FIG. 2.
NF-Y is required for transcription of the ODF gene and recruitment of RNAPII to the promoter. (A) Chromatin immunoprecipitation analysis of NF-Y binding to the mouse ODF gene in vivo. ST-2 cells were exposed to 30 nM vitamin D3 (lane 3) for 24 h and were cross-linked with 1% formaldehyde. Nuclear extracts were immunoprecipitated with anti-NF-YB antibody (lanes 2 and 3) or preimmune goat immunoglobulin G (IgG) (lane 1). Eluates were subjected to Western blot (WB) analysis with anti-NF-YB antibody or PCR analysis with appropriate primers. PCR products were separated on 1.5% agarose gels and were stained with EtBr. (B) ST-2 cells were transfected with 2 nM siRNA for the NF-YA mRNA for 48 h. Transfectants were treated for 0, 12, 24, or 48 h with 30 nM vitamin D3. The upper panel (lanes 1 to 8) shows Western blot analysis for NF-YA or RNAPII using 10 μg of total cell lysates. The middle panel (lanes 9 to 16) shows RT-PCR analysis for ODF and GAPDH expression. The bottom panel (lanes 17 to 24) shows ChIP assays for the ODF core promoter region (−105 to +55) using indicated antibodies. (C) Quantitative PCR analysis for ChIP materials used in panel B. Results shown are the averages (means ± standard errors of the means) of three independent experiments. IP, immunoprecipitation. AcH4, acetylated histone H4.