1. http://cs273a.stanford.edu [Bejerano Spr06/07] 1 TTh 11:00-12:15 in Clark S361 Profs: Serafim Batzoglou, Gill Bejerano TAs: George Asimenos, Cory McLean

2. http://cs273a.stanford.edu [Bejerano Spr06/07] 2 Lecture 10 Transcription Regulation in Vertebrates contd.

3. http://cs273a.stanford.edu [Bejerano Spr06/07] 3 unicellular multicellular Unicellular vs. Multicellular

4. http://cs273a.stanford.edu [Bejerano Spr06/07] 4 Pol II Transcription Key components: Proteins DNA sequence DNA epigenetics Protein components: General Transcription factors Activators Co-activators

5. http://cs273a.stanford.edu [Bejerano Spr06/07] 5 Activators & Co-Activators Protein - DNA Protein - Protein

6. http://cs273a.stanford.edu [Bejerano Spr06/07] 6 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)

7. http://cs273a.stanford.edu [Bejerano Spr06/07] 7 Chromatin Remodeling “off” “on”

8. http://cs273a.stanford.edu [Bejerano Spr06/07] 8 Tx Factors Binding Sites

9. http://cs273a.stanford.edu [Bejerano Spr06/07] 9 Distal Transcription Regulatory Elements

10. http://cs273a.stanford.edu [Bejerano Spr06/07] 10 Enhancers

11. http://cs273a.stanford.edu [Bejerano Spr06/07] 11 Basal factors RNAP II Enhancer with bound protein promoter Enhancers: action over very large distances

12. http://cs273a.stanford.edu [Bejerano Spr06/07] 12 Transient Transgenic Enhancer Assay Reporter Gene Minimal Promoter Conserved Element Construct is injected into 1 cell embryos Taken out at embryonic day 10.5-14.5 Assayed for reporter gene activity in situ transgenic

13. http://cs273a.stanford.edu [Bejerano Spr06/07] 13 Enhancer verification Matched staining in genital eminence Matched staining in dorsal apical ectodermal ridge (part of limb bud)

14. http://cs273a.stanford.edu [Bejerano Spr06/07] 14 Fly Enhancer Combinatorics

15. http://cs273a.stanford.edu [Bejerano Spr06/07] 15 Vertebrate Enhancer Combinatorics

16. http://cs273a.stanford.edu [Bejerano Spr06/07] 16 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]

17. http://cs273a.stanford.edu [Bejerano Spr06/07] 17 Repressors / Silencers

18. http://cs273a.stanford.edu [Bejerano Spr06/07] 18 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] Verifying repressors is trickier [loss vs. gain of function]. How do you predict an enhancer from a repressor? Duh... repressors Repressors

19. http://cs273a.stanford.edu [Bejerano Spr06/07] 19 Insulators

20. http://cs273a.stanford.edu [Bejerano Spr06/07] 20 Gene Expression Domains: Independent

21. http://cs273a.stanford.edu [Bejerano Spr06/07] 21 Gene Expression Domains: Dependent

22. http://cs273a.stanford.edu [Bejerano Spr06/07] 22 Correlation with Human Disease [Wang et al, 2000]

23. http://cs273a.stanford.edu [Bejerano Spr06/07] 23 Other Positional Effects [de Kok et al, 1996]

24. http://cs273a.stanford.edu [Bejerano Spr06/07] 24 Chromatin Structure

25. http://cs273a.stanford.edu [Bejerano Spr06/07] 25 Histone Code

26. http://cs273a.stanford.edu [Bejerano Spr06/07] 26 Epigenetics [Goldberg et al, 2007]

27. http://cs273a.stanford.edu [Bejerano Spr06/07] 27 More Functional Assays In vitro / in vivo Fragment / BAC Gain / Loss BAC cut and paste

28. http://cs273a.stanford.edu [Bejerano Spr06/07] 28 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.

29. http://cs273a.stanford.edu [Bejerano Spr06/07] 29 Gene Regulatory Networks [Davidson & Erwin, 2006]

30. http://cs273a.stanford.edu [Bejerano Spr06/07] 30 The Hox Paradox [Wray, 2003]

31. http://cs273a.stanford.edu [Bejerano Spr06/07] 31 The Great Vertebrate-Invertebrate Divide

32. http://cs273a.stanford.edu [Bejerano Spr06/07] 32 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)

33. http://cs273a.stanford.edu [Bejerano Spr06/07] 33 GRN Kernel properties 1.Network subcircuits that consist of regulatory genes (i.e., TFs). 2.They execute the developmental patterning functions required to specify the embryo spatial domain/s in which body part/s will form. 3.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). 4.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. 5.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.

34. http://cs273a.stanford.edu [Bejerano Spr06/07] 34 Kernel example [Davidson & Erwin, 2006]

35. http://cs273a.stanford.edu [Bejerano Spr06/07] 35 Kernels and Phyla t now

36. http://cs273a.stanford.edu [Bejerano Spr06/07] 36 Deciphering the cis-regulatory code

37. http://cs273a.stanford.edu [Bejerano Spr06/07] 37 [Blanchette et al., 2006] CRM prediction algorithm (Overview)