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Nat Methods. 2014 Feb;11(2):203-9. doi: 10.1038/nmeth.2766. Epub 2013 Dec 15.

High-resolution mapping of transcription factor binding sites on native chromatin.

Author information

1
1] Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [2] Medical Scientist Training Program, University of Washington School of Medicine, Seattle, Washington, USA. [3] Molecular & Cellular Biology Graduate Program, University of Washington, Seattle, Washington, USA.
2
1] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. [2] Centre National de la Recherche Scientifique UMR 218 and Institut Curie, Centre de Recherche, Paris, France.
3
Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
4
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
5
1] Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [2] Howard Hughes Medical Institute, Seattle, Washington, USA.

Abstract

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

PMID:
24336359
PMCID:
PMC3929178
DOI:
10.1038/nmeth.2766
[Indexed for MEDLINE]
Free PMC Article
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