The RFA regulatory sequence-binding protein in the promoter of

The prostate-specific antigen (PSA), which plays an important role during the liquefaction process of semen, is a differentiation marker for human prostate. It has become the most sensitive marker for monitoring and detecting prostate cancer. PSA as a serine protease can activate some growth factors that might be related to the advancement of prostate cancer by hydrolyzing growth factor-binding protein. The PSA gene is expressed specifically in prostate epithelial cells. The ex-pression of…     

Identification of two novel cis-elements in the promoter of the prostate-specific antigen gene that are required to enhance androgen receptor-mediated transactivation.

A monomeric androgen responsive element (ARE) is not sufficient to mediate significant androgen induction of the prostate-specific antigen (PSA) gene. Co-transfection experiments using a series of 5'deletion fragments of the proximal promoter region of the PSA gene linked to bacterial chloramphenicol acetyltransferase (CAT) as a reporter have identified two motif sequences which are indispensable for androgen receptor (AR)-mediated transactivation of the PSA promoter and have been designated as motifs A and B respectively. Of note, motif B alone has very little independent enhancer activity regardless of the presence or absence of androgen, whereas multi-copies of motif A exert androgenic inducibility for a heterologous promoter independent of the presence of ARE. Nucleotide substitutions in either motif significantly decrease the androgen inducibility and the nuclear protein binding ability. Furthermore, gel band shift experiments consistently demonstrate that nuclear proteins can bind these motifs, and they are non-receptor factors. Our data indicate that these two DNA motifs are novel cis -regulatory elements and exhibit different mechanisms in cooperation with ARE for AR-mediated transactivation.     

Dependence of selective gene activation on the androgen receptor NH2- and COOH-terminal interaction

The agonist-induced androgen receptor NH(2)- and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH(2)-terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH(2)-terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence (433)WHTLF(437) contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation.     

Modulation of androgen receptor transactivation by the SWI3-related gene product (SRG3) in multiple ways

The SWI3-related gene product (SRG3), a component of the mouse SWI/SNF complex, has been suggested to have an alternative function. Here, we demonstrate that in the prostate transactivation of the androgen receptor (AR) is modulated by SRG3 in multiple ways. The expression of SRG3, which is developmentally regulated in the prostate, is induced by androgen through AR. SRG3 in turn enhances the transactivation of AR, providing a positive feedback regulatory loop. The SRG3 coactivation of AR transactivation is achieved through the recruitment of coactivator SRC-1, the protein level of which is upregulated by SRG3, providing another pathway of positive regulation. Interestingly, SRG3 coactivation of AR transactivation is fully functional in BRG1/BRM-deficient C33A cells and the AR/SRG3/SRC-1 complex formed in vivo contains neither BRG1 nor BRM protein, suggesting the possibility of an SRG3 function independent of the SWI/SNF complex. Importantly, the AR/SRG3/SRC-1 complex occupies androgen response elements on the endogenous SRG3 and PSA promoter in an androgen-dependent manner in mouse prostate and LNCaP cells, respectively, inducing gene expression. These results suggest that the multiple positive regulatory mechanisms of AR transactivation by SRG3 may be important for the rapid proliferation of prostate cells during prostate development and regeneration.     

Androgen receptor-associated protein complex binds upstream of the androgen-responsive elements in the promoters of human prostate-specific antigen and kallikrein 2 genes.

An increasing number of proteins which bind to hormone-dependent nuclear receptors and mediate their effects on gene expression are being identified. The human prostate-specific antigen (PSA) and kallikrein 2 (KLK2) genes are regulated by the androgen receptor (AR). Using electrophoresis mobility shift assays (EMSA), a common nuclear protein(s) which binds upstream of the androgen-responsive elements (AREs) in the PSA and KLK2 promoters was identified. Binding occurred between bp -539 and -399 and bp -349 and -224 in the PSA and KLK2 promoters respectively, which were shown previously to be necessary for AR-mediated transactivation. Glutathione S-transferase (GST)-AR fusion proteins were constructed to determine whether the AR interacted directly with this protein or protein complex. Specific interactions were observed with AR fusion proteins containing the DNA binding domain. EMSA supershift experiments and GST-AR pull-down experiments followed by Western blotting identified a Fos-related protein(s) of approximately 40 kDa as part of this complex. Competition experiments with a double-stranded oligonucleotide containing an AP-1 binding site demonstrated that DNA binding was not mediated by AP-1. These results indicate that a Fos-containing protein complex distinct from AP-1 binds upstream of the AREs in the PSA and KLK2 promoters, interacts with the AR and may participate in regulation of these two androgen-responsive genes.     

Direct interactions of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed-early promoter and stimulating viral reactivation

The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.     

p53 represses androgen-induced transactivation of prostate-specific antigen by disrupting hAR amino- to carboxyl-terminal interaction

Prostate-specific antigen (PSA) is highly overexpressed in prostate cancer. One important regulator of PSA expression is the androgen receptor (AR), the nuclear receptor that mediates the biological actions of androgens. AR is able to up-regulate PSA expression by directly binding and activating the promoter of this gene. We provide evidence here that that this AR activity is repressed by the tumor suppressor protein p53. p53 appears to exert its inhibition of human AR (hAR) by disrupting its amino- to carboxyl-terminal (N-to-C) interaction, which is thought to be responsible for the homodimerization of this receptor. Consistent with this, p53 is also able to block hAR DNA binding in vitro. Our previous data have shown that c-Jun can mediate hAR transactivation, and this appears to result from a positive effect on hAR N-to-C interaction and DNA binding. Interestingly, c-Jun is able to relieve the negative effects of p53 on hAR transactivation, N-to-C interaction, and DNA binding, demonstrating antagonistic activities of these two proteins. Importantly, a p53 mutation found in metastatic prostate cancer severely disrupts the p53 negative activity on hAR, suggesting that the inability of p53 mutants to down-regulate hAR is, in part, responsible for the metastatic phenotype.     

Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins.

Expression from a human cytomegalovirus early promoter (E1.7) has been shown to be activated in trans by the IE2 gene products (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281-290, 1989). Using wild-type and mutant viral proteins, we have defined the protein regions required for transactivation of the E1.7 promoter in IE2 and for augmentation of transactivation in the IE1 protein. Two regions of the IE2 proteins were found to be essential for transactivation. One near the amino terminus is within 52 amino acids encoded by exon 3. The second comprises the carboxyl-terminal 85 amino acids encoded by exon 5. The IE2 protein encoded by an mRNA which lacks the intron within exon 5 and the IE2 protein encoded by exon 5 had no activity for transactivation of the E1.7 promoter. Although the IE1 gene product alone had no effect on this early viral promoter, maximal early promoter activity was detected when both IE1 and IE2 gene products were present. The IE1 protein positively regulated its enhancer-containing promoter-regulatory region. The IE1 protein alone increased the steady-state level of IE2 mRNA; therefore, IE1 and IE2 are synergistic for expression from the E1.7 promoter. Like the IE2 proteins, the IE1 protein requires for activity 52 amino acids encoded by exon 3. IE1 also requires amino acids encoded by exon 4. Since the IE1 and IE2 proteins have 85 amino acids in common at the amino-terminal end encoded by exons 2 and 3, the difference between these specific transactivators resides in their carboxyl-terminal amino acids encoded by exons 4 and 5, respectively.