Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1 KM Hannan 1,3 , RD Hannan 1,4 , SD Smith 1 , LS Jeerson 2 , Mingyue Lun 1 and LI Rothblum* ,1 1 Henry Hood Research Program, Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, Pennsylvania, PA 17822 USA; 2 Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania, PA 17033, USA We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBF- mediated transcription. In the present study, we have examined the mechanism by which Rb represses UBF- dependent rDNA transcription and determined if other Rb-like proteins have similar eects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb. Oncogene (2000) 19, 4988 – 4999. Keywords: retinoblastoma protein; ribosomal RNA; transcription; UBF; DNA-binding Introduction Rb and Rb-like proteins have been implicated in the regulation of transcription of specific genes by RNA polymerase II (Qin et al., 1992; Weinberg 1995; Taya 1997). Recent studies have extended this role, at least for Rb, to the regulation of RNA polymerase I and polymerase III transcription (Shan et al., 1992; Cava- naugh et al., 1995; White et al., 1996; Voit et al., 1997; White 1997; Hannan et al., 2000). Rb is the 110 kDa protein product of the retinoblastoma susceptibility gene which functions as a tumor suppresser and negative growth regulator (Friend et al., 1986; Lee et al., 1987; Qin et al., 1992; Hamel et al., 1992; 1993; Levine, 1993; Claudio et al., 1994; Chen et al., 1996; White 1997; Hannan et al., 2000). Two additional proteins have been classified as Rb-like proteins, p107 and p130 (Pertile, 1995; Weinberg, 1995; Baldi et al., 1996). All three proteins are structurally related as indicated by the presence of a typical Rb A/B pocket (Levine, 1993; Chen et al., 1996). The A/B pocket of Rb is composed of an A domain (amino acids 379 – 572) and B domain (amino acids 646 – 772) separated by a spacer sequence (Knudsen and Wang, 1997; Herwig and Strauss, 1997). The pocket binds a number of viral and cellular proteins each containing an LxCxE motif, such as UBF, hBrm, large T antigen, and E1A (Hamel et al., 1993; Copenhaver et al., 1994; Beckmann et al., 1995; Weinberg 1995; Hannan et al., 2000). Typically, both the A and B regions of the pocket are necessary for these interactions. Thus, they are highly conserved between species and related proteins, unlike the spacer region (Knudsen and Wang, 1997). In addition, to sharing similar structural domains with Rb, the Rb-like proteins, p107 and p130, share some functional characteristics. For example, all three proteins can induce cell cycle arrest when over expressed in the appropriate cell line (Claudio et al., 1994). On the other hand, dierences between these proteins have been reported. For example, p107 has not been found to be mutated in tumors or cell lines as has been reported for Rb (Levine, 1993; Claudio et al., 1994; Chen et al., 1996; LeCouter et al., 1996) and p130 (Helin et al., 1997). Although Rb has been shown to regulate the transcription of genes transcribed by all three classes of DNA dependent RNA polymerases (Cavanaugh et al., 1995; White et al., 1996; Chu et al., 1997; Taya, 1997; White, 1997), p130 and p107 have been shown to regulate transcription by RNA polymerases II and III (Claudio et al., 1994; Weinberg 1995; Sutclie et al., 1999.) They can also dier with respect to the proteins with which they interact. For example, E2F-1, -2, and -3 interact preferentially with Rb, E2F-4 with either p107 or p130 and E2F-5 interacts only with p130 (Moberg et al., 1996; Smith et al., 1996; Beijersbergen et al., 1994). Although there are a number of structural and functional similarities between the members of the Rb family, they are not necessarily surrogates for one another. In vivo and in vitro (Cavanaugh et al., 1995; Voit et al., 1997; Hannan et al., 2000) experiments have demonstrated that Rb can regulate transcription from the 45S gene promoter by RNA polymerase I (ribosomal DNA (rDNA) transcription). Several lines of evidence indicate that the eect of Rb on rDNA transcription is mediated by the interaction of Rb with the rDNA transcription factor, UBF (Upstream Binding Factor) (Cavanaugh et al., 1995; Voit et al., 1997). UBF is a highly conserved protein which purifies as two polypeptides, UBF1 (97 kD) and UBF2 Oncogene (2000) 19, 4988 – 4999 ª 2000 Macmillan Publishers Ltd All rights reserved 0950 – 9232/00 $15.00 www.nature.com/onc *Correspondence: LI Rothblum Current addresses: 3 Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett Street, Melbourne, Victoria 8006, Australia; 4 Baker Medical Research Institute, P.O. Box 6492, Melbourne, Victoria 8008, Australia Received 29 February 2000; revised 10 August 2000; accepted 17 August 2000