Thank you for the midterm feedback! http://cs273a.stanford.edu [Bejerano Fall09/10] 1 Lecture 12 Cis-Regulation Cellular Signaling http://cs273a.stanford.edu [Bejerano Fall09/10] 2 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] 3 Unicellular vs. Multicellular unicellular multicellular http://cs273a.stanford.edu [Bejerano Fall09/10] 4 Vertebrate Transcription Regulation http://cs273a.stanford.edu [Bejerano Fall09/10] 5 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] 6 Activators & Co-Activators Protein - Protein Protein - DNA http://cs273a.stanford.edu [Bejerano Fall09/10] 7 The Core Promoter http://cs273a.stanford.edu [Bejerano Fall09/10] 8 Chromatin Remodeling “off” “on” http://cs273a.stanford.edu [Bejerano Fall09/10] 9 CpG islands http://cs273a.stanford.edu [Bejerano Fall09/10] 10 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. • 11 http://cs273a.stanford.edu [Bejerano Fall09/10] 12 Transcription Factor (TF) Binding Sites http://cs273a.stanford.edu [Bejerano Fall09/10] 13 TFs in the Human Genome http://cs273a.stanford.edu [Bejerano Fall09/10] 14 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] 15 Enhancers http://cs273a.stanford.edu [Bejerano Fall09/10] 16 Enhancers: action over very large distances Basal factors promoter RNAP II Enhancer with bound protein http://cs273a.stanford.edu [Bejerano Fall09/10] 17 Transient Transgenic Enhancer Assay in situ Conserved Element transgenic 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 http://cs273a.stanford.edu [Bejerano Fall09/10] 18 Vertebrate Enhancer Combinatorics limb neural tube http://cs273a.stanford.edu brain [Bejerano Fall09/10] Sall1 19 Vertebrate Enhancer Combinatorics http://cs273a.stanford.edu [Bejerano Fall09/10] 20 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] 21 Gene Expression Domains: Independent http://cs273a.stanford.edu [Bejerano Fall09/10] 22 Vertebrate Gene Regulation distal: in 106 letters gene (how to) control region (when & where) DNA DNA binding proteins proximal: in 103 letters http://cs273a.stanford.edu [Bejerano Fall09/10] 23 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] 24 Many non-coding elements tested are cis-regulatory http://cs273a.stanford.edu [Bejerano Fall09/10] 25 Gene Expression Domains: Dependent http://cs273a.stanford.edu [Bejerano Fall09/10] 26 Distal Transcription Regulatory Elements http://cs273a.stanford.edu [Bejerano Fall09/10] 27 Repressors / Silencers http://cs273a.stanford.edu [Bejerano Fall09/10] 28 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... http://cs273a.stanford.edu [Bejerano Fall09/10] 29 Insulators http://cs273a.stanford.edu [Bejerano Fall09/10] 30 Disease Implications: Genes gene genome protein Limb Malformation Over 300 genes already implicated in limb malformations. http://cs273a.stanford.edu [Bejerano Fall09/10] 31 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] 32 Transcription Regulation & Human Disease [Wang et al, 2000] http://cs273a.stanford.edu [Bejerano Fall09/10] 33 Critical regulatory sequences Lettice et al. HMG 2003 12: 1725-35 Single base changes Knock out http://cs273a.stanford.edu [Bejerano Fall09/10] 34 Other Positional Effects [de Kok et al, 1996] http://cs273a.stanford.edu [Bejerano Fall09/10] 35 Genomewide Association Studies point to non-coding DNA http://cs273a.stanford.edu [Bejerano Fall09/10] 36 WGA Disease http://cs273a.stanford.edu [Bejerano Fall09/10] 37 Rapid TFBS turnover http://cs273a.stanford.edu [Bejerano Fall09/10] 38 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] 39 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] 40 Britten & Davidson Hypothesis: Repeat to Rewire! [Davidson & Erwin, 2006] [Britten & Davidson, 1971] http://cs273a.stanford.edu [Bejerano Fall09/10] 41 Modular: Most Likely to Evolve? Chimp http://cs273a.stanford.edu [Bejerano Fall09/10] Human 42 Human Accelerated Regions • Human-specific substitutions in conserved sequences Human [Pollard, K. et al., Nature, 2006] Chimp [Beniaminov, A. et al., RNA, 2008] 43 [Prabhakar, S. et al., Science, 2008] Signal Transduction http://cs273a.stanford.edu [Bejerano Fall09/10] 44 Cell Communication Lodish, 20-1 45 Wnt and Hedgehog signaling Jacob & Lum Science 2007 46 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 47