Mapping of the late promoter of simian virus 40.

Mapping of the simian virus 40 (SV40) late promoter was carried out in the absence of the viral early protein, large tumor (T) antigen, and replication of the viral DNA template. SV40 late control region DNA fragments, containing specific deletions, were cloned directly upstream from the coding region of the herpes simplex virus-1 (HSV-1) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The promoter activities of the fragments were determined by measuring the tk transformation frequencies of the chimeric tk constructs in mouse L TK- APRT- (adenine phosphoribosyltransferase-negative) and human 143B TK- cell lines. The following results were obtained. (i) The SV40 control region functions with equal efficiencies in the early and late promoter orientations. (ii) A major late control element has been localized within the G+C-rich 21-base-pair (bp) repeat. Thus, in conjunction with our earlier results, the 21-bp repeat is a bidirectional promoter element functioning as a major component of both the early and late promoters and is an element that enhances the replication efficiency of SV40 DNA. (iii) Minor late promoters have been localized within the minimal replication origin and the 72-bp repeat. (iv) The minimal replication origin is not per se a constituent of the major late promoter; however, both the minimal replication origin and the 21-bp repeat are required for obtaining high levels of late gene expression observed at late times after infection by SV40. (v) The 72-bp repeat exerts a 4- to 5-fold enhancement of late promoter expression.     

Down-regulation of PTEN expression due to loss of promoter activity in human hepatocellular carcinoma cell lines

AIM: To investigate the regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression in human hepatocellular carcinoma (HCC) cell lines. METHODS: The mRNA and protein levels of PTEN were detected by Northern blot and Western blot in HCC cell lines, respectively. Plasmids containing different fragments of PTEN promoter with Luciferase reporter were constructed and transiently transfected into HCC cell lines to study the promoter activity. DNA analysis and RT-PCR were performed to detect the mutation of PTEN promoter and PTEN cDNA. RESULTS: Either protein or mRNA levels of PTEN in L02 cells (as a control) were significantly higher than that in HCC cell lines. The profile of PTEN promoter activity in 8 cell lines was closely correlated with levels of PTEN mRNA and PTEN protein. Furthermore, the sequence analysis of 8 cells lines showed no mutation in the region of PTEN promoter and PTEN cDNA. CONCLUSION: PTEN expression is down-regulated in HCC cell lines probably due to loss of activity of PTEN promoter.     

Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.     

Human potassium chloride cotransporter 1 (SLC12A4) promoter is regulated by AP-2 and contains a functional downstream promoter element

Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5' end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region.     

Androgen receptor signalling: comparative analysis of androgen response elements and implication of heat-shock protein 90 and 14-3-3eta

Androgen receptor (AR) signalling was analysed using as models the cysteine-rich secretory protein-1 (CRISP-1) and CRISP-3 gene promoters, which are differentially regulated by androgen in vivo and contain multiple potential androgen response elements. Using electrophoretic mobility shift assay, we identified several elements with differing affinities for the AR at positions -3706, -1270, -1253 and -350 of the CRISP-1 promoter and at positions -369 and -349 of the CRISP-3 promoter. The strongest binding was observed for the -1253 element of CRISP-1. In transactivation assays using a PC-3 cell line stably transfected with the AR (PC-3/AR), the -1253 element placed as two or four copies upstream of the TK minimal promoter yielded a strong induction of luciferase reporter gene activity in the presence of the androgen methyltrienolone (R1881). In the context of the CRISP promoters a 2-fold induction by R1881 was measured for the CRISP-3 upstream region whereas only limited effects were noted for the CRISP-1 upstream region. The androgenic stimulation of the p(-1253 ARE)(4x)-TK-luciferase reporter construct was dose-dependently inhibited by geldanamycin and radicicol, two compounds that selectively interact with the chaperone protein, heat-shock protein 90. Cotransfection with an expression vector for the 14-3-3eta protein markedly enhanced the androgen-dependent stimulation. These results emphasize the influence of promoter context on androgen regulation and the importance of AR-associated proteins.     

Progesterone receptor regulates Bcl-2 gene expression through direct binding to its promoter region in uterine leiomyoma cells

CONTEXT: Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. OBJECTIVE: Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. RESULTS: The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. CONCLUSION: Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.