HIRA and CABIN1 regulate overlapping sets of genes. (A) HeLa cells were nucleofected with siRNAs to HIRA, CABIN1, or the nontargeting (NTG) control as indicated. Three independent nucleofections were preformed with Smartpool siRNAs to HIRA or CABIN1. Lysates were Western blotted to detect HIRA, CABIN1, and β-actin as indicated. (B) Pie charts showing significantly upregulated and downregulated probes after HIRA or CABIN1 knockdown. Green indicates upregulated probes, and red indicates downregulated probes. (C) qRT-PCR analysis to confirm expression changes detected by microarray after siHIRA knockdown. Fold change is calculated by dividing normalized (to housekeeping gene) expression in siHIRA cells by normalized (to housekeeping gene) expression in siNTG cells. *, P > 0.05. P < 0.05 for the other 9 genes. Values are means ± SEM of four independent experiments. (D) Table showing overlap of changing probes after HIRA and CABIN1 knockdown. (E) Heat maps showing hierarchical clustering of genes whose expression changes after three independent siHIRA or siCABIN1 knockdowns, compared to three independent siNTG nucleofections. Green indicates upregulated probes, and red indicates downregulated probes. (F) Sonicated chromatin from HeLa cells was immunoprecipitated with antibodies to GFP or HIRA, and the indicated target genes were detected by qPCR. Expression of HPD and MALL increased on HIRA knockdown, while expression of all others decreased. Genes were selected at random from a list of genes with a fold change (FC) of >1.2-fold and P < 0.05 from siHIRA knockdown cells. Values are means ± SEM of three independent ChIP experiments. P < 0.05 for all comparisons.