MW  11:00-12:15 in Redwood G19
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MW  11:00-12:15 in Redwood G19 Profs: Serafim Batzoglou, Gill Bejerano TA: Cory McLean PowerPoint PPT Presentation


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MW  11:00-12:15 in Redwood G19 Profs: Serafim Batzoglou, Gill Bejerano TA: Cory McLean. Lecture 12. Vertebrate Gene Cis-Regulation contd. Vertebrate Gene Regulation. gene (how to) control region (when & where). distal: in 10 6 letters. DNA. DNA binding proteins.

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MW  11:00-12:15 in Redwood G19 Profs: Serafim Batzoglou, Gill Bejerano TA: Cory McLean

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Mw 11 00 12 15 in redwood g19 profs serafim batzoglou gill bejerano ta cory mclean

MW  11:00-12:15 in Redwood G19

Profs: Serafim Batzoglou, Gill Bejerano

TA: Cory McLean

http://cs273a.stanford.edu [Bejerano Aut07/08]


Lecture 12

Lecture 12

  • Vertebrate Gene Cis-Regulation contd.

http://cs273a.stanford.edu [Bejerano Aut07/08]


Vertebrate gene regulation

Vertebrate Gene Regulation

  • gene (how to)

  • control region(when & where)

distal: in 106 letters

DNA

DNA binding

proteins

proximal: in 103 letters

http://cs273a.stanford.edu [Bejerano Aut07/08]


Vertebrate transcription regulation

Vertebrate Transcription Regulation

http://cs273a.stanford.edu [Bejerano Aut07/08]


Mw 11 00 12 15 in redwood g19 profs serafim batzoglou gill bejerano ta cory mclean

Unicellular vs. Multicellular

unicellular

multicellular

http://cs273a.stanford.edu [Bejerano Aut07/08]


Pol ii transcription

Pol II Transcription

  • Key components:

  • Proteins

  • DNA sequence

  • DNA epigenetics

  • Protein components:

  • General Transcription factors

  • Activators

  • Co-activators

http://cs273a.stanford.edu [Bejerano Aut07/08]


Activators co activators

Activators & Co-Activators

Protein - Protein

Protein - DNA

http://cs273a.stanford.edu [Bejerano Aut07/08]


Tfs in the human genome

TFs in the Human Genome

Not a lot…

http://cs273a.stanford.edu [Bejerano Aut07/08]


Signal transduction

Signal Transduction

http://cs273a.stanford.edu [Bejerano Aut07/08]


The core promoter

The Core Promoter

http://cs273a.stanford.edu [Bejerano Aut07/08]


Cpg islands

CpG islands

http://cs273a.stanford.edu [Bejerano Aut07/08]


Cis regulatory components

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)

http://cs273a.stanford.edu [Bejerano Aut07/08]


Chromatin remodeling

Chromatin Remodeling

“off”

“on”

http://cs273a.stanford.edu [Bejerano Aut07/08]


Tx factors binding sites

Tx Factors Binding Sites

http://cs273a.stanford.edu [Bejerano Aut07/08]


Distal transcription regulatory elements

Distal Transcription Regulatory Elements

http://cs273a.stanford.edu [Bejerano Aut07/08]


Enhancers

Enhancers

http://cs273a.stanford.edu [Bejerano Aut07/08]


Mw 11 00 12 15 in redwood g19 profs serafim batzoglou gill bejerano ta cory mclean

Enhancers: action over very large distances

RNAP II

Basal factors

promoter

Enhancer with bound protein

http://cs273a.stanford.edu [Bejerano Aut07/08]


Transient transgenic enhancer assay

Transient Transgenic Enhancer Assay

in situ

Conserved

Element

Minimal Promoter

Reporter Gene

Construct is injected into 1 cell embryos

Taken out at embryonic day 10.5-14.5

Assayed for reporter gene activity

transgenic

http://cs273a.stanford.edu [Bejerano Aut07/08]


Enhancer verification

Enhancer verification

Matched staining in dorsal apical ectodermal ridge (part of limb bud)

Matched staining in genital eminence

http://cs273a.stanford.edu [Bejerano Aut07/08]


Fly enhancer combinatorics

Fly Enhancer Combinatorics

http://cs273a.stanford.edu [Bejerano Aut07/08]


Vertebrate enhancer combinatorics

Vertebrate Enhancer Combinatorics

http://cs273a.stanford.edu [Bejerano Aut07/08]


What are enhancers

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]

http://cs273a.stanford.edu [Bejerano Aut07/08]


Repressors silencers

Repressors / Silencers

http://cs273a.stanford.edu [Bejerano Aut07/08]


What are enhancers1

What are Enhancers?

Repressors

  • 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

http://cs273a.stanford.edu [Bejerano Aut07/08]


Insulators

Insulators

http://cs273a.stanford.edu [Bejerano Aut07/08]


Gene expression domains independent

Gene Expression Domains: Independent

http://cs273a.stanford.edu [Bejerano Aut07/08]


Gene expression domains dependent

Gene Expression Domains: Dependent

http://cs273a.stanford.edu [Bejerano Aut07/08]


Correlation with human disease

Correlation with Human Disease

[Wang et al, 2000]

http://cs273a.stanford.edu [Bejerano Aut07/08]


Other positional effects

Other Positional Effects

[de Kok et al, 1996]

http://cs273a.stanford.edu [Bejerano Aut07/08]


Chromatin structure

Chromatin Structure

http://cs273a.stanford.edu [Bejerano Aut07/08]


Histone code

Histone Code

http://cs273a.stanford.edu [Bejerano Aut07/08]


Epigenetics

Epigenetics

[Goldberg et al, 2007]

http://cs273a.stanford.edu [Bejerano Aut07/08]


More functional assays

More Functional Assays

In vitro / in vivo

Fragment / BAC

Gain / Loss

BAC cut and paste

http://cs273a.stanford.edu [Bejerano Aut07/08]


Protein chromatin assays

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.

http://cs273a.stanford.edu [Bejerano Aut07/08]


Gene regulatory networks

Gene Regulatory Networks

[Davidson & Erwin, 2006]

http://cs273a.stanford.edu [Bejerano Aut07/08]


The hox paradox

The Hox Paradox

[Wray, 2003]

http://cs273a.stanford.edu [Bejerano Aut07/08]


The great vertebrate invertebrate divide

The Great Vertebrate-Invertebrate Divide

http://cs273a.stanford.edu [Bejerano Aut07/08]


Gene regulatory network grn components

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)

http://cs273a.stanford.edu [Bejerano Aut07/08]


Grn kernel properties

GRN Kernel properties

  • Network subcircuits that consist of regulatory genes (i.e., TFs).

  • They execute the developmental patterning functions required to specify the embryo spatial domain/s in which body part/s will form.

  • 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).

  • 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.

  • 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.

http://cs273a.stanford.edu [Bejerano Aut07/08]


Kernel example

Kernel example

[Davidson & Erwin, 2006]

http://cs273a.stanford.edu [Bejerano Aut07/08]


Kernels and phyla

Kernels and Phyla

t

now

http://cs273a.stanford.edu [Bejerano Aut07/08]


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