Thank you for the midterm feedback! Projects will be assigned this weekend. http://cs273a.stanford.edu [Bejerano Fall11/12]

Mid Term Feedback Feedback Lecture 12 Mid Term Feedback Feedback Cis-Regulation Cellular Signaling http://cs273a.stanford.edu [Bejerano Fall11/12]

MidTerm Feedback Feedback You like us! You like your teachers and love your TAs We like you too ;) We also like recruiting (at all levels) from this class Talk to us if you’re interested http://cs273a.stanford.edu [Bejerano Fall11/12]

Big Picture Reminder We are surveying the functional classes of elements encoded by the human genome. We have previously discussed: Repetitive sequences (mobile elements & low complexity) Protein coding genes (and pseudogenes) Non coding RNAs (structural, micro, long, anti-sense etc.) Today we’ll cover the last (known :) large class of functional elements, concluding the functional tour. http://cs273a.stanford.edu [Bejerano Fall11/12]

Gene Regulation gene (how to) control region (when & where) DNA Protein coding RNA gene DNA DNA binding proteins http://cs273a.stanford.edu [Bejerano Fall11/12]

Unicellular vs. Multicellular http://cs273a.stanford.edu [Bejerano Fall11/12]

Broad View of Transcription Chromatin / Proteins Extracellular signals DNA / Proteins http://cs273a.stanford.edu [Bejerano Fall11/12]

Vertebrate Transcription Regulation http://cs273a.stanford.edu [Bejerano Fall11/12]

Pol II Transcription Key components: Proteins DNA sequence DNA epigenetics Protein components: General Transcription factors Activators Co-activators http://cs273a.stanford.edu [Bejerano Fall11/12]

Activators & Co-Activators Protein - Protein Protein - DNA http://cs273a.stanford.edu [Bejerano Fall11/12]

The Core Promoter http://cs273a.stanford.edu [Bejerano Fall11/12]

Chromatin Remodeling “off” “on” http://cs273a.stanford.edu [Bejerano Fall11/12]

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 Fall11/12]

CpG islands Functionally: Unmethylated Evolutionary: CpGs are rare outside “CpG islands” http://cs273a.stanford.edu [Bejerano Fall11/12]

Transcription Factor (TF) Binding Sites http://cs273a.stanford.edu [Bejerano Fall11/12]

TFs in the Human Genome http://cs273a.stanford.edu [Bejerano Fall11/12]

Synergy / non-linear additivity Gene DNA http://cs273a.stanford.edu [Bejerano Fall11/12]

Combinatorial Regulatory Code 2,000 different proteins can bind specific DNA sequences. DNA Proteins 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 Fall11/12]

Distal Transcription Regulatory Elements http://cs273a.stanford.edu [Bejerano Fall11/12]

Enhancers http://cs273a.stanford.edu [Bejerano Fall11/12]

Enhancers: action over very large distances Basal factors RNAP II promoter Enhancer with bound protein http://cs273a.stanford.edu [Bejerano Fall11/12]

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 Fall11/12]

Vertebrate Enhancer Combinatorics limb neural tube brain Sall1 http://cs273a.stanford.edu [Bejerano Fall11/12]

Vertebrate Enhancer Combinatorics http://cs273a.stanford.edu [Bejerano Fall11/12]

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 Fall11/12]

Most Non-Coding Elements likely work in cis… “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 Every orange tick mark is roughly 100-1,000bp long, each evolves under purifying selection, and does not code for protein. http://cs273a.stanford.edu [Bejerano Fall11/12]

Many non-coding elements tested are cis-regulatory http://cs273a.stanford.edu [Bejerano Fall11/12]

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 Fall11/12]

Gene Expression Domains: Independent http://cs273a.stanford.edu [Bejerano Fall11/12]

Repressors / Silencers http://cs273a.stanford.edu [Bejerano Fall11/12]

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 Fall11/12]

Insulators http://cs273a.stanford.edu [Bejerano Fall11/12]

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: Epigenetic domains / signatures Gene expression domains Gene regulatory networks (GRN) http://cs273a.stanford.edu [Bejerano Fall11/12]

Disease Implications: Genes genome protein Limb Malformation Over 300 genes already implicated in limb malformations. http://cs273a.stanford.edu [Bejerano Fall11/12]

Disease Implications: Cis-Reg gene genome NO protein made Limb Malformation Growing number of cases (limb, deafness, etc). http://cs273a.stanford.edu [Bejerano Fall11/12]

Transcription Regulation & Human Disease [Wang et al, 2000] http://cs273a.stanford.edu [Bejerano Fall11/12]

Critical regulatory sequences Lettice et al. HMG 2003 12: 1725-35 Single base changes Knock out http://cs273a.stanford.edu [Bejerano Fall11/12]

Other Positional Effects [de Kok et al, 1996] http://cs273a.stanford.edu [Bejerano Fall11/12]

Genomewide Association Studies point to non-coding DNA http://cs273a.stanford.edu [Bejerano Fall11/12]

WGA Disease http://cs273a.stanford.edu [Bejerano Fall11/12]

9p21 Cis effects Follow up study: http://cs273a.stanford.edu [Bejerano Fall11/12]

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 Fall11/12]

Britten & Davidson Hypothesis: Repeat to Rewire! [Davidson & Erwin, 2006] [Britten & Davidson, 1971] http://cs273a.stanford.edu [Bejerano Fall11/12]

Modular: Most Likely to Evolve? Chimp Human With the vast genomic resources available nowadays, we can isolate both genotypic and phenotypic differences between species. However, a gap still exists in our ability to associate genotype with phenotype, and an overarching goal in biology today is to start bridging that gap, to be able to say: This inversion causes foot shape alterations This duplication and that substitution both play a role in brain size Or going from phenotype to genotype, This sweat-related gene has some change in chimpanzee relative to human The study I am talking about today tries to link genotype to phenotype for a single type of genomic rearrangement, human-specific deletions. http://cs273a.stanford.edu [Bejerano Fall11/12] 45

Human Accelerated Regions Human-specific substitutions in conserved sequences Human Chimp HAR1 expressed in Cajal-Retzius neurons at border of marginal zone 46 [Pollard, K. et al., Nature, 2006] [Prabhakar, S. et al., Science, 2008] [Beniaminov, A. et al., RNA, 2008] 46

Signal Transduction http://cs273a.stanford.edu [Bejerano Fall11/12]

Cell Communication Lodish, 20-1

Wnt and Hedgehog signaling Jacob & Lum Science 2007

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

Giant State Machine… Chromatin / Proteins Extracellular signals DNA / Proteins http://cs273a.stanford.edu [Bejerano Fall11/12]