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![Page 1: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/1.jpg)
Comparative genomics, ChIP-chip and transfections to find
cis-regulatory modules
Penn State University, Center for Comparative Genomics and Bioinformatics: Webb Miller, Francesca Chiaromonte, Ross Hardison
Children’s Hospital of Philadelphia: Mitch Weiss, Lou Dore
NimbleGen: Roland Green, Xinmin ZhangCold Spring Harbor, March 2007
What is conservation good for??
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Ideal cases for interpretation by comparative genomics
Neutral DNASimilarity
Human vs mouse
Position along chromosome
DNA segments with a function common to divergent species.
DNA segments in which change is beneficial to at least one of the two species.
Negative selection(purifying)
P (not neutral)Neutral DNA
Similarity
Positive selection(adaptive)
Neutral DNA
Human vs rhesus
![Page 3: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/3.jpg)
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
![Page 4: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/4.jpg)
Functional classes show distinctive trends in phylogenetic depth of
conservation
![Page 5: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/5.jpg)
Genes likely regulated by clade-specific pTRRs are enriched for
distinctive functions
310
450
91
173
Millions ofyears
Percentage of pTRRs that align no further than:Primates: 3%
Eutherians: 71%
Marsupials: 21%
Tetrapods: 4%
Vertebrates: 1%
David King
Enriched GO categories
q-value for FDR
Immune response
Protease inhibition
Mitosis and cell cycleTranscriptional regulation
0.0006
0.0005
0.0005
0.004
0.012Ion transport
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Regulatory potential (RP) captures pattern, composition and constraint
in alignments
Genome Research 16:1585 (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
PSU’s Galaxy
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High RP plus conserved consensus motif is a good predictor of CRMs around
GATA-1 regulated genes
Genome Research 16:1480 (2006)
![Page 8: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/8.jpg)
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-1Rylski et al., Mol Cell Biol. 2003
![Page 9: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/9.jpg)
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 SuccessPositive 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
1. Non-overlapping tiling array with 50bp probe and 100bp resolution (NimbleGen)
2. Cover range Mouse chr7:57225996-123812258 (~70Mbp)3. Antibody against the ER portion of
GATA-1-ER protein in rescued G1E-ER4 cells
50 50
100
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
1) Cluster of high signals2) “hill” shape of the signals
HS1 HS2 HS3
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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
![Page 15: Comparative genomics, ChIP-chip and transfections to find cis-regulatory modules Penn State University, Center for Comparative Genomics and Bioinformatics:](https://reader036.fdocuments.in/reader036/viewer/2022081506/56649d9d5503460f94a867fd/html5/thumbnails/15.jpg)
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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0
1
2
3
4
GHP181GHP10GHP205GHP7GHP182GHP309
GHP1GHP186GHP204
GHP4GHP314GHP172GHP167GHP74GHP193GHP25GHP27GHP9
GHP170GHP18GHP16GHP243GHP15GHP28GHP17GHP31GHP11GHP198GHP169GHP14GHP173GHP29GHP199GHP12GHP3GHP2GHP24GHP164GHP13GHP30GHP19GHP26GHP161GHP191GHP197GHP183GHP184GHP22GHP6GHP23GHP206GHP194GHP202GHP0GHP200
GHP8GHP185GHP118GHP20 GHN037GHN534GHN006GHN133GHN322GHN478GHN159
YC3
GHN240GHN391GHN419GHN213
Mean fold change
GATA-1 occupied sites by ChIP-chip No GATA-1
21 out of 59 ChIP-chip hits increase activity of HBGpr-Luc in K562 cells.
36% of ChIP-chip hits act as enhancers in K562 cells
14.55.7
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0
1
2
3
4
5
GHP7GHP172GHP198GHP10GHP25GHP14GHP15GHP181GHP13GHP186GHP170
GHP1GHP182GHP16GHP169GHP184GHP18GHP9
GHP164GHP24GHP173GHP4
GHP197GHP193GHP167GHP30GHP183GHP185GHP23GHP26GHP2GHP29GHP199GHP28GHP161GHP31GHP191GHP194GHP3GHP12GHP200GHP206
GHP0GHP11GHP27GHP22GHP8GHP118GHP21GHP20 GHN534GHN037GHN391GHN159GHN478GHN240GHN006GHN419GHN133GHN322GHN213
Mean fold change
GATA-1 occupied sites by ChIP-chip No GATA-1
15 out of 50 ChIP-chip hits increase activity of HBGpr-Luc in MEL cells.
30% of ChIP-chip hits act as enhancers in MEL cells
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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
0
1
2
3
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K562_1 K562_2 MEL
Fold change over parent
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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 …
B:Yong Cheng, Ross, Yuepin Zhou, David KingF:Ying Zhang, Joel Martin, Christine Dorman, Hao Wang
PSU Database crew: Belinda Giardine, Cathy Riemer, Yi Zhang, Anton Nekrutenko
Alignments, chains, nets, browsers, ideas, …Webb Miller, Jim Kent, David Haussler
RP scores and other bioinformatic input:Francesca Chiaromonte, James Taylor, Shan Yang, Diana Kolbe, Laura Elnitski
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
Mpeak TAMALPAIS
275 hits in both 276 hits in both216 6059
321 total ChIP-chip hits
Technical replicates of ChIP-chip with antibody against GATA1-ER
19 ChIP-chip hits were tested by qPCR:13 were validated: ~70%