FIG. 6.

NAP1, Alien, and VDR are corecruited in vivo to the vitamin D3 responsive chromatin site. ChIP and re-ChIP experiments were performed on the CYP24 promoter known to be repressed by VDR in the absence of ligand and activated in the presence of 1,25(OH)₂D₃. MCF-7 cells were transfected with the expression plasmid encoding myc-NAP1 and were untreated or treated with hormone for 60 min prior to ChIP experiments. The upper panel shows the recruitment to the VDREs with a direct repeat spaced by 3 nucleotides whereas the lower control panel shows PCR results from the same material amplifying an upstream chromosomal location of the CYP24 promoter lacking VDREs. (A) ChIP and re-ChIP experiments were performed with the anti-Alien antibody for the first immunoprecipitation. These immunoprecipitates were subjected to a second immunoprecipitation with the antibodies against the indicated factors. Anti-myc antibody was used to precipitate myc-tagged NAP1. As a specificity control, inactivated anti-Alien antibody or IgG was used. (B) The same experimental setup as in panel A was used except that the anti-VDR antibody was employed in the first immunoprecipitation. α, anti.