Figure 5

Mybbp1a associates with and stabilizes rDNA promoter occupancy of HDAC1/2. (A) Co-immunoprecipitation of HDAC1 and HDAC2 with endogenous Mybbp1a. Immunoprecipitation (IP) was done with control IgG or antibody against Mybbp1a (M). Immunoprecipiates and lysate input (Inpt, 1/40 of IP) were probed for HDAC1 and HDAC2. (B) & (C) HDAC1 and HDAC2 binding to the rRNA promoter (amplicon 0 in Figure 2A) were examined in the control (−) and Myc-Mybbp1a-overexpressing (+) HeLa cells (B) or the control (−) and Mybbp1a-knockdown (+) HeLa cells (C). Chromatin fragments were prepared from these cells and subjected to ChIP with control, HDAC1, or HDAC2 antibody. Quantitative determination of the bound DNA, carried out with real-time PCR, is shown by the bar graphs. Data presented are normalized to the IgG values, with the ratio for each control group set to 1 (*p < 0.05; **p < 0.01). (D) Myc-Mybbp1a-overexpressing (Myc-Mybbp1a) HeLa cells were treated with solvent (−), deacetylase inhibitor TSA, or DNMT inhibitor 5-AzaC, prior to qRT-PCR analysis of pre-rRNA expression. For bar graphs, data presented are normalized to GAPDH values, with the mean ± SD values from at least three experiments also shown (*p < 0.05).