Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules

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Comparative genomics, ChIP-chip and transfections
to find cis-regulatory modules
What is conservation good for??

• Penn State University, Center for Comparative
  Genomics and Bioinformatics Webb Miller,
  Francesca Chiaromonte, Ross Hardison
• Childrens Hospital of Philadelphia Mitch Weiss,
  Lou Dore
• NimbleGen Roland Green, Xinmin Zhang

Cold Spring Harbor, March 2007
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Ideal cases for interpretation by comparative
genomics
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Putative transcriptional regulatory regions
pTRRs

• Antibodies vs 10 sequence-specific factors
• Sp1, Sp3, E2F1, E2F4, cMyc, STAT1, cJun, CEBPe,
  PU1, RA Receptor A
• High resolution ChIP-chip platforms Affymetrix
  and NimbleGen
• Data from several different labs in ENCODE
  consortium
• High likelihood hits for ChIP-chip
• 5 false discovery rate
• Supported by chromatin modification data
• Modified histones in chromatin H4Ac, H3Ac,
  H3K4me, H3K4me2, H3K4me3, etc.
• DNase hypersensitive sites (DHSs) or nucleosome
  depleted sites
• Result set of 1369 pTRRs

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Functional classes show distinctive trends in
phylogenetic depth of conservation
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Genes likely regulated by clade-specific pTRRs
are enriched for distinctive functions
Percentage of pTRRs that align no further than
David King
Primates 3
Millions of years
91
Eutherians 71
173
310
Marsupials 21
450
Tetrapods 4
Vertebrates 1
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Regulatory potential (RP) captures pattern,
composition and constraint in alignments
Genome Research 161585 (2006)

• High RP for an aligned sequence means it contains
  patterns similar to those found in gene
  regulatory regions
• Positive training set Alignments of known
  regulatory regions
• Negative training set Alignments of likely
  neutral DNA (ancestral repeats)
• Human and mouse RP scores are on UCSC Genome
  Browser and PSUs Galaxy

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High RP plus conserved consensus motif is a good
predictor of CRMs around GATA-1 regulated genes
Genome Research 161480 (2006)
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Genes Co-expressed in Late Erythroid Maturation
G1E cells proerythroblast line lacking the
transcription factor GATA-1. G1E-ER cells
rescued by expressing an estrogen-responsive form
of GATA-1 Rylski et al., Mol Cell Biol. 2003
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Predict CRMs based on alignment and expression of
nearby genes

• Gene is up- or down-regulated by GATA-1
• Noncoding DNA sequence
• Aligns between mouse and other mammals and has a
  positive RP score
• Contains a conserved consensus binding site motif
  for GATA-1

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preCRMs with conserved consensus GATA-1 BS tend
to be active on transfected plasmids
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DNA segments with positive RP and a GATA-1
binding motif validate as enhancers at a good
rate
RP consensus motif Tested Validated
Success Positive conserved 44 23
52 Positive mouse 6 4
67 Negative conserved 6 1
17 Negative none 17 0 0
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Design of ChIP-chip for occupancy by GATA-1

• Non-overlapping tiling array with 50bp probe and
  100bp resolution (NimbleGen)
• Cover range
• Mouse chr757225996-123812258 (70Mbp)
• 3. Antibody against the ER portion of
  GATA-1-ER protein in rescued G1E-ER4 cells

Yong Cheng (PSU), with Mitch Weiss Lou Dore
(CHoP), Roland Green, Xinmin Zhang(NimbleGen)
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Signals in known occupied sites in Hbb LCR
HS1
HS2
HS3
1) Cluster of high signals 2) hill shape of the
signals
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ChIP-chip hits are high quality and tend to have
GATA-1 binding motifs

• Peak calling by Mpeak (Ren) and Tamalpais (Beida
  and Farnham) gave 321 ChIP-chip hits
• 19 hits were tested by qPCR
• 13 were validated 70
• 267 out of the 321 (83) have WGATAR motifs,
  binding site for GATA-1
• Random sampling on average gives 102 DNA segments
  with the motif
• The ChIP-chip hits are 2.6-fold enriched for the
  GATA-1 binding site motif

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Only HALF the GATA-1 binding site motifs are
conserved outside rodents

• Of the GATA-1 binding motifs in those 249 hits,
  112 (45) are conserved between mouse and at
  least one non-rodent species.

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Distribution of ChIP-chip hits on 70Mb of mouse
chr7
Yong Cheng, Yuepin Zhou and Christine Dorman
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36 of ChIP-chip hits act as enhancers in K562
cells
14.5
5.7
21 out of 59 ChIP-chip hits increase activity of
HBGpr-Luc in K562 cells.
GATA-1 occupied sites by ChIP-chip
No GATA-1
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30 of ChIP-chip hits act as enhancers in MEL
cells
15 out of 50 ChIP-chip hits increase activity of
HBGpr-Luc in MEL cells.
GATA-1 occupied sites by ChIP-chip
No GATA-1
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Validated ChIP hit, enhancer, deep conservation
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Validated ChIP hit, enhancer, limited conservation
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ChIP-chip hit, enhancer, rodent specific
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Test of neutrality using polymorphism and
divergence data
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A promoter distal to the beta-like globin genes
has a signal for recent purifying selection
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The distal promoter is close to the locus control
region for beta-globin genes
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Evolutionary approaches to predicting and
analyzing regulatory regions

• Sequence comparison alone will not detect all
  regulatory regions
• Need comprehensive protein-binding data
• Comparative genomics can help interpret the
  binding data
• Aspects of regulation of some functional groups
  are clade-specific
• Depth of conservation may correlate with certain
  types of function
• Strong constraint on basal mechanisms?
• Lineage-specific fine tuning?
• A majority of sites occupied by GATA-1 in G1E-ER
  cells have some function other than enhancement
  (by our assays)
• Incorporation of pattern and composition
  information along with with conservation can lead
  to effective discrimination of functional classes
  (regulatory potential).

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Many thanks
PSU Database crew Belinda Giardine, Cathy
Riemer, Yi Zhang, Anton Nekrutenko
BYong Cheng, Ross, Yuepin Zhou, David
King FYing Zhang, Joel Martin, Christine Dorman,
Hao Wang
RP scores and other bioinformatic
input Francesca Chiaromonte, James Taylor, Shan
Yang, Diana Kolbe, Laura Elnitski
Alignments, chains, nets, browsers, ideas, Webb
Miller, Jim Kent, David Haussler
Funding from NIDDK, NHGRI, Huck Institutes of
Life Sciences at PSU
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Categories of Tested DNA Segments
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Regulatory potential (RP) to distinguish
functional classes
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Examples of validated preCRMs
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ChIP-chip hits for GATA-1 occupancy
Technical replicates of ChIP-chip with antibody
against GATA1-ER
Mpeak
TAMALPAIS
275 hits in both
276 hits in both
216
60
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321 total ChIP-chip hits
19 ChIP-chip hits were tested by qPCR 13 were
validated 70