High-throughput biochemical analysis of in vivo location data reveals novel distinct classes of POU5F1(Oct4)/DNA complexes.
The transcription factor POU5F1 is a key regulator of embryonic stem (ES) cell pluripotency and a known oncoprotein. We have developed a novel high-throughput binding assay called MEGAshift (microarray evaluation of genomic aptamers by shift) that we use to pinpoint the exact location, affinity, and stoichiometry of the DNA-protein complexes identified by chromatin immunoprecipitation studies. We consider all genomic regions identified as POU5F1-ChIP-enriched in both human and mouse. Compared with regions that are ChIP-enriched in a single species, we find these regions more likely to be near actively transcribed genes in ES cells. We resynthesize these genomic regions as a pool of tiled 35-mers. This oligonucleotide pool is then assayed for binding to recombinant POU5F1 by gel shift. The degree of binding for each oligonucleotide is accurately measured on a custom oligonucleotide microarray. We explore the relationship between experimentally determined and computationally predicted binding strengths, find many novel functional combinations of POU5F1 half sites, and demonstrate efficient motif discovery by incorporating binding information into a motif finding algorithm. In addition to further refining location studies for transcription factors, this method holds promise for the high-throughput screening of promoters, SNP regions, and epigenetic modifications for factor binding.
Tantin
Dean
D
Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.
Gemberling
Matthew
M
Callister
Catherine
C
Fairbrother
William G
WG
Fairbrother
William
W
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D053595:Embryonic Stem Cells / Q000378:metabolism; D023281:Genomics; D006801:Humans; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D050436:Regulatory Elements, Transcriptional
D050814:Octamer Transcription Factor-3; D015345:Oligonucleotide Probes; C494173:POU5F1 protein, human; D004247:DNA