The Retinoblastoma Tumor Suppressor Protein (Rb) is a critical regulator of cellular proliferation. In the canonical model, Rb regulates the G1-to-S phase transition by repressing RNA polymerase II transcription of genes necessary for DNA replication and cell cycle progression. Rb also represses RNA polymerase III-transcribed genes, including those of the U6 snRNA family; however, the contribution of this regulatory pathway to cellular physiology is not well understood. Saos2-tet-Rb osteosarcoma cells harboring an inducible Rb transgene exhibited diminished U6 snRNA levels in early G1 upon RB expression. Interestingly, Rb occupancy of the RNU6-1 locus was distributed around a promoter proximal nucleosome, suggesting that chromatin states are altered during Rb repression. Indeed, HDAC1 and HDAC2 histone deacetylases associated with the endogenous RNU6-1 locus in an Rb-dependent manner. Consistently, histone deacetylation was important for Rb repression of U6-1 in vitro transcription from chromatin templates but not templates lacking histones. Rb also enhanced DNMT1 and DNMT3a DNA methyltransferase recruitment to the RNU6-1 locus, although methylation was not required for repression. These data show that Rb represses RNA polymerase III transcription through the cell cycle by altering the chromatin architecture of the U6 promoter through recruitment of histone deacetylases and chromatin remodeling complexes.
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