Post on 25-Feb-2016
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MW 11:00-12:15 in Redwood G19Profs: Serafim Batzoglou, Gill BejeranoTA: Cory McLean
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Lecture 12
Vertebrate Gene Cis-Regulation contd.
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Vertebrate Gene Regulation
gene (how to)control region(when & where)
DNA
proximal: in 103 letters
distal: in 106 letters
DNA bindingproteins
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Vertebrate Transcription Regulation
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unicellular
multicellular
Unicellular vs. Multicellular
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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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TFs in the Human Genome
Not a lot…
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Signal Transduction
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The Core Promoter
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CpG islands
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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:• Gene Expression Domains• Gene Regulatory Networks (GRN)
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Chromatin Remodeling
“off”
“on”
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Tx Factors Binding Sites
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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 embryosTaken out at embryonic day 10.5-14.5Assayed for reporter gene activity
in situ
transgenic
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Enhancer verification
Matched staining in genital eminence
Matched staining in dorsal apical
ectodermal ridge (part of limb bud)
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Fly Enhancer Combinatorics
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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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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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Gene Expression Domains: Independent
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Gene Expression Domains: Dependent
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Correlation with Human Disease
[Wang et al, 2000]
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Other Positional Effects
[de Kok et al, 1996]
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Chromatin Structure
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Histone Code
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Epigenetics
[Goldberg et al, 2007]
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More Functional Assays
In vitro / in vivoFragment / BACGain / LossBAC cut and paste
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Protein & Chromatin Assays
Protein binding assays: Electrophoretic mobility shift assays (EMSA) / Gel Shift DNAseI protection SELEX & CASTing Chromatin immuno-precipitation (ChIP), ChIP-chipand various chromatin assays.
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Gene Regulatory Networks
[Davidson & Erwin, 2006]
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The Hox Paradox
[Wray, 2003]
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The Great Vertebrate-Invertebrate Divide
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Gene Regulatory Network (GRN) Components
Davidson & Erwin (2006): 4 classes of GRN components:• ‘‘kernels’’ evolutionarily inflexible subcircuits that perform
essential upstream functions in building given body parts.• ‘‘plug-ins’’ certain small subcircuits that have been
repeatedly co-opted to diverse developmental purposes(regulatory, inc. signal transduction systems)
• “I/O switches” that allow or disallow developmental subcircuits to function in a given context (e.g., control of size of homologous body parts, many hox genes)
• differentiation gene batteries (execute cell-type specific function, end-players)
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GRN Kernel properties
1. Network subcircuits that consist of regulatory genes (i.e., TFs).2. They execute the developmental patterning functions required to
specify the embryo spatial domain/s in which body part/s will form. 3. Kernels are dedicated to given developmental functions and are not
used elsewhere in development of the organism (though individual genes of the kernel are likely used in many different contexts).
4. They have a particular form of structure in that the products of multiple regulatory genes of the kernel are required for function of each of the participating cis-regulatory modules of the kernel.
5. Interference with expression of any one kernel gene will destroy kernel function altogether and is likely to produce the catastrophic phenotype of lack of the body part.
The result is extraordinary conservation of kernel architecture.
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Kernel example
[Davidson & Erwin, 2006]
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Kernels and Phyla
t
now