P300 Marks Active Enhancers

Ruijuan Li Chao He Rui Fu

Main contents

• Background and conclusion

• Experimental approaches

• Data processing

• Summary and discussion

Background

• ProblemsEnhancers

• Previous researchesExisting work

• Authors introductionResearch interests

Problems

• Enhancers prediction AccurateWhen and where enhancers are active in vivo

Previous researches• Comparative genome methods

Evolutionary sequence constraint failed to reveal when and where enhancers are active in vivo

Some regulatory elements are not sufficiently conserved to be detectable.

• Conservation-independent approachChIP-seq with an antibody specific for an enhancer-

binding protein• P300 has been showed to be associated with

enhancers

Authors introduction• Axel Visel

– Staff scientist in the Genomics Division, Lawrence Berkeley National Laboratory

– Comparative genomics, sequencing-based chromatin studies (ChIP-seq), and transgenic reporter assays

– Systematic identification and functional characterization of distant-acting enhancers

• Matthew J. Blow– Comparative genomics, RNA editing

• Prabhakar S, Visel A, ..., Pennacchio LA, Rubin EM, Noonan JP (13 authors). Human-specific gain of function in a developmental enhancer. Science 2008

• Visel A, ..., Rubin EM, Pennacchio LA (10 authors). Ultraconservation identifies a small subset of extremely constrained developmental enhancers. Nature Genet 2008

• Rahimov F, Marazita ML, Visel A, ..., Murray JC (23 authors). Disruption of an AP-2alpha binding site in an IRF6 enhancer is associated with cleft lip. Nature Genet 2008

• De Val S, ..., Visel A, ..., Black BL (15 authors). Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. Cell 2008

• Lein ES, Hawrylycz MJ, ..., Visel A, ..., Jones AR (108 authors). Genome-wide atlas of gene expression in the adult mouse brain. Nature 2007

• Pennacchio LA, ..., Visel A, Rubin EM (19 authors). In vivo enhancer analysis of human conserved non-coding sequences. Nature 2006

Corresponding Author• Len A. Pennacchio

• Molecular biologist, Senior staff scientist in the Genomics Division, Lawrence Berkeley National Laboratory

• Head of the Genetic Analysis Program and the Genomic Technologies Program, Joint Genome Institute

• 2007 White House Presidential Early Career Award for Scientists and Engineers (PECASE);

• Contributed to the human genome project with an analysis of human chromosome 16.

• Gene regulation, the genetic basis of differences in body shape between different individuals, conserved sequences in the genome, and connections between junk and heart disease

Conclusion

• P300 binding sites accurately identifies enhancers and their associated activities in vivo.

• The data will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

Experimental approaches

ChIP-seq: map p300 binding sequences in vivo

Transgenic mouse enhancer assay: test the activity of predicted p300 peaks

Workflow of ChIP-seq

http://en.wikipedia.org/wiki/Chip-Sequencing

ChIP-seq to predict putative enhancer sites

Comparison with previous method

ChIP-seq to predict putative enhancer sites

Workflow oftransgenic mouse enhancer assay

Examples of successful prediction

Transgenic mouse enhancer assay

• Advantageaccurate

• Disadvantageeffect of endogenous regulatory elements

Data processing

• Peak Calling to identify p300 binding sites

• Validate p300 binding sites are active enhancers

Peak Calling1. Reads extend to 300bp

2. Identify candidate peak

3. Merge nearer peaks

4. Discard artefact peaks

Dr Wang’s PPT for molecular computational biology

Validation

Evidence1: Transgenic Experiment Validated

ValidationEvidence2: Functional Elements are more likely to be Conserved

ValidationEvidence3: Active enhancer are nearer to active gene

Summary

• p300 binding sites are probably to be cell-type specific enhancer.

• Most p300-bound regions are conserved

• p300 binding sites are significant nearer to expressed genes than random sites.

Improvement

• Peak CallingArbitrary to extend the reads to 300bpNo control to test peak quality

• Active enhancer predictionSensitivity is lowStill some errors

Nowadays

• More marks: H3K4me1, H3K27ac

• Enhancers are transcribing bidirectional smRNA

• Locate enhancers target promoters by new experiments

Thank you