The estrogen-related receptor ␣ (ERR␣) is an orphan member of the nuclear receptor superfamily. We show that the major isoform of the human ERR␣ gene, ERR␣1, can sequence-specifically bind a consensus palindromic estrogen response element (ERE) and directly compete with estrogen receptor ␣ (ER␣) for binding. ERR␣1 activates or represses ERE-regulated transcription in a cell type-dependent manner, repressing in ER-positive MCF-7 cells while activating in ER-negative HeLa cells. Thus, ERR␣1 can function both as a modulator of estrogen responsiveness and as an estrogen-independent activator. Repression likely occurs in the absence of exogenous ligand since charcoal treatment of the serum had no effect on silencing activity. Mutational analysis revealed that repression is not simply the result of competition between ER␣ and ERR␣1 for binding to the DNA. Rather, it also requires the presence of sequences within the carboxyl-terminal E/F domain of ERR␣1. Thus, ERR␣1 can function as either an active repressor or a constitutive activator of ERE-dependent transcription. We hypothesize that ERR␣1 can play a critical role in the etiology of some breast cancers, thereby providing a novel therapeutic target in their treatment. The nuclear receptor (NR)1 superfamily is comprised of hundreds of transcription factors that regulate a vast array of genes and physiological responses (1-9). Most nuclear receptors share a similar structural organization (Fig. 1A). The amino-terminal A/B domain can function as a hormone-independent activator of transcription. The highly conserved C domain contains the DNA binding domain (DBD) that confers sequence-specific DNA binding activity. A hinge region, called the D domain, bridges the C domain with the carboxyl-terminal E/F domain that includes the receptor-specific ligand binding domain (LBD) of the protein. The binding of appropriate ligands results in conformation changes leading to alterations in the transcriptional properties of the receptor, including the exposure of a transcriptional activation region within the carboxyl end. Although many nuclear receptor superfamily members bind known ligands (e.g., steroids, retinoids, thyroid hormones), some, termed orphan receptors, share significant sequence similarity in their LBDs with their ligand binding family members but lack as-yet known naturally occurring ligands (7-10).Among the first orphan receptors identified were the estrogen-related receptors ERR␣ and ERR␤ (officially named NR3B1 and NR3B2, respectively) (10). They were cloned by low stringency screening of cDNA libraries with probes corresponding to the DBD of estrogen receptor ␣ (ER␣) (10). Subsequently, ERR␣1 was identified as the major isoform present in HeLa cells (Fig. 1A) (11, 12). The DBD of human ERR␣1 shares 70% amino acid similarity with the DBD of human ER␣; the LBD shares 35% amino acid identity. A third member of the ERR family, ERR␥ (NR3B3), has also been identified (13-15). These three ERRs are closely related by sequence similarity but encoded by different genes.De...

We previously showed that (a) estrogen-related receptor A1 (ERRA1) down-modulates estrogen receptor (ER) -stimulated transcription in low ErbB2 -expressing MCF-7 mammary carcinoma cells, and (b) ERRA and ErbB2 mRNA levels positively correlate in clinical breast tumors. We show here that ERRA1 represses ERA-mediated activation in MCF-7 cells because it failed to recruit the coactivator glucocorticoid receptor interacting protein 1 (GRIP1) when bound to an estrogen response element. In contrast, ERRA1 activated estrogen response element -and ERR response element -mediated transcription in ERA-positive, high ErbB2 -expressing BT-474 mammary carcinoma cells, activation that was enhanced by overexpression of GRIP1. Likewise, regulation of the endogenous genes pS2, progesterone receptor, and ErbB2 by ERRA1 reflected the cell type -specific differences observed with our reporter plasmids. Importantly, overexpression of activated ErbB2 in MCF-7 cells led to transcriptional activation, rather than repression, by ERRA1. Two-dimensional PAGE of radiophosphate-labeled ERRA1 indicated that it was hyperphosphorylated in BT-474 relative to MCF-7 cells; incubation of these cells with anti-ErbB2 antibody led to reduction in the extent of ERRA1 phosphorylation. Additionally, mitogen-activated protein kinases (MAPK) and Akts, components of the ErbB2 pathway, phosphorylated ERRA1 in vitro. ERRA1-activated transcription in BT-474 cells was inhibited by disruption of ErbB2/epidermal growth factor receptor signaling with trastuzumab or gefitinib or inactivation of downstream components of this signaling, MAPK kinase/MAPK, and phosphatidylinositol-3-OH kinase/ Akt, with U0126 or LY294002, respectively. Thus, ERRA1 activities are regulated, in part, via ErbB2 signaling, with ERRA1 likely positively feedback-regulating ErbB2 expression. Taken together, we conclude that ERRA1 phosphorylation status shows potential as a biomarker of clinical course and antihormonal-and ErbB2-based treatment options, with ERRA1 serving as a novel target for drug development. (Mol Cancer Res 2007;5(1):71 -85)

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (␥HV68). Introduction of a translation stop codon in ␥HV68 gene 50 (gene 50.stop ␥HV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop ␥HV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop ␥HV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type ␥HV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68

The late genes of SV40 are not expressed at significant levels until after the onset of viral DNA replication. We previously identified two hormone response elements (HREs) in the late promoter that contribute to this delay. Mutants defective in these HREs overexpress late RNA at early, but not late, times after transfection of CV-1PD cells. Overexpression of nuclear receptors (NRs) that recognize these HREs leads to repression of the late promoter in a sequence-specific and titratable manner, resulting in a delay in late gene expression. These observations led to a model in which the late promoter is repressed at early times after infection by NRs, with this repression being relieved by titration of these repressors through simian virus 40 (SV40) genome replication to high copy number. Here, we tested this model in the context of the viral life cycle. SV40 genomes containing mutations in either or both HREs that significantly reduce NR binding without altering the coding of any proteins were constructed. Competition for replication between mutant and wild-type viruses in low-multiplicity coinfections indicated that the ؉1 HRE offered a significant selective advantage to the virus within a few cycles of infection in African green monkey kidney cell lines CV-1, CV-1P, TC-7, MA-134, and Vero but not in CV-1PD cells. Interestingly, the ؉55 HRE offered a selective disadvantage in MA-134 cells but had no effect in CV-1, CV-1P, TC-7, Vero, and CV-1PD cells. Thus, we conclude that these HREs are biologically important to the virus, but in a cell type-specific manner.Simian virus 40 (SV40) is a member of the primate polyomavirus family. Its small 5.2-kbp double-stranded DNA genome codes for two sets of proteins: the regulatory proteins, large T antigen and small t antigen, and the structural proteins, leader protein 1 (LP1, also called agnoprotein) (27) and virion protein 1 (VP1), VP2, and VP3 (23). The genes encoding the regulatory and structural proteins are separated and controlled by a bidirectional promoter-regulatory region that spans ϳ400 bp and that can be bound by numerous factors (30). Expression of these two sets of genes is temporally regulated. The regulatory genes are expressed at early times in the lytic cycle of infection. Their products interact with numerous cellular regulatory factors (2, 44), resulting in the infected cell being pushed into an unchecked proliferation loop. Large T antigen also directly plays multiple roles in transcriptional control and replication of the viral genome. The expression of the structural genes is delayed until after the onset of viral DNA replication (1).How does SV40 manage this early-to-late switch in gene expression when both sets of genes are controlled from the same bidirectional promoter-regulatory region? Two non-mutually exclusive mechanisms contribute to this temporal regulation. The replication-independent mechanism involves transactivation of the late promoter by large T antigen (7,21,31). Evidence that transactivation is, in part, independent of DNA replicat...