Figure 2

Preferential association of Mybbp1a with chromatin regulatory region of the methylated, inactive rDNA. (A) Schematic representation of a single human rDNA repeat. The gene structure and locations of the amplicons (black lines, in kb relative to the transcription start site) are shown. (B) & (C) ChIP analysis was done to profile Mybbp1a binding on the rDNA gene. Chromatin was prepared from HeLa cells (B) or HeLa cells transfected with empty vector or a Myc-tagged Mybbp1a-encoding construct (C). Enrichment of rDNA fragments precipitated with anti-Mybbp1a (B) or anti-Myc (C) antibody was quantitatively analyzed by real-time PCR using primers denoted in (A). For each amplicon, binding was normalized to the values of either IgG (B) or anti-Myc binding in the control transfection (C). ChIP, followed by end-point PCR analysis of amplicon 0 (A), was performed to confirm the binding of Mybbp1a to rDNA promoter (bottom). Representative results from one of three biological replicates are shown. (D) Schematic representation of the rRNA promoter region amplified in the site-specific methylation analysis and ChIP-chop assays (Primer-F and Primer-R denote respectively the forward and reverse primers) and the locations of HpaII/MspI sites (see Methods). (E) Quantitative analysis and graphical representation of the association of Mybbp1a, PAF49, and H4K20me3 with rDNA promoter. ChIP-chop assay was done as described in the Methods. Extent of methylation in the bound promoter rDNA is assessed by signals derived from the HpaII digestion, and is shown relative to level of unmethylated promoter.