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DNA-protein interactions are crucial in directing cellular activities. Transcription Factors (TFs) are proteins that regulate gene expression and play a major role in maintaining a pluripotent, or undifferentiated, cell state in Human Embryonic Stem Cells (hESCs). The NANOG TF is known to coordinate gene regulation critical for pluripotency and its binding sites have been well-studied. However, its temporal activity has yet to be thoroughly explored. A new assay termed Repli-ChIP was developed to quantify TF-bound DNA during DNA replication. Two hESC cultures were treated with a synthetic nucleoside called BrdU, which labels nascent DNA. In one culture, immunoprecipitation (IP) using anti-NANOG antibodies was used to extract NANOG TFs at 1-hr post-BrdU treatment, termed the “early” or “0-hr” time point. In the second culture, BrdU treatment was stopped at 1 hour and NANOG was immunoprecipitated after an additional 16 hours of incubation in new media, termed the “late” or “16-hr” time point. Next, IP with anti-BrdU antibodies was used to isolate BrdU-labeled DNA. Quantitative PCR measured the amount of nascent DNA at the LHFP gene, which was used to infer the level of NANOG binding. Results imply decreased NANOG binding and activity late in replication. Findings for NANOG support the validity of Repli-ChIP in determining temporal TF binding changes.
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