Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G 0 and early G 1 , but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid-to late G 1 phase, when RB becomes phosphorylated by cyclin D-and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence.The retinoblastoma protein RB is a highly abundant tumor suppressor that can bind and regulate a variety of transcription factors (reviewed in Refs. 1-4). One example that has been added recently to the growing list of RB-binding proteins is the RNA polymerase (pol) 1 III-specific factor TFIIIB (5, 6). Recombinant RB was shown to bind to TFIIIB in vitro and repress its activity (5, 6). Furthermore, coimmunoprecipitation and cofractionation experiments demonstrated a stable association between endogenous cellular RB and TFIIIB (6). The functional significance of this interaction was shown in studies of RB knockout mice, since primary fibroblasts from RbϪ/Ϫ mice display elevated TFIIIB activity relative to fibroblasts derived from their wild-type siblings (6). These results establish TFIIIB as a bona fide target for repression by RB. Similar approaches have shown that TFIIIB is also bound and repressed by the RB-related proteins p107 and p130 (7).TFIIIB is required for the expression of all pol III templates (reviewed in Refs. 8 and 9). It serves to recruit the polymerase to a promoter and position it over the transcription start site (10). By interacting with this general factor, RB appears able to regulate the expression of all pol III-transcribed genes, including tRNA, 5 S rRNA, U6 small nuclear RNA, VA1, and Alu genes (5, 6, 11). Since a high rate of tRNA and rRNA synthesis is required to sustain rapid growth, it has been speculated that the inhibition of pol III transcription may contribute to the growth suppression capacity of RB (12)(13)(14).RB function is regulated by cyclin-dependent kinases (reviewed in Refs. 2 and 3 and Ref. 15). The cyclin D-dependent kinases CDK4 and CDK6 phosphorylate RB partially and the process is completed by cyclin E-CDK2 (16, 17). The action of cyclin E-CDK2 appears to depend on prior phosphorylation by the cyclin D-dependent kinases (17). At least 10 serine and threonine residues can become phosp...

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb ؊/؊ p130 ؊/؊ fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

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S U M M A R Y Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber typerestricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.

RNA polymerase III transcription is down-regulated when F9 embryonal carcinoma cells differentiate into parietal endoderm. This reflects a decrease in the activity of TFIIIB, a multisubunit complex that is required for all class III gene expression. Two essential components of TFIIIB are the TATA-binding protein (TBP) and an associated polypeptide called BRF that is specific to this complex. The abundance of both TBP and BRF decreases during F9 cell differentiation. Whereas the amount of TBP assembled into TFIIIB is down-regulated, this is not the case for all TBP-containing complexes. BRF levels show a more dramatic decline that appears sufficient to account for the overall change in transcriptional activity. Developmental regulation of a specific class of genes may therefore be achieved through changes in the availability of a TBP-associated factor.The rapidly changing patterns of gene expression required for early development demand tight transcriptional control. In situ hybridization has revealed that RNA polymerase (pol) 1 III transcription is regulated strongly during mouse embryogenesis (1). After fertilization, the steady-state levels of pol III transcripts increase severalfold, and labeling is seen in the pronuclei and polar bodies, consistent with de novo transcription (1). Indeed, microinjection experiments using single cell embryos have demonstrated that the pol III machinery is active even before cell cleavage (2). The blastomeres of two-and four-cell morulae and of eight-cell blastocysts are heavily labeled (1). Hybridization decreases in the trophectoderm cells of expanding blastocysts but remains strong in the inner cell mass (1). By the late primitive streak stage, high expression is restricted to the ectoderm and mesoderm but has decreased substantially in the embryonic and extra-embryonic endoderm (1).The decrease in pol III activity during differentiation into endoderm can be reproduced accurately using embryonal carcinoma (EC) cells, which mimic events within the early embryo (3-5). For example, pol III transcription decreases substantially both in vitro and in intact nuclei when F9 EC cells differentiate into parietal endoderm (PE) (4, 5). Crude fractions containing the general pol III factor TFIIIB are sufficient to restore expression in PE cell extracts to undifferentiated levels (4). This suggested that the down-regulation of pol III during PE formation is achieved by a specific decrease in the availability of active TFIIIB (4). However, the molecular details of this event remain to be determined, since TFIIIB was largely uncharacterized at the time of the previous study.It has since become clear that TFIIIB is a multisubunit complex that contains the TATA-binding protein TBP and at least two TBP-associated factors (TAFs) (reviewed in .One of these TAFs is related in both structure and function to the general pol II factor TFIIB (9 -14). This TAF has been variously named TDS4, PCF4, BRF, and TFIIIB90, but we shall refer to it as BRF, an acronym for TFIIB-related factor. Although yeast TF...