We further focused on the rDNA promoter region and confirmed binding by end point PCR. Based on the distinct nature of rDNA repeats the all or none occurrence of DNA methylation that is directly coupled to the transcriptional status, we next ana lyzed the relationship between Mybbp1a promoter occu pancy and rDNA methylation. To address STI 571 this issue, we performed the ChIP chop assay that allows differentiation between the methylated and unmethylated promoter in the precipitated DNA. In the first step, the input and precipitated DNA were digested with the iso schizomers MspI or HpaII. A region of the rRNA promoter that harbors four HpaII/MspI sites was subsequently amp lified by the specific primers.

Using quantita tive real time PCR, the HpaII resistant PCR products generated from the input and precipitated DNA measures the level of the methylated rRNA promoter, whereas the difference between mock digested and HpaII digested sig nal reflects the level of the rRNA promoters that are unmethylated. Our results revealed that the DNA methy lation levels measured at the promoter was about 60% in the HeLa cells. As controls to the experiment, we also monitored methylation extent of the DNA bound by PAF49 and a known repressive mark H4K20me3. As expected, PAF49 was predominantly associated with the non methylated promoters, correlating with active transcrip tion. Conversely, analysis of the H4K20me3 modification revealed it distribution with methylated DNA, thus correlating with inactive rRNA genes.

Finally, since the rRNA promoter immunoprecipitated by anti Mybbp1a antibody was mostly HpaII resistant, our data suggest that Mybbp1a is mainly associated with the methylated promoter. Together, these findings pinpoint the selective association of Mybbp1a with the inactive rDNA promoter in the chromatin context and correlate well with its inhibitory effect on rRNA expression. Mybbp1a is important for maintaining the epigenetic status of rDNA promoter Having established a potential link of Mybbp1a to the repressed GSK-3 rDNA genes, we next examined whether its suppression of rDNA transcription is mediated through a DNA methylation dependent mechanism. Toward this end, we monitored the local DNA methylation status of rDNA promoter in the control vs. knockdown cells. Equivalent amounts of DNA from these cells were digested either with the HpaII or Msp1. PCR analysis, with primers that specifically amplified the region of rDNA harboring 4 HpaII/MspI sites, revealed that 60% of the rDNA promoters in the con trol HeLa cells were methylated. However, promoter genomic DNA from Mybbp1a knockdown cells were more easily digested by HpaII, which was equivalent to a methylation rate of 36%.