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Thank you for the midterm feedback!

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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.

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unicellular

multicellular

Unicellular vs. Multicellular

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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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CpG islands

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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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Transcription Factor (TF) Binding Sites

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TFs in the Human Genome

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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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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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Gene Expression Domains: Independent

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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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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

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Many non-coding elements tested are cis-regulatory

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Gene Expression Domains: Dependent

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Distal Transcription Regulatory Elements

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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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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

HANDFUL of cases known (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

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Rapid TFBS turnover

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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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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

43[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