MW 11:00-12:15 in Redwood G19 Profs: Serafim Batzoglou, Gill Bejerano TA: Cory McLean

1. MW  11:00-12:15 in Redwood G19 Profs: Serafim Batzoglou, Gill Bejerano TA: Cory McLean http://cs273a.stanford.edu [Bejerano Aut07/08]

2. Lecture 12 • Vertebrate Gene Cis-Regulation contd. http://cs273a.stanford.edu [Bejerano Aut07/08]

3. 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 Aut07/08]

4. Vertebrate Transcription Regulation http://cs273a.stanford.edu [Bejerano Aut07/08]

5. Unicellular vs. Multicellular unicellular multicellular http://cs273a.stanford.edu [Bejerano Aut07/08]

6. Pol II Transcription • Key components: • Proteins • DNA sequence • DNA epigenetics • Protein components: • General Transcription factors • Activators • Co-activators http://cs273a.stanford.edu [Bejerano Aut07/08]

7. Activators & Co-Activators Protein - Protein Protein - DNA http://cs273a.stanford.edu [Bejerano Aut07/08]

8. TFs in the Human Genome Not a lot… http://cs273a.stanford.edu [Bejerano Aut07/08]

9. Signal Transduction http://cs273a.stanford.edu [Bejerano Aut07/08]

10. The Core Promoter http://cs273a.stanford.edu [Bejerano Aut07/08]

11. CpG islands http://cs273a.stanford.edu [Bejerano Aut07/08]

12. 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: • Gene Expression Domains • Gene Regulatory Networks (GRN) http://cs273a.stanford.edu [Bejerano Aut07/08]

13. Chromatin Remodeling “off” “on” http://cs273a.stanford.edu [Bejerano Aut07/08]

14. Tx Factors Binding Sites http://cs273a.stanford.edu [Bejerano Aut07/08]

15. Distal Transcription Regulatory Elements http://cs273a.stanford.edu [Bejerano Aut07/08]

16. Enhancers http://cs273a.stanford.edu [Bejerano Aut07/08]

17. Enhancers: action over very large distances RNAP II Basal factors promoter Enhancer with bound protein http://cs273a.stanford.edu [Bejerano Aut07/08]

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 Aut07/08]

19. Enhancer verification Matched staining in dorsal apical ectodermal ridge (part of limb bud) Matched staining in genital eminence http://cs273a.stanford.edu [Bejerano Aut07/08]

20. Fly Enhancer Combinatorics http://cs273a.stanford.edu [Bejerano Aut07/08]

21. Vertebrate Enhancer Combinatorics http://cs273a.stanford.edu [Bejerano Aut07/08]

22. 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 Aut07/08]

23. Repressors / Silencers http://cs273a.stanford.edu [Bejerano Aut07/08]

24. 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 Aut07/08]

25. Insulators http://cs273a.stanford.edu [Bejerano Aut07/08]

26. Gene Expression Domains: Independent http://cs273a.stanford.edu [Bejerano Aut07/08]

27. Gene Expression Domains: Dependent http://cs273a.stanford.edu [Bejerano Aut07/08]

28. Correlation with Human Disease [Wang et al, 2000] http://cs273a.stanford.edu [Bejerano Aut07/08]

29. Other Positional Effects [de Kok et al, 1996] http://cs273a.stanford.edu [Bejerano Aut07/08]

30. Chromatin Structure http://cs273a.stanford.edu [Bejerano Aut07/08]

31. Histone Code http://cs273a.stanford.edu [Bejerano Aut07/08]

32. Epigenetics [Goldberg et al, 2007] http://cs273a.stanford.edu [Bejerano Aut07/08]

33. More Functional Assays In vitro / in vivo Fragment / BAC Gain / Loss BAC cut and paste http://cs273a.stanford.edu [Bejerano Aut07/08]

34. Protein & Chromatin Assays • Protein binding assays: • Electrophoretic mobility shift assays (EMSA) / Gel Shift • DNAseI protection • SELEX & CASTing • Chromatin immuno-precipitation (ChIP), ChIP-chip • and various chromatin assays. http://cs273a.stanford.edu [Bejerano Aut07/08]

35. Gene Regulatory Networks [Davidson & Erwin, 2006] http://cs273a.stanford.edu [Bejerano Aut07/08]

36. The Hox Paradox [Wray, 2003] http://cs273a.stanford.edu [Bejerano Aut07/08]

37. The Great Vertebrate-Invertebrate Divide http://cs273a.stanford.edu [Bejerano Aut07/08]

38. Gene Regulatory Network (GRN) Components • Davidson & Erwin (2006): 4 classes of GRN components: • ‘‘kernels’’ evolutionarily inflexible subcircuits that perform essential upstream functions in building given body parts. • ‘‘plug-ins’’ certain small subcircuits that have been repeatedly co-opted to diverse developmental purposes(regulatory, inc. signal transduction systems) • “I/O switches” that allow or disallow developmental subcircuits to function in a given context (e.g., control of size of homologous body parts, many hox genes) • differentiation gene batteries (execute cell-type specific function, end-players) http://cs273a.stanford.edu [Bejerano Aut07/08]

39. GRN Kernel properties • Network subcircuits that consist of regulatory genes (i.e., TFs). • They execute the developmental patterning functions required to specify the embryo spatial domain/s in which body part/s will form. • Kernels are dedicated to given developmental functions and are not used elsewhere in development of the organism (though individual genes of the kernel are likely used in many different contexts). • They have a particular form of structure in that the products of multiple regulatory genes of the kernel are required for function of each of the participating cis-regulatory modules of the kernel. • Interference with expression of any one kernel gene will destroy kernel function altogether and is likely to produce the catastrophic phenotype of lack of the body part. • The result is extraordinary conservation of kernel architecture. http://cs273a.stanford.edu [Bejerano Aut07/08]

40. Kernel example [Davidson & Erwin, 2006] http://cs273a.stanford.edu [Bejerano Aut07/08]

41. Kernels and Phyla t now http://cs273a.stanford.edu [Bejerano Aut07/08]