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Thank you for the midterm feedback!

Projects will be assigned this weekend.

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Lecture 12

Mid Term Feedback Feedback

Cis-Regulation

Cellular Signaling

MidTerm Feedback Feedback

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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.

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Gene Regulation

•gene (how to)•control region(when & where)

DNA

DNA binding

proteins

RNA geneProtein coding

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unicellular

multicellular

Unicellular vs. Multicellular

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Broad View of Transcription

Chromatin / Proteins

DNA / Proteins

Extracellular signals

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Vertebrate Transcription Regulation

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Pol II Transcription

Key components:• Proteins• DNA sequence• DNA epigenetics

Protein components:• General Transcription factors• Activators• Co-activators

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Activators & Co-Activators

Protein - DNA

Protein - Protein

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The Core Promoter

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Chromatin Remodeling

“off”

“on”

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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 .

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CpG islands

Functionally:Unmethylated

Evolutionary:CpGs are rareoutside “CpG islands”

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Transcription Factor (TF) Binding Sites

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TFs in the Human Genome

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Synergy / non-linear additivity

Gene

DNA

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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

DNA

Protein binding site

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Distal Transcription Regulatory Elements

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Enhancers

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Basal factors RNAP II

Enhancer with bound protein

promoter

Enhancers: action over very large distances

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Transient Transgenic Enhancer Assay

Reporter GeneMinimal PromoterConservedElement

Construct is injected into 1 cell embryos

Taken out at embryonic day 10.5-14.5

Assayed for reporter gene activity

in situ

transgenic

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Vertebrate Enhancer Combinatorics

Sall1limbneuraltube brain

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Vertebrate Enhancer Combinatorics

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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]

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Most Non-Coding Elements likely work in cis…

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

Every orange tick mark is roughly 100-1,000bp long, each evolves under purifying selection, and does not code for protein.

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Many non-coding elements tested are cis-regulatory

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Vertebrate Gene Regulation

gene (how to)

control region(when & where)

DNA

proximal: in 103 letters

distal: in 106 letters

DNA binding

proteins

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Gene Expression Domains: Independent

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Repressors / Silencers

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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

repressorsRepressors

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Insulators

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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)

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Disease Implications: Genes

genome

gene

protein

Limb Malformation

Over 300 genes already

implicated in limb malformations.

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Disease Implications: Cis-Reg

genome

gene

NO protein

made

Limb Malformation

Growing number of cases (limb, deafness, etc).

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Transcription Regulation & Human Disease

[Wang et al, 2000]

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Critical regulatory sequences

Lettice et al. HMG 2003 12: 1725-35

Single base changes

Knock out

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Other Positional Effects

[de Kok et al, 1996]

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Genomewide Association Studies point to non-coding DNA

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WGA Disease

9p21 Cis effects

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Follow up study:

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Cis-Regulatory Evolution: E.g., obile Elements

[Yass is a small town in New South Wales, Australia.]

Gene

Gene

What settings make these“co-option” events happen?

Gene

Gene

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Britten & Davidson Hypothesis: Repeat to Rewire!

[Britten & Davidson, 1971]

[Davidson & Erwin, 2006]

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Modular: Most Likely to Evolve?

Chimp Human

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Human Accelerated Regions• Human-specific substitutions in conserved

sequences

46[Pollard, K. et al., Nature, 2006] [Prabhakar, S. et al., Science, 2008][Beniaminov, A. et al., RNA, 2008]

Human Chimp

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Signal Transduction

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Cell Communication

Lodish, 20-1

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Wnt and Hedgehog signaling

Jacob & Lum Science 2007

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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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Giant State Machine…

Chromatin / Proteins

DNA / Proteins

Extracellular signals