Recruitment of the mSin3A-HDAC complex to the Nanog promoter
during ES cell proliferation. A, qPCR analysis of ES cells in the
presence of 5 μm retinoic acid (RA) showing a decrease
in pluripotency markers Oct4, Nanog, and Sox2 and
an increase in lineage markers Hand1, HoxA5, and
Nestin. B, Western blot showing levels of Oct4, Nanog, Sox2,
HDAC1, HDAC2, and mSin3A upon RA treatment (5 μm). Tubulin was
used as a loading control. C, schematic of the proximal
Nanog promoter showing positions of Oct4-Sox2 and p53 binding sites
relative to the transcription start site. Arrows denote location of
primers used for chromatin immunoprecipitation (ChIP) relative to the
transcription start site.D and E, ChIP assays were performed
for histone modifications, transcription factors, and deacetylase complex
members as indicated using protein extracts of proliferating and 5-day
RA-treated ES cells, with primers amplifying the Oct4-Sox2 binding site of the
Nanog promoter. Values are expressed as -fold enrichment relative to
IgG control ChIP. Results shown are the average of three independent PCR
reactions. F, ChIP from proliferating and 5-day RA-treated ES cells
using antibodies to mSin3A with primers amplifying the p53 binding site of the
Nanog promoter.