Distinct Mechanisms for Repression of RNA Polymerase III Transcription by the Retinoblastoma Tumor Suppressor Protein

Abstract

The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAP C and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.The retinoblastoma (RB) protein controls the cell cycle, differentiation, apoptosis, and general growth. Typically, RB functions to repress RNA polymerase II transcription of genes that act at control points in these key cellular processes (10,11,14). The classical understanding of RB function is exemplified by its ability to control the cell cycle (reviewed in reference 45). For example, RB can target genes that are required to enter into the S phase of the cell cycle and whose promoters contain binding sites for the E2F family of transcriptional activator proteins (4,17,31,46). RB can directly block the transactivation function of E2F (34) or alter chromatin structure to repress transcription by recruiting coregulatory proteins, such as histone deacetylases (HDACs) (2, 25, 26), ATP-dependent chromatin remodeling complexes (39,43,53), and histone methyltransferases (32).RB is related to two other pocket domain proteins, p107 and p130, that have also been shown to repress E2F target genes and inhibit cell growth when overexpressed (reviewed in references 12 and 29). The different pocket domain proteins are utilized at distinct phases of the cell cycle to control transcription of E2F target genes (27, 42), suggesting that RB, p107, and p130 have specialized nonredundant functions for controlling gene expression. Interestingly, the minimal regions of RB required for RNA polymerase II transcriptional repression are different from those essential for tumor suppression. RB contains 928 amino acids and can be functionally divided into at least three regions: the N-terminal region between amino acids 1 and 378, ...

PTEN Represses RNA Polymerase III-Dependent Transcription by Targeting the TFIIIB Complex

“…Consistent with the idea that a high translational capacity is necessary for the rapid growth and proliferation of tumor cells, RNA Pol III transcription products are elevated in transformed and tumor cells (3,4,15,31,49). Accordingly, the tumor suppressors p53 (6) and Rb (19,35) repress, while oncogenic c-myc (15) induces, RNA Pol III-dependent transcription. The ability of these proteins to deregulate RNA Pol III-dependent transcription occurs through their capacity to directly associate with the TFIIIB complex and modify its function.…”

“…The function of PTEN as a lipid phosphatase that inhibits the PI3K signal transduction pathway distinguishes it from the tumor suppressors p53 and Rb, which function as transcription factors to directly regulate RNA Pol III-dependent transcrip- (19,35), while p53 interacts with TBP (6), resulting in a dysfunctional TFIIIB complex that is unable to associate with TFIIIC or RNA Pol III (6,19,35). In contrast, PTEN indirectly targets TFIIIB, inducing the dissociation between Brf1 and TBP and preventing the formation of a functional TFIIIB complex.…”

“…PTEN targets the TFIIIB complex to repress the formation of TBP-Brf1 complexes. To elucidate the mechanism for PTEN-mediated repression of RNA Pol III transcription, we examined potential changes in TFIIIB, which was previously shown to be targeted by the tumor suppressors Rb (19,35) and p53 (6). Neither a reduced expression of PTEN in LN18 cells nor the expression of PTEN in U87 cells affected the expression of the TFIIIB components TBP, Brf1, and Bdp1 or the TFIIIC subunit TFIIIC 102 (Fig.…”

Co-expression of multiple subunits enables recombinant SNAPC assembly and function for transcription by human RNA polymerases II and III

“…In vitro transcription assays of human U1 and U6 snRNA genes were performed for 1 h at 30 °C using the depleted extracts as described previously [6,[27][28][29]. For the U6 transcription experiment shown in Fig.…”

The JNKs differentially regulate RNA polymerase III transcription by coordinately modulating the expression of all TFIIIB subunits

“…The tumor suppressors RB and p53 bind directly to TFIIIB and prevent its recruitment to promoters (8)(9)(10). PTEN negatively regulates Brf1 phosphorylation and its ability to associate with TBP to form functional TFIIIB complexes (7).…”