has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.The retinoblastoma susceptibility gene encodes the important tumor suppressor retinoblastoma protein (RB) (12,15,37,57). Inactivating mutations in this gene are found in many human cancers, including retinoblastomas, many sarcomas, and bladder and small-cell lung carcinomas (12,15,37,57). In a large proportion of other human malignancies the Rb gene is of the wild type, but its function is disrupted. For example, the cyclin-dependent kinases that switch off RB are hyperactive in many tumors (12,15,37,57). Indeed, it has been suggested that the regulatory pathway involving RB may be compromised in all human malignancies (57). It is therefore of considerable importance to obtain a clear understanding of the mechanisms used by RB to influence cellular activity.RB is a highly abundant protein that has been shown to bind and regulate a variety of transcription factors (12,15,37,48). The best-characterized example is the factor E2F, which controls several genes that are transcribed by RNA polymerase (Pol) II (11,14). Indeed, RB was thought for some time to control only Pol II-transcribed genes. However, recent advances have demonstrated that RB can also regulate transcription by Pols I and III (7,58,62). Pol I synthesizes large rRNA, whereas Pol III synthesizes a variety of small stable RNAs, including 5S rRNA and tRNA; together Pols I and III can be responsible for up to 80% of all nuclear transcription (39).Experiments using knockout mice revealed a major role for endogenous RB in regulating Pol III. Primary fibroblasts from Rb Ϫ/Ϫ mice were found to have a fivefold higher Pol III transcriptional activity than equivalent cells from wild-type mice, when assayed in vitro or in vivo (28, 62). Furthermore, overexpression of RB can inhibit Pol III transcription in transfected cells or in a system reconstituted with partially purified factors (8,28,62). This repression involves binding of RB to the Pol III-specific factor TFIIIB (8, 28). TFIIIB is a multisubunit complex which contains the TATA-binding protein (TBP), a TBP-associated factor (TAF) called BRF, and at least one other esse...
