17. There is still much to learn Ting Wang

  18. Histone Modifications Ting Wang

  19. Histone Modifications

  20. Outline • Epigenetic phenomena • What is epigenetics? • Overview of the types of epigenetic marks • Technologies for Surveying the marks • What are we learning?

  21. How does one measure histone marks? Ting Wang

  22. How does one measure methylation?

  23. HPAII CCGG Ting Wang

  24. Bisulfite sequencing Ting Wang

  25. MSP1 = CCGG Ting Wang

  26. Ting Wang

  27. Ting Wang

  28. Which method should I use?

  29. MethylMap: methylation analysis of microdissected tissue

  30. Current efforts 1-2 mm x 1-2 mm area from FFPE sections (~1000-4500 cells) Ciliary Body Uveal Melanoma

  31. What are we learning? • How is the basal methylation pattern established? • Promoter associated CpG islands are unmethylated, all other CpGsmethylated

  32. Background: H3K4 me3 blocks de novo methylation

  33. Histone Modifications

  34. How are methylation patterns in differentiated cells established? • Many patterns of methylation do not vary between cell type: basal pattern • CpGs are methylated, CpG islands, the regulatory regions upstream of promoters are not.

  35. How does this occur? • Wave of de novo methylation at implantation stage • 1) Blocked by SP1 = GGCGG • 2) RNA pol II -> H3K4 Me which blocks DMNT3L binding. (or combination? But SP1 blocking does not require transcription)

  36. What is known about locus specific methylation (pluripotency genes)? • Oct 3 / 4 -- pluripotency gene, one of the Yamanaka 4. Need to turn off during differentiation. • Repressed by TF • TF recruites the Histone Methyl Transferase G9a complex which contains HDACs. • Remove acetyl groups from lysines and G9a methylates H3K9 • H3K9me recruits HP1 which forms heterochromatin • G9a recruits DNMT 3a and 3b for methylation and stable repression

  37. What is known about gene specific methylation?

  38. What is known about locus specific methylation (satellite repeats)? • Transciption form dsRNA • Dicer chops up • RISC complex takes the pieces and targets to repeats • Recruits SUV39H1 and H2 which are SET containing Histonemethyltransferases • Trimethylate H3K9 and recruit HP1. Also SUV39H1 and H2 recruit DNMT3a and 3b.

  39. Abberantmethylation in Cancer Ting Wang

  40. Ting Wang

  41. Ting Wang

  42. Ting Wang

  43. DNA repair protein O6-methylguanine-methyl- transferase Ting Wang

  44. Acknowledgement • Ting Wang

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