FIG. 4.

SET1A and ASH2 bind to the Myc and BRCA1 promoters and the H19 DMR, and shRNA knockdown of SET1A decreases both H3K4 dimethylation and gene expression. (A) SET1A and ASH2 bind to the promoter regions of Myc and BRCA1 as well as the H19 DMR. HCT116 cells were harvested and subjected to IP with either an anti-BORIS, an anti-SET1A (Santa Cruz, Inc.), or an anti-ASH2 (Santa Cruz, Inc.) antibody. DNA was eluted and purified before analysis with primers to BRCA1, Myc, or the H19 DMR. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. (B) HCT116 cells were transfected with either vector only (Cont) or a SET1A shRNA and then harvested, separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and immunoblotted using an anti-SET1A antibody (Bethyl Laboratories). (C and D) shRNA knockdown of SET1A decreases SET1A and ASH2, but not BORIS or BAT3, binding to the Myc (C) or BRCA1 (D) promoter. HCT116 cells were treated with nonsense (Cont) or SET1A shRNA, and ChIP was done with an anti-BORIS, anti-BAT3, anti-SET1A, or anti-ASH2 antibody followed by QPCR (as described above). (E) shRNA knockdown of SET1A decreases BRCA1, Myc, and H19 gene expression. HCT116 cells were treated with nonsense (Cont) or SET1A shRNA (in duplicate), RNA was isolated, and BRCA1, Myc, and H19 RNA levels were determined by RT-PCR that was done in triplicate. Error bars represent one standard deviation about the arithmetic mean. *, P < 0.05 by t test. (F) shRNA knockdown of SET1A decreases promoter histone H3K4 dimethylation. HCT116 cells were treated with nonsense (Cont) or SET1A shRNA, harvested for ChIP with either an H3K4 or H3K9 antibody, and analyzed by QPCR with primers to either the Myc or BRCA1 promoter. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. Statistical significance was established by Student's t test. *, P < 0.05.