CS273A Computational Tour of the Human Genome, Gill Bejerano, Spring 2006/07

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TTh  1100-1215 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 vs. Multicellular
unicellular
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 - Protein
Protein - DNA
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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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Enhancers action over very large distances
RNAP II
Basal factors
promoter
Enhancer with bound protein
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Transient Transgenic Enhancer Assay
in situ
Reporter Gene
Minimal Promoter
Conserved Element
Construct is injected into 1 cell embryos Taken
out at embryonic day 10.5-14.5 Assayed for
reporter gene activity
transgenic
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Enhancer verification
Matched staining in dorsal apical ectodermal
ridge (part of limb bud)
Matched staining in genital eminence
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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?
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
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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

• 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.

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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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CRM prediction algorithm (Overview)
Blanchette et al., 2006