P300 Marks Active Enhancers
Ruijuan Li
Chao He
Rui Fu
Main contents
• Background and conclusion
• Experimental approaches
• Data processing
• Summary and discussion
Background
• Problems
Enhancers
• Previous researches
Existing work
• Authors introduction
Research interests
Problems
• Enhancers prediction
 Accurate
 When 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 approach
ChIP-seq with an antibody specific for an enhancerbinding 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). Genomewide 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
comparative
genomic
method
ChIP-seq
method
ChIP-seq
to predict putative enhancer sites
Workflow of
transgenic mouse enhancer assay
Selection of
86 candidate regions for testing
Cloning human genomic sequences
into Hsp68-LacZ reporter vector
Pronuclear injection and staining at E11.5
Examples of successful prediction
Transgenic mouse enhancer assay
• Advantage
accurate
• Disadvantage
effect of endogenous regulatory elements
Data processing
• Peak Calling to identify p300 binding sites
• Validate p300 binding sites are active
enhancers
Peak Calling
1. 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
Validation
Evidence2: Functional Elements are more likely to be Conserved
Validation
Evidence3: Active enhancer are nearer to active gene
Summary
•
p300 binding sites are probably to be celltype specific enhancer.
•
Most p300-bound regions are conserved
•
p300 binding sites are significant nearer to
expressed genes than random sites.
Improvement
• Peak Calling
Arbitrary to extend the reads to 300bp
No control to test peak quality
• Active enhancer prediction
Sensitivity is low
Still some errors
Nowadays
• More marks: H3K4me1, H3K27ac
• Enhancers are transcribing bidirectional
smRNA
• Locate enhancers target promoters by new
experiments
Thank you