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1. Thank you for the midterm feedback! http://cs273a.stanford.edu [Bejerano Fall09/10]

2. Lecture 12 Cis-Regulation Cellular Signaling http://cs273a.stanford.edu [Bejerano Fall09/10]

3. Big Picture Context We are surveying the functional classes of elements encoded by the human genome. We have previously discussed: • Protein coding genes • Repetitive sequences • Non coding RNAs Today we’ll cover the last (known :) large class of functional elements. http://cs273a.stanford.edu [Bejerano Fall09/10]

4. Unicellular vs. Multicellular unicellular multicellular http://cs273a.stanford.edu [Bejerano Fall09/10]

5. Vertebrate Transcription Regulation http://cs273a.stanford.edu [Bejerano Fall09/10]

6. Pol II Transcription • Key components: • Proteins • DNA sequence • DNA epigenetics • Protein components: • General Transcription factors • Activators • Co-activators http://cs273a.stanford.edu [Bejerano Fall09/10]

7. Activators & Co-Activators Protein - Protein Protein - DNA http://cs273a.stanford.edu [Bejerano Fall09/10]

8. The Core Promoter http://cs273a.stanford.edu [Bejerano Fall09/10]

9. Chromatin Remodeling “off” “on” http://cs273a.stanford.edu [Bejerano Fall09/10]

10. CpG islands http://cs273a.stanford.edu [Bejerano Fall09/10]

11. Nucleosome tail modifications • Lysine acetylations. • Histone Acetyl-Transferases (HAT) & Histone Deacetylases (HDAC). • Lysine and Argenine Metylations. • Modified by histone-metyl-transferase. • Phosphorilation. • Ubiquitination. • H2A ubiquitination affects 10-15% of this histone in most eukaryotic cells • ADP-ribosylation.

12. http://cs273a.stanford.edu [Bejerano Fall09/10]

13. Transcription Factor (TF) Binding Sites http://cs273a.stanford.edu [Bejerano Fall09/10]

14. TFs in the Human Genome http://cs273a.stanford.edu [Bejerano Fall09/10]

15. Combinatorial Regulatory Code 2,000 different proteins can bind specific DNA sequences. Proteins DNA Protein binding site Gene DNA A regulatory region encodes 3-10 such protein binding sites. When all are bound by proteins the regulatory region turns “on”,and the nearby gene is activated to produce protein. http://cs273a.stanford.edu [Bejerano Fall09/10]

16. Enhancers http://cs273a.stanford.edu [Bejerano Fall09/10]

17. Enhancers: action over very large distances RNAP II Basal factors promoter Enhancer with bound protein http://cs273a.stanford.edu [Bejerano Fall09/10]

18. Transient Transgenic Enhancer Assay in situ Conserved Element Minimal Promoter Reporter Gene Construct is injected into 1 cell embryos Taken out at embryonic day 10.5-14.5 Assayed for reporter gene activity transgenic http://cs273a.stanford.edu [Bejerano Fall09/10]

19. Vertebrate Enhancer Combinatorics brain limb neural tube Sall1 http://cs273a.stanford.edu [Bejerano Fall09/10]

20. Vertebrate Enhancer Combinatorics http://cs273a.stanford.edu [Bejerano Fall09/10]

21. What are Enhancers? • What do enhancers encode? • Surely a cluster of TF binding sites. • [but TFBS prediction is hard, fraught with false positives] • What else? DNA Structure related properties? • So how do we recognize enhancers? • Sequence conservation across multiple species • [weak but generic] http://cs273a.stanford.edu [Bejerano Fall09/10]

22. Gene Expression Domains: Independent http://cs273a.stanford.edu [Bejerano Fall09/10]

23. Vertebrate Gene Regulation • gene (how to) • control region(when & where) distal: in 106 letters DNA DNA binding proteins proximal: in 103 letters http://cs273a.stanford.edu [Bejerano Fall09/10]

24. Most Non-Coding Elements are likely cis-regulatory “IRX1 is a member of the Iroquois homeobox gene family. Members of this family appear to play multiple roles during pattern formation of vertebrate embryos.” gene deserts regulatory jungles 9Mb http://cs273a.stanford.edu [Bejerano Fall09/10]

25. Many non-coding elements tested are cis-regulatory http://cs273a.stanford.edu [Bejerano Fall09/10]

26. Gene Expression Domains: Dependent http://cs273a.stanford.edu [Bejerano Fall09/10]

27. Distal Transcription Regulatory Elements http://cs273a.stanford.edu [Bejerano Fall09/10]

28. Repressors / Silencers http://cs273a.stanford.edu [Bejerano Fall09/10]

29. What are Enhancers? Repressors • What do enhancers encode? • Surely a cluster of TF binding sites. • [but TFBS prediction is hard, fraught with false positives] • What else? DNA Structure related properties? • So how do we recognize enhancers? • Sequence conservation across multiple species • [weak but generic] • Verifying repressors is trickier [loss vs. gain of function]. • How do you predict an enhancer from a repressor? Duh... repressors repressors http://cs273a.stanford.edu [Bejerano Fall09/10]

30. Insulators http://cs273a.stanford.edu [Bejerano Fall09/10]

31. Disease Implications: Genes gene genome protein Limb Malformation Over 300 genes already implicated in limb malformations. http://cs273a.stanford.edu [Bejerano Fall09/10]

32. Disease Implications: Cis-Reg gene genome NO protein made Limb Malformation HANDFUL of cases known (limb, deafness, etc). http://cs273a.stanford.edu [Bejerano Fall09/10]

33. Transcription Regulation & Human Disease [Wang et al, 2000] http://cs273a.stanford.edu [Bejerano Fall09/10]

34. Critical regulatory sequences Lettice et al. HMG 2003 12: 1725-35 Single base changes Knock out http://cs273a.stanford.edu [Bejerano Fall09/10]

35. Other Positional Effects [de Kok et al, 1996] http://cs273a.stanford.edu [Bejerano Fall09/10]

36. Genomewide Association Studies point to non-coding DNA http://cs273a.stanford.edu [Bejerano Fall09/10]

37. WGA Disease http://cs273a.stanford.edu [Bejerano Fall09/10]

38. Rapid TFBS turnover http://cs273a.stanford.edu [Bejerano Fall09/10]

39. Cis-Regulatory Components • Low level (“atoms”): • Promoter motifs (TATA box, etc) • Transcription factor binding sites (TFBS) • Mid Level: • Promoter • Enhancers • Repressors/silencers • Insulators/boundary elements • Cis-regulatory modules (CRM) • Locus control regions (LCR) • High Level: • Epigenetic domains / signatures • Gene expression domains • Gene regulatory networks (GRN) http://cs273a.stanford.edu [Bejerano Fall09/10]

40. Cis-Regulatory Evolution: E.g., obile Elements Gene Gene Gene Gene What settings make these“co-option” events happen? [Yass is a small town in New South Wales, Australia.] http://cs273a.stanford.edu [Bejerano Fall09/10]

41. Britten & Davidson Hypothesis: Repeat to Rewire! [Davidson & Erwin, 2006] [Britten & Davidson, 1971] http://cs273a.stanford.edu [Bejerano Fall09/10]

42. Modular: Most Likely to Evolve? Chimp Human http://cs273a.stanford.edu [Bejerano Fall09/10]

43. Human Accelerated Regions Human Chimp Human-specific substitutions in conserved sequences 43 [Pollard, K. et al., Nature, 2006] [Prabhakar, S. et al., Science, 2008] [Beniaminov, A. et al., RNA, 2008]

44. Signal Transduction http://cs273a.stanford.edu [Bejerano Fall09/10]

45. Cell Communication Lodish, 20-1

46. Wnt and Hedgehog signaling Jacob & Lum Science 2007

47. Signaling Pathways Important in Developmental Biology • Wnt/Frizzled through b-catenin • Hedgehog • TGF-b family through Smads • Growth factors via JAK-STATs • Notch • Integrin • TNF