The X gene of human hepatitis B virus encodes the polypeptide HBx which transactivates viral and host genes through a variety of cis‐acting enhancer elements present in RNA polymerases I, II and III promoters. To better understand the mechanism of X transactivation, we cloned cDNAs of proteins that bind HBx. Here we demonstrate that one of these cDNAs is a full‐length cDNA of human RPB5, a subunit shared by RNA polymerases. The HBx transactivation domain and the central region of human RPB5 were necessary for the specific binding of the two proteins as shown by: (i) in vitro assays using deletion mutants of fusion proteins; (ii) in vivo assays which detect associated proteins by co‐immunoprecipitation of the non‐fused proteins from transfected HepG2 cells. Over‐expressed HBx seemed to associate with assembled forms of endogenous human RPB5 in HBx‐transfected cells, since the endogenous RPB5 co‐immunoprecipitated with HBx. The HBx binding region of human RPB5 by itself stimulated chloramphenicol acetyltransferase activities from several different reporters having X‐responsive element(s). Our results support the idea that the interaction of HBx and human RPB5 can facilitate HBx transactivation and that human RPB5 has a domain which can communicate with transcriptional regulators.

Many transcription coactivators interact with nuclear receptors in a ligand-and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor ␣, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the liganddependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fide transcription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref.1). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, 1 and retinoids as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (reviewed in Ref.2). Functional analysis of nuclear receptors has shown that there are two major activation domains. The activation function-2 (AF-2) at the extreme C-terminal region of the ligandbinding domain (LBD) exhibits ligand-dependent transactivation, whereas the N-terminal activation function-1 contains a ligand-independent transactivation domain. The AF-2 region is conserved among nuclear receptors, and deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities. X-ray crystallographic studies of the LBD of nuclear receptors revealed that the ligand binding induces a major conformational change in the AF-2 region (3-7), suggesting that this region may play a critical role in mediating transactivation by a ligand-dependent interaction with coactivators. As expected, many coactivators fail to interact with AF-2 mutants of nuclear receptors (8 -10). Transcriptional activation of most nuclear receptors involves at least two separate processes as follows: derepression and activation. Repression is mediated in part by interaction of unliganded receptors with corepressors such as N-CoR (11) and SMRT (12). H...

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G 1 upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G 1 to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.

Silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is known to interact with Sin3 and recruit the histone deacetylases (HDACs) that lead to hypoacetylation of histones and transrepression of target transcription factors. Herein, we found that coexpression of SMRT significantly repressed transactivations by activating protein-1 (AP-1), nuclear factor-B (NFB), and serum response factor (SRF) in a dose-dependent manner, but not in the presence of trichostatin A, a specific inhibitor of HDAC. Similarly, coexpression of HDAC1 and mSin3A also showed repressive effects. Consistent with these results, the C-terminal region of SMRT directly interacted with SRF, the AP-1 components c-Jun and c-Fos, and the NFB components p50 and p65, as demonstrated by the yeast and mammalian two hybrid tests as well as the glutathione S-transferase pull down assays. Thus, we concluded that SMRT serves to recruit Sin3/HDACs to SRF, NFB, and AP-1 in vivo and modulate their transactivation.Retinoic acid and thyroid hormone receptor bind to their target genes and repress transcription (for a review see Ref. 1). The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) 1 (2) and the nuclear hormone receptor corepressors (N-CoR) (3) were originally isolated as factors associated with the hinge and ligand binding D/E domains of unliganded receptors. These corepressors interact with Sin3 and recruit the histone deacetylases (HDAC1 or Rpd-3⅐HDAC2) that lead to hypoacetylation of the histones, consistent with the concept that histone hypoacetylation correlates with gene repression (4 -7). Two groups have recently reported the isolation of histone deacetylase core complexes (mSin3 and the NuRD complex) that are critically involved in this transcriptional repression (8,9). A few components of the NuRD complex are also present in the mSin3 complex that consists of seven polypeptides. In particular, SAP30 directly interacts with NCoR, although N-CoR does not appear to be stably associated with this mSin3 complex. Antibody-blocking experiments and studies with histone deacetylase inhibitors also supported the idea that N-CoR/SMRT may serve as an adapter molecule between the core mSin3 complex and sequence-specific transcriptional repressors such as unliganded nuclear receptors (8, 9). However, recent evidence also showed that N-Cor/SMRT and Sin3 directly interact with the key components of the transcriptional initiation process (TFIIB, and the TBP-associating factors) to inhibit basal transcription, suggesting an alternative repression pathway (10, 11). Interestingly, the N-CoR⅐Sin3⅐HDAC complex is also known to mediate transcriptional repression from a wide variety of other nonreceptormediated pathways. These include MyoD (12), the bHLH-LZ proteins Mad and Mxi that mediate repression of myc activities and tumor suppression (13), E2F-repressive retinoblastoma protein (14), and the oncoproteins PLZF-RAR (15) and LAZ3/ BCL6 (16), which are involved in acute promyelocytic leukemia and non-Hodgkin lymphomas, respect...

Hepatitis C virus (HCV) infection is often associated with hepatic steatosis and yet the molecular mechanisms of HCVassociated steatosis are poorly understood. Because sterol regulatory element-binding proteins (SREBPs) are the major transcriptional factors in lipogenic gene expression including fatty acid synthase (FAS), we examined the effects of HCV nonstructural proteins on the signaling pathways of SREBP. In this study, we demonstrated that HCV nonstructural 4B (NS4B) protein increased the transcriptional activities of SREBPs. We also showed that HCV NS4B enhanced the protein expression levels of SREBPs and FAS. This was further confirmed in the context of viral RNA replication and HCV infection. The up-regulation of both SREBP and FAS by NS4B protein required phosphatidylinositol 3-kinase activity. We also demonstrated that NS4B protein induced a lipid accumulation in hepatoma cells. In addition, NS4B protein synergistically elevated the transcriptional activity of HCV core-mediated SREBP-1. These results strongly suggest that NS4B may play an important role in HCV-associated liver pathogenesis by modulating the SREBP signaling pathway.