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 Zhang Cold Spring Harbor, March 2007 What is conservation good for??

Ideal cases for interpretation by comparative genomics Neutral DNA Similarity 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

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

Functional classes show distinctive trends in phylogenetic depth of conservation

Genes likely regulated by clade-specific pTRRs are enriched for distinctive functions Millions of years 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 cycle Transcriptional regulation Ion transport

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

High RP plus conserved consensus motif is a good predictor of CRMs around GATA-1 regulated genes Genome Research 16:1480 (2006)

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

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

preCRMs with conserved consensus GATA-1 BS tend to be active on transfected plasmids

DNA segments with positive RP and a GATA-1 binding motif validate as enhancers at a good rate RP consensus motifTested Validated Success Positiveconserved % Positivemouse % Negativeconserved % Negativenone %

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: (~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)

Signals in known occupied sites in Hbb LCR 1) Cluster of high signals 2) “hill” shape of the signals HS1 HS2 HS3

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

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.

Distribution of ChIP-chip hits on 70Mb of mouse chr7 Yong Cheng, Yuepin Zhou and Christine Dorman

GATA-1 occupied sites by ChIP-chipNo 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

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

Validated ChIP hit, enhancer, deep conservation

Validated ChIP hit, enhancer, limited conservation

ChIP-chip hit, enhancer, rodent specific

Test of neutrality using polymorphism and divergence data

A promoter distal to the beta-like globin genes has a signal for recent purifying selection

The distal promoter is close to the locus control region for beta-globin genes

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).

Many thanks … B:Yong Cheng, Ross, Yuepin Zhou, David King F: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

Categories of Tested DNA Segments

Regulatory potential (RP) to distinguish functional classes

Examples of validated preCRMs

ChIP-chip hits for GATA-1 occupancy MpeakTAMALPAIS 275 hits in both276 hits in both 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%