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