MW 11:00-12:15 in Beckman B302 Prof: Gill Bejerano TAs: Jim Notwell & Harendra Guturu

1. CS173 Lecture 7: Transcriptional activation I MW  11:00-12:15 in Beckman B302 Prof: Gill Bejerano TAs: Jim Notwell & Harendra Guturu http://cs173.stanford.edu [BejeranoWinter12/13]

2. Announcements • Piazza is getting livelier. Thank you :) • HW1 due this coming Monday. All well? http://cs173.stanford.edu [BejeranoWinter12/13]

3. TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGACTAAATCTCATTCAGAAGAAGTGATTGTACCTGAGTTCAATTCTAGCGCAAAGGAATTACCAAGACCATTGGCCGAAAAGTGCCCGAGCATAATTAAGAAATTTATAAGCGCTTATGATGCTAAACCGGATTTTGTTGCTAGATCGCCTGGTAGAGTCAATCTAATTGGTGAACATATTGATTATTGTGACTTCTCGGTTTTACCTTTAGCTATTGATTTTGATATGCTTTGCGCCGTCAAAGTTTTGAACGATGAGATTTCAAGTCTTAAAGCTATATCAGAGGGCTAAGCATGTGTATTCTGAATCTTTAAGAGTCTTGAAGGCTGTGAAATTAATGACTACAGCGAGCTTTACTGCCGACGAAGACTTTTTCAAGCAATTTGGTGCCTTGATGAACGAGTCTCAAGCTTCTTGCGATAAACTTTACGAATGTTCTTGTCCAGAGATTGACAAAATTTGTTCCATTGCTTTGTCAAATGGATCATATGGTTCCCGTTTGACCGGAGCTGGCTGGGGTGGTTGTACTGTTCACTTGGTTCCAGGGGGCCCAAATGGCAACATAGAAAAGGTAAAAGAAGCCCTTGCCAATGAGTTCTACAAGGTCAAGTACCCTAAGATCACTGATGCTGAGCTAGAAAATGCTATCATCGTCTCTAAACCAGCATTGGGCAGCTGTCTATATGAATTAGTCAAGTATACTTCTTTTTTTTACTTTGTTCAGAACAACTTCTCATTTTTTTCTACTCATAACTTTAGCATCACAAAATACGCAATAATAACGAGTAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTTTCCTACGCATAATAAGAATAGGAGGGAATATCAAGCCAGACAATCTATCATTACATTTAAGCGGCTCTTCAAAAAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGACCTTTGCGCCAAATAATGTGGATTTGGAAAAAGAGTATAAGTCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATCGAGCTTTGAAGAAAAAGTAAGCTCAGAAAAACCTCAATACAGCTCATTCTGGAAGAAAATCTATTATGAATATGTGGTCGTTGACAAATCAATCTTGGGTGTTTCTATTCTGGATTCATTTATGTACAACCAGGACTTGAAGCCCGTCGAAAAAGAAAGGCGGGTTTGGTCCTGGTACAATTATTGTTACTTCTGGCTTGCTGAATGTTTCAATATCAACACTTGGCAAATTGCAGCTACAGGTCTACAACTGGGTCTAAATTGGTGGCAGTGTTGGATAACAATTTGGATTGGGTACGGTTTCGTTGGTGCTTTTGTTGTTTTGGCCTCTAGAGTTGGATCTGCTTATCATTTGTCATTCCCTATATCATCTAGAGCATCATTCGGTATTTTCTTCTCTTTATGGCCCGTTATTAACAGAGTCGTCATGGCCATCGTTTGGTATAGTGTCCAAGCTTATATTGCGGCAACTCCCGTATCATTAATGCTGAAATCTATCTTTGGAAAAGATTTACAATGATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATAAAGTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGACTAAATCTCATTCAGAAGAAGTGATTGTACCTGAGTTCAATTCTAGCGCAAAGGAATTACCAAGACCATTGGCCGAAAAGTGCCCGAGCATAATTAAGAAATTTATAAGCGCTTATGATGCTAAACCGGATTTTGTTGCTAGATCGCCTGGTAGAGTCAATCTAATTGGTGAACATATTGATTATTGTGACTTCTCGGTTTTACCTTTAGCTATTGATTTTGATATGCTTTGCGCCGTCAAAGTTTTGAACGATGAGATTTCAAGTCTTAAAGCTATATCAGAGGGCTAAGCATGTGTATTCTGAATCTTTAAGAGTCTTGAAGGCTGTGAAATTAATGACTACAGCGAGCTTTACTGCCGACGAAGACTTTTTCAAGCAATTTGGTGCCTTGATGAACGAGTCTCAAGCTTCTTGCGATAAACTTTACGAATGTTCTTGTCCAGAGATTGACAAAATTTGTTCCATTGCTTTGTCAAATGGATCATATGGTTCCCGTTTGACCGGAGCTGGCTGGGGTGGTTGTACTGTTCACTTGGTTCCAGGGGGCCCAAATGGCAACATAGAAAAGGTAAAAGAAGCCCTTGCCAATGAGTTCTACAAGGTCAAGTACCCTAAGATCACTGATGCTGAGCTAGAAAATGCTATCATCGTCTCTAAACCAGCATTGGGCAGCTGTCTATATGAATTAGTCAAGTATACTTCTTTTTTTTACTTTGTTCAGAACAACTTCTCATTTTTTTCTACTCATAACTTTAGCATCACAAAATACGCAATAATAACGAGTAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTTTCCTACGCATAATAAGAATAGGAGGGAATATCAAGCCAGACAATCTATCATTACATTTAAGCGGCTCTTCAAAAAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGACCTTTGCGCCAAATAATGTGGATTTGGAAAAAGAGTATAAGTCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATCGAGCTTTGAAGAAAAAGTAAGCTCAGAAAAACCTCAATACAGCTCATTCTGGAAGAAAATCTATTATGAATATGTGGTCGTTGACAAATCAATCTTGGGTGTTTCTATTCTGGATTCATTTATGTACAACCAGGACTTGAAGCCCGTCGAAAAAGAAAGGCGGGTTTGGTCCTGGTACAATTATTGTTACTTCTGGCTTGCTGAATGTTTCAATATCAACACTTGGCAAATTGCAGCTACAGGTCTACAACTGGGTCTAAATTGGTGGCAGTGTTGGATAACAATTTGGATTGGGTACGGTTTCGTTGGTGCTTTTGTTGTTTTGGCCTCTAGAGTTGGATCTGCTTATCATTTGTCATTCCCTATATCATCTAGAGCATCATTCGGTATTTTCTTCTCTTTATGGCCCGTTATTAACAGAGTCGTCATGGCCATCGTTTGGTATAGTGTCCAAGCTTATATTGCGGCAACTCCCGTATCATTAATGCTGAAATCTATCTTTGGAAAAGATTTACAATGATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATAAAG Genome Content http://cs173.stanford.edu [BejeranoWinter12/13]

4. Gene Products long non-coding RNA reverse transcription microRNA rRNA, snRNA, snoRNA

5. Gene Regulation Different cells in our body hold copies of (essentially) the same genome. Yet they express very different repertoires of proteins and non-coding RNAs. How do cells do it? A: like they do everything else: using their proteins & ncRNAs… http://cs173.stanford.edu [BejeranoWinter12/13]

6. Gene Regulatory Switches • Gene = genomic substring that encodes HOW to make a protein. • Genomic switch = genomic substring that encodes WHEN, WHERE & HOW MUCH of a protein to make. [0,1,1,1] B Gene H Gene Gene H N Gene N B [1,0,0,1] [1,1,0,0] http://cs173.stanford.edu [BejeranoWinter12/13]

7. Transcription Activation http://cs173.stanford.edu [BejeranoWinter12/13]

8. RNA Polymerase • Transcription = Copying a segment of DNA into (non/coding) RNA • Gene transcription starts at the (aptly named) TSS, orgene transcription start site • Transcription is done be RNA polymerase, a complex of 10-12 subunit proteins. • There are three types of RNA polymerases in human: • RNA pol I synthesizes ribosomal RNAs • RNA pol II synthesizes pre-mRNAs and most microRNAs • RNA pol III synthesizes tRNAs, rRNAand other ssRNAs TSS RNA Polymerase http://cs173.stanford.edu [BejeranoWinter12/13]

9. RNA Polymerase is General Purpose • RNA Polymerase is the general purpose transcriptional machinery. • It generally does not recognize gene transcription start sites by itself, and requires interactions with multiple additional proteins. generalpurpose contextspecific http://cs173.stanford.edu [BejeranoWinter12/13]

10. Terminology • Transcription Factors (TF): Proteins that return to the nucleus, bind specific DNA sequences there, and affect transcription. • There are 1,200-2,000 TFs in the human genome (out of 20-25,000 genes) • Only a subset of TFs may be expressed in a given cell at a given point in time. • Transcription Factor Binding Sites: 4-20bp stretches of DNA where TFs bind. • There are millions of TF binding sites in the human genome. • In a cell at a given point in time, a site can be either occupied or unoccupied. http://cs173.stanford.edu [BejeranoWinter12/13]

11. Terminology • Promoter: The region of DNA 100-1,000bp immediately “upstream” of the TSS, which encodes binding sites for the general purpose RNA polymerase associated TFs, and at times some context specific sites. • There are as many promoters as there are TSS’s in the human genome. Many genes have more than one TSS. • Enhancer: A region of 100-1,000bp up to 1Mb or more upstream or downstream from the TSS that includes binding sites for multiple TFs. When bound by (the right) TFs an enhancer turns on/accelerates transcription. • Note how an enhancer (E) very far away in sequence can in fact get very close to the promoter (P) in space. promoter TSS gene http://cs173.stanford.edu [BejeranoWinter12/13]

12. Terminology • Gene regulatory domain: the full repertoire of enhancers that affect the expression of a (protein coding or non-coding) gene, at some cells under some condition. • Gene regulatory domains do not have to be contiguous in genome sequence. • Neither are they disjoint: One or more enhancers may well affect the expression of multiple genes (at the same or different times). TSS promoter enhancers for different contexts http://cs173.stanford.edu [BejeranoWinter12/13]

13. Imagine a giant state machine Transcription factors bind DNA, turn on or off different promoters and enhancers, which in-turn turn on or off different genes, some of which may themselves be transcription factors, which again changes the presence of TFs in the cell, the state of active promoters/enhancers etc. Proteins DNA transcription factorbinding site Gene DNA http://cs173.stanford.edu [BejeranoWinter12/13]

14. One nice hypothetical example requires active enhancers to function functions independently of enhancers http://cs173.stanford.edu [BejeranoWinter12/13]

15. The State Space Discrete, but very large. All states served by same genome(!) 1012cells 1cell http://cs173.stanford.edu [BejeranoWinter12/13]

16. Transcription Activation: Some measurements and observations http://cs173.stanford.edu [BejeranoWinter12/13]

17. Transcription Factor Binding Sites (TFBS) • An antibodyis a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria. • Antibodies can be raised that instead recognize specific transcription factors. • Chromatin Immunoprecipitation: Take DNA (region or whole genome) bound by TFs, crosslink DNA-TFs, shear DNA, select DNA fragments bound by TF of interest using antibody, get rid of TF and antibody, sequence pool of DNA.  Obtain genomic regions bound by TF. http://cs173.stanford.edu [BejeranoWinter12/13]

18. TFBS Position Weight Matrix (PWM) Note the strong independence assumption between positions. Holds for most transcription binding profiles in the human genome. http://cs173.stanford.edu [BejeranoWinter12/13]

19. Transcription Factors have Large “fan outs” • We could have had one TF regulate two TFS, each of which regulates two other TFs, etc. and each of those contributing to the regulation of a modest number of target genes (that do the real work). • Instead TFs reproducibly bind to thousands of genomic locations almost anywhere we’ve looked. • Gene regulation forms a dense network. http://cs173.stanford.edu [BejeranoWinter12/13]

20. Transfections As far as we’ve seen, enhancers work “the same” irrespective of distance (or orientation) to TSS, or identity of target gene. enhancer reporter gene minimalpromoter in cellular contextof choice • Which enhancers work in what contexts? • What if you mutate enhancer bases (disrupt or introduce binding sites) and run the experiment again? • What if you co-transfect a TF you think binds to this enhancer? • What if you instead add siRNA for that TF? http://cs173.stanford.edu [BejeranoWinter12/13]

21. Synergy / non-linear additivity Gene DNA http://cs173.stanford.edu [BejeranoWinter12/13]

22. Transgenics enhancer reporter gene minimalpromoter Observe enhancer behavior in vivo. Qualitative (not quantitative) assay. Can section and stain to obtain more specific cell-type information. http://cs173.stanford.edu [BejeranoWinter12/13]

23. BAC transgenics: necessity vs sufficiency You can take 100-200kb segments out of the genome, insert a reporter gene in place of gene X, and measure regulatory domain expression. You can then continue to delete or mutate individual enhancers. http://cs173.stanford.edu [BejeranoWinter12/13]

24. Gene Regulation: Enhancers are modular and additive brain limb neural tube Sall1 Temporal gene expression pattern “equals” sum of promoter and enhancers expression patterns. http://cs173.stanford.edu [BejeranoWinter12/13]

25. Genome Engineering • Technologies are in fact constantly improving that allow us to edit the nuclear genome itself. • Edit the genome of an embryonic stem cell, breed homozygous modified animals. http://cs173.stanford.edu [BejeranoWinter12/13]

26. Chromosome conformation capture (3C) • People are also developing methods to detect when two genomic regions far in sequence are in fact interacting in space. • Ultimately this will allow to determine experimentally the regulatory domain of each gene (likely condition dependent). http://cs173.stanford.edu [BejeranoWinter12/13]

27. Gene Regulation is HOT • Despite its complexity gene regulation is currently one of the hottest topics in the study of the human genome. • Large projects are pouring tons of money to generate huge descriptive datasets. • The challenge now is to glean logic from these piles. To be continued… http://cs173.stanford.edu [BejeranoWinter12/13]