Regulation of the human U6 small nuclear RNA transcription by the retinoblastoma tumor suppressor protein

The Retinoblastoma tumor suppressor (RB) protein restricts unregulated cell division by repressing transcription of genes whose products are essential for cell growth and division. RB represses a subset of RNA Polymerase (Pol) II-transcribed genes that contain E2F binding sites and are important for progression into S-phase. RB also acts as a general repressor of Pol I and Pol III transcription. Considering that Pol I and Pol III transcribed products are essential for cell growth and division, repression of transcription by Pol I and Pol III can be an important aspect of the growth-suppressive function of RB. The growth inhibitory function of RB is linked to its anti-tumorigenic potential, and it is likely that understanding RB repression of Pol I and Pol III transcription can elucidate some important aspects of the RB tumor suppression mechanism. My study examines the mechanism for RB repression of U6 transcription by Pol III.;Sequence comparison among the nine U6 copies in humans revealed that all the functional U6 copies are enriched in CpG dinucleotides at the promoter regions compared to the non-functional copies, and as the CpG sequence is the primary target for methylation in humans, this indicated a potential involvement of DNA methylation in regulation of U6 transcription. Existing evidence indicating a repressive role for DNA methylation on transcription, led to the hypothesis that DNA methylation contributes to U6 repression. In support, in vitro transcription from pre-methylated U6 templates demonstrated that DNA methylation has an inhibitory effect on U6 transcription.;Earlier studies indicated that RB interacts with DNA methyltransferase (DNMT) 1 and DNMT1 activity contributes to RB repression of E2F-transactivated Pol II transcription. Consistently, RB was found to direct recruitment of DNA methylation and DNA methyltransferases 1 and 3A to the U6 promoter during repression of Pol III transcription. Also, siRNA-mediated depletion of DNMT1, 3A and 3B in RB positive cells resulted in enhanced U6 transcription, suggesting a repressive role for DNA methyltransferases in U6 transcription. The results presented here implicate RB-directed promoter DNA methylation as an important aspect of the mechanism for RB-mediated repression of human U6 transcription.