Resynthesizing 316 pluripotency control regions (PluCRs). (A) Regions identified by chromatin immunoprecipitation (ChIP) as binding sites for POU5F1 (Oct4), SOX2, and NANOG were resynthesized as an oligonucleotide pool. The pool tiles through the regions, resulting in a population of 45,793 overlapping oligonucleotides that can be amplified by a universal primer pair. The P32 radiolabeled oligonucleotide pool was incubated in untreated J1 embryonic cell extract and in extract from RA-treated J1 cells and separated into a bound and unbound fraction by two methods. Ligands for specific factors were separated by coimmunoprecipitation. Ligands for unknown cellular DNA binding proteins were identified by their retention in nitrocellulose filter (see below). (B) Morphological changes associated with RA differentiation are recorded in the left panels. Pluripotency markers POU5F1, SOX2, and NANOG were assayed by Western before (lane 1) and after (lane 2) RA treatment.
