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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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slide1

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]

slide5

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]

slide17

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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