Transcript 1 Distant-Acting Enhancers
Genome-wide Identification of Craniofacial Transcriptional Enhancers
Axel Visel
Scientist, Genomics Division Lawrence Berkeley National Laboratory
Outline
1 Distant-Acting Enhancers
Why are they important?
How can we find them in the genome (and determine their function)?
2 Finding Developmental Face/Palate Enhancers
Data from preliminary ChIP-seq and transgenic mouse studies
3 FaceBase – Contributions and Expectations
Data and reagents Interactions
promoter protein coding Enhancers are Required for Development distant-acting enhancers
wild-type limb enhancer deleted 1 megabase limb enhancer Shh gene human Shh enhancer point mutations mouse embryo
Noncoding Sequences in Human Disease
Meta-Analysis of Genome-Wide Association Studies (GWAS): Distant Enhancers?
60% Linked to Exons 40% Noncoding LD blocks
Of 1,200 disease-associated SNPs, 40% are not linked to any coding gene
Visel/Pennacchio/Rubin 2009 (
Nature
461:199)
How do we find enhancers?
Approach A: Extreme Conservation of Non-coding sequences
mouse fugu inject into fertilized mouse egg PCR amplify clone P LacZ
pHsp68LacZ >500 enhancers identified to date see http://enhancer.lbl.gov
reimplant collect at e11.5
Major limitation: can’t find enhancers active in a particular process, e.g. face development
minimum reproducibility: 3 embryos
How do we find enhancers?
Approach B: ChIP-seq with the enhancer-associated p300 protein microdissection midbrain tissue forebrain tissue limb tissue p300 ChIP-Seq mouse embryo (e11.5) limb
2,400,000 reads
2,100 peaks forebrain
3,600,000 reads
2,400 peaks midbrain
3,500,000 reads
600 peaks
Test in transgenic mouse assay (Nature 457:854, 2009)
p300 ChIP-Seq Predicts in vivo Enhancer Activity
ChIP-seq forebrain midbrain limb
transgenic mouse assay
80%-90% success rate (n>100) 8/8 locations AND activity of enhancers 11/12 11/11
(Nature 457:854, 2009)
Outline
1 Distant-Acting Enhancers
Why are they important?
How can we find them in the genome (and determine their function)?
2 Finding Developmental Face/Palate Enhancers
Data from preliminary ChIP-seq and transgenic mouse studies
3 FaceBase – Contributions and Expectations
Data and reagents Interactions
Enhancers Play a Role in Clefting Disorders enhancer SNP disrupts a single AP-2a binding site x SNP IRF6 gene human cleft lip and palate associated with cleft lip/palate human chr1 virtual section of mouth region enhancer activity in ectoderm of fusing facial prominences Rahimov et al. 2008 (Nature Genetics 40:1341) Jeff Murray Lab, OPT data: David FitzPatrick
ChIP-seq for Craniofacial Enhancer Discovery
Example: Enhancer near known clefting gene MSX1
mx mx mx
Msx1 gene expression in maxillary component of 1 st branchial arch (Mackenzie et al., Development 111:269)
Three-dimensional imaging of enhancer activity Optical Projection Tomography
(Sharpe et al., Science 296:541) OPT of Enhancer Browser embryos:
David FitzPatrick/Harris Morrison, MRC Edinburgh
Three-dimensional imaging of enhancer activity Optical Projection Tomography
(Sharpe et al., Science 296:541) OPT of Enhancer Browser embryos:
David FitzPatrick/Harris Morrison, MRC Edinburgh
In vivo validation of ChIP-seq predictions
OPT scans:
David FitzPatrick/Harris Morrison, MRC Edinburgh
Outline
1 Distant-Acting Enhancers
Why are they important?
How can we find them in the genome (and determine their function)?
2 Finding Developmental Face/Palate Enhancers
Data from preliminary ChIP-seq and transgenic mouse studies
3 FaceBase – Contributions and Expectations
Data and reagents Interactions
Visel Lab – FaceBase Aims
Genome-wide identification of enhancer candidates
p300 ChIP-seq: timepoints (e11.5 – e15.5), better spatial resolution RNA-seq data
large-scale sequence based data Transgenic validation and characterization
test 30 candidate sequences/year in transgenic mice whole-mount photos and OPT data (collaboration with FitzPatrick lab) provide validated vectors as reagents to other FaceBase investigators
image/video/3D data Follow-up of human genetic studies
test risk alleles of clefting-associated craniofacial enhancers in mice
integration of enhancer data with human genetic data
Visel Lab – FaceBase Expectations
Developmental biology and expression imaging groups
Intersect with gene expression data Please approach us with regions of interest!
Human genetics groups
Please approach us with non-coding cleft-associated regions!
Use ChIP-seq and transgenics to search for enhancers Transgenic testing of cleft-associated risk variants
Acknowledgments
Lawrence Berkeley National Lab and DOE Joint Genome Institute Len Pennacchio Eddy Rubin Matt Blow Shyam Prabhakar Mouse Transgenics Malak Shoukry Jennifer Akiyama Veena Afzal Amy Holt Ingrid Plaijzer-Frick Roya Hosseini Collaborators/Contributors: Terri Beaty, Robert Cornell, Michael Dixon, David FitzPatrick, Rulang Jiang, Michael Lovett, Mary Marazita, Jeff Murray, Stephen Murray, Leif Oxburgh, Bing Ren, John Rubenstein, Brian Schutte, Alan Scott, Douglas Spicer
http://enhancer.lbl.gov
Next-Gen Sequencing Tao Zhang Feng Chen Crystal Wright Enhancer Browser Inna Dubchak Simon Minovitsky
NIDCR (FaceBase)
DOE, NHGRI