The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A Paul Lavender, Laurence Vandel, Andrew J Bannister and Tony Kouzarides Wellcome/CRC Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK A yeast two-hybrid screen has identi®ed HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1 ± a high anity binding site, mediated by an LXCXE motif and a separate low anity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N- and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP co- activator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture. Keywords: HMG-box; retinoblastoma; E1A Introduction The pocket proteins, RB, p107, p130, represent a family of transcriptional regulators which have a highly conserved transcription factor-binding `pocket' do- main. The prototype of this family, RB, has been found mutated in all Retinoblastomas as well as in many other tumours and this has led to its characterization as a tumour suppressor protein (Weinberg, 1995). Although p107 and p130 have functions which overlap those of RB, these two genes have not been found to be mutated in tumours. Perhaps, as a consequence, RB still remains the most studied member of this family. The pocket proteins (most commonly RB) have been shown to bind and regulate a number of dierent transcription factors (Kouzarides, 1995). Their most characterized target is the E2F family of transcriptional activators, E2F1-5 (Weinberg, 1995). The three pocket proteins bind and regulate a distinct subset of E2F family members but the reason or mechanism behind this speci®city is not yet clear. The E2F1 protein has the ability to promote G1 to S phase transition by stimulating the activity of S-phase speci®c genes (Nevins, 1992; Johnson et al., 1993). The binding of RB to E2F1 leads to a suppression of these activation functions, which correlates with RB's ability to induce growth arrest. Only a subset of E2F family members (E2F1, 2, 3) possess the ability to stimulate S-phase. The fact that this subset of E2F proteins is precisely the subset regulated by RB in vivo (rather than p107 or p130) may explain why RB is the only pocket protein mutated in tumours (Lukas et al., 1996). Another function of RB which also correlates with its ability to induce growth arrest, is the repression of genes involved in protein biosynthesis. RB can repress most RNA polymerase III (Pol III) transcribed genes in vivo (White et al., 1996) and can regulate RNA polymerase I (pol I) transcription by repressing the UBF transcription factor (Cavanaugh et al., 1995). Repression of pol I, pol III and E2F regulated pol II genes requires the pocket domain of RB (which is found mutated in tumours) and is alleviated by viral transforming proteins such as E1A binding to RB. The ability of RB to recognize and repress a diverse array of factors which regulate pol I, pol II and pol III genes may be related to the sequence similarity displayed between the RB pocket domain and the two general factors TBP and TFIIB (Hagemeier et al., 1993a). Although the mechanism of this repression is not yet understood, masking of TBP binding capacity may be involved for both E2F (Hagemeier et al., 1993b) and UBF (Cavanaugh et al., 1995). However, in addition, RB must contain independent repressive capacity, which acts directly to silence the transcriptional machinery (Weintraub et al., 1992, 1995). A growing body of evidence suggests that RB not only functions to regulate cell proliferation but is also intimately involved in regulating cell dierentiation. Indirect evidence has come from the generation of RB de®cient mice which survive until 14.5 days post- coitum, a time when dierentiation of a number of dierent cell types is known to be initiated (Jacks et al., 1992; Lee et al., 1992; Clarke et al., 1992). More direct evidence comes from the analysis of RB-interacting proteins (reviewed in Kouzarides, 1995). A number of these (myoD, C/EBP, Elf1) are transcription factors which have a role in regulating the dierentiation of speci®c cell lineages (Gu et al., 1993; Chen et al., 1996; Wang et al., 1993). Thus RB may not only regulate the cell cycle, but may also be involved in regulating cell dierentiation. Here we report that HBP1, an HMG-box transcrip- tion factor, is bound and regulated by RB and the other pocket proteins. We show that HBP1 has the capacity to induce morphological transformation of cells in culture and that its transcriptional activation capacity is regulated by the viral E1A protein. Correspondence: T Kouzarides Received 6 February 1997; accepted 1 May 1997 Oncogene (1997) 14, 2721 ± 2728  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00