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

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

4 unicellular multicellular Unicellular vs. 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 - DNA Protein - Protein

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

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.

http://cs273a.stanford.edu [Bejerano Fall09/10] 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 Gene 2,000 different proteins can bind specific DNA sequences. 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. Proteins DNA Protein binding site

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

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

http://cs273a.stanford.edu [Bejerano Fall09/10] 18 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

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

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) DNA proximal: in 10 3 letters distal: in 10 6 letters DNA binding proteins

http://cs273a.stanford.edu [Bejerano Fall09/10] 24 Most Non-Coding Elements are likely cis-regulatory 9Mb “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

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? 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 genome gene protein Limb Malformation Over 300 genes already implicated in limb malformations.

http://cs273a.stanford.edu [Bejerano Fall09/10] 32 Disease Implications: Cis-Reg genome gene 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 [Yass is a small town in New South Wales, Australia.] Gene What settings make these “co-option” events happen? Gene

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

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

43 Human Accelerated Regions 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] Human Chimp

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

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  -catenin Hedgehog TGF-  family through Smads Growth factors via JAK-STATs Notch Integrin TNF

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