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Thank you for the midterm feedback!

Projects will be assigned shortly.

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Lecture 12

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Cis-Regulation

Cellular Signaling

MidTerm Feedback Feedback

General comments:•Programming requirements

–CS106A enough?

•Coverage: Depth vs. Breadth•UCSC Primer Fit•Homework vs. lectures•You’re enjoying this class :)

Let’s also reflect about doing your homework briefly.

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Big Picture Context

We are surveying the functional classes of elements encoded by the human genome.

We have previously discussed:•Repetitive sequences•Protein coding genes•Non coding RNAs

Today we’ll cover the last (known :) large class of functional elements.

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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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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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Synergy / non-linear additivity

Gene

DNA

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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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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