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P300 Marks Active Enhancers

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.

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P300 Marks Active Enhancers

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  1. P300 Marks Active Enhancers Ruijuan Li Chao He Rui Fu

  2. Main contents • Background and conclusion • Experimental approaches • Data processing • Summary and discussion

  3. Background • Problems • Enhancers • Previous researches • Existing work • Authors introduction • Research interests

  4. Problems • Enhancers prediction • Accurate • When and where enhancers are active in vivo

  5. 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 enhancer-binding protein • P300 has been showed to be associated with enhancers

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Experimental approaches ChIP-seq: map p300 binding sequences in vivo Transgenic mouse enhancer assay: test the activity of predicted p300 peaks

  11. Workflow of ChIP-seq http://en.wikipedia.org/wiki/Chip-Sequencing

  12. ChIP-seq to predict putative enhancer sites

  13. Comparison with previous method

  14. ChIP-seq to predict putative enhancer sites

  15. Workflow oftransgenic mouse enhancer assay

  16. Examples of successful prediction

  17. Transgenic mouse enhancer assay • Advantage accurate • Disadvantage effect of endogenous regulatory elements

  18. Data processing • Peak Calling to identify p300 binding sites • Validate p300 binding sites are active enhancers

  19. 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 computationalbiology

  20. Validation Evidence1: Transgenic Experiment Validated

  21. Validation Evidence2: Functional Elements are more likely to be Conserved

  22. Validation Evidence3: Active enhancer are nearer to active gene

  23. 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.

  24. 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

  25. Nowadays • More marks: H3K4me1, H3K27ac • Enhancers are transcribing bidirectional smRNA • Locate enhancers target promoters by new experiments

  26. Thank you

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