http://cs273a.stanford.edu [Bejerano Spr06/07] 1

TTh  11:00-12:15 in Clark S361

Profs: Serafim Batzoglou, Gill Bejerano

TAs: George Asimenos, Cory McLean

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Lecture 10

Transcription Regulation in Vertebrates contd.

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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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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 embryos

Taken out at embryonic day 10.5-14.5

Assayed 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

repressors

Repressors

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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 vivo

Fragment / BAC

Gain / Loss

BAC 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-chip

and 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

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Deciphering the cis-regulatory code

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[Blanchette et al., 2006]

CRM prediction algorithm (Overview)