http://cs273a.stanford.edu [bejerano fall09/10] 1 thank you for the midterm feedback!
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http://cs273a.stanford.edu [Bejerano Fall09/10] 1
Thank you for the midterm feedback!
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.
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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