Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.     

A role for A/T-rich sequences and Pit-1/GHF-1 in a distal enhancer located in the human growth hormone locus control region with preferential pituitary activity in culture and transgenic mice.

A region located remotely upstream of the human pituitary GH (GH-N) gene and required for efficient GH-N gene expression in the pituitary of transgenic mice was cloned as a 1.6-kb Bg/II (1.6G) fragment. The 1.6G fragment in the forward or reverse orientation increased -496GH-N promoter activity significantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment was also able to stimulate activity from a minimal thymidine kinase (TK) promoter which, unlike -496GH-N, lacked any Pit-1/GHF-1 element. Enhancer activity was localized by deletion analysis to a 203-bp region in the 3'-end of the 1.6G fragment and was characterized by the presence of a diffuse 136-bp nuclease-protected site, observed with pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein. A major low-mobility complex was observed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear protein, and the pattern was distinct from that seen with a HeLa cell extract. The nuclease-protected region contains three A/T-rich Pit-1/ GHF-1-like elements, and their disruption, in the context of the 203-bp region fused to the TK promoter, reduced enhancer activity significantly in pituitary cells in culture. A mutation in this region was also shown to decrease enhancer activity in transgenic mice and correlated with a decrease in the 203-bp enhancer region complex observed by EMSA. The participation of Pit-1/GHF-1 in this complex is indicated by competition studies with Pit-1/GHF-1 elements and antibodies, and direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by EMSA using recombinant protein. These studies link the A/T-rich sequences to the distal enhancer activity associated with the GH locus control region in vitro and in vivo.     

Enhancer-blocking activity is associated with hypersensitive site V sequences in the human growth hormone locus control region

Activation of the human growth hormone gene (hGH-N) is linked to a locus control region (LCR) containing four (I-III, V) hypersensitive sites (HS). Pit-1 binding to HS I/II is required for efficient pituitary expression. However, inclusion of HS III and V, located about 28 and 32 kb upstream of the hGH-N gene, respectively, is also required for consistent hGH-N expression levels in vivo. HS V is referred to as a boundary for the hGH LCR, but no specific enhancer blocking or barrier function is reported. We examined a 547 bp fragment containing HS V sequences (nucleotides -32,718/-32,172 relative to hGH-N) for enhancer-blocking activity using a well-established transient gene transfer system and assessed these sequences for CCCTC binding factor (CTCF), which is linked to enhancer-blocking activity. The 547 bp HS V fragment decreased enhancer activity with a reverse-orientation preference when inserted between HS III enhancer sequences and a minimal thymidine kinase promoter (TKp). These sequences are associated with CTCF in human pituitary and nonpituitary chromatin. Enhancer-blocking activity with an orientation preference was further localized to a 45 bp sub-fragment, with evidence of CTCF and upstream binding factor 1 (USF1) binding; USF1 is linked more closely with barrier function. The presence of yin and yang 1 (Yy1) that cooperates with CTCF in the regulation of X-chromosome inactivation was also seen. A decrease in CTCF and Yy1 RNA levels was associated with a significant reduction in enhancer-blocking activity. Assessment of CpG-dinucleotides in the TKp indicates that the presence of HS V sequences are associated with an increased incidence of CpG-dinucleotide methylation of the GC box region. These data support association of CTCF and enhancer-blocking activity with HS V that is consistent with a role as a (LCR) boundary element and also implicates Yy1 in this process.     

A retroviral promoter and a cellular enhancer define a bipartite element which controls env ERVWE1 placental expression

The HERV-W family contains hundreds of loci diversely expressed in several physiological and pathological contexts. A unique locus termed ERVWE1 encodes an envelope glycoprotein (syncytin) involved in hominoid placental physiology. Here we show that syncytin expression is regulated by a bipartite element consisting of a cyclic AMP (cAMP)-inducible long terminal repeat (LTR) retroviral promoter adjacent to a cellular enhancer conferring a high level of expression and placental tropism. Deletion mutant analysis showed that the ERVWE1 5' LTR contains binding sites essential for basal placental activity in the region from positions +1 to +125. The region from positions +125 to +310 represents a cAMP-responsive core HERV-W promoter active in all cell types. Site-directed mutagenesis analysis highlighted the complexity of U3 regulation. ERVWE1 placenta-specific positive (e.g., T240) and negative (e.g., G71) regulatory sites were identified, as were essential sites required for basic activity (e.g., A247). The flanking sequences of the ERVWE1 provirus contain several putative regulatory elements. The upstream HERV-H and HERV-P LTRs were found to be inactive. Conversely, the 436-bp region located between the HERV-P LTR and ERVWE1 was shown to be an upstream regulatory element (URE) which is significantly active in placenta cells. This URE acts as a tissue-specific enhancer. Genetic and functional analyses of hominoid UREs revealed large differences between UREs of members of the Hominidae and the Hylobatidae. These data allowed the identification of a positive regulatory region from positions -436 to -128, a mammalian apparent LTR retrotransposon negative regulatory region from positions -128 to -67, and a trophoblast-specific enhancer (TSE) from positions -67 to -35. Putative AP-2, Sp-1, and GCMa binding sites are essential constituents of the 33-bp TSE.     

Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen.

The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.     

Regulation of growth hormone secretion in human trophoblastic cells in culture

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10(5) cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10(-6) M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.     

Inducibility of the HS II enhancer depends on binding of an erythroid specific nuclear protein.

An erythroid specific, inducible enhancer associated with hypersensitive site II (HS II) plays a central role in the function of the human beta globin dominant control region. The HS II enhancer consists of tandem AP-1 binding sites and has been shown to bind members of the ubiquitous jun and fos families of proteins. The same sites are now shown to bind the erythroid specific protein, NF-E2. Inducibility of the HS II enhancer depends on NF-E2 binding, even in the presence of another hypersensitive site. Further, increased activity of the enhancer in induced K562 cells correlates with the presence of NF-E2, which appears to be present in a modified form. NF-E2 is distinct from some enhancer binding proteins in K562 nuclear extracts, in that it does not contain Fos or Fra-1 protein. Thus, binding by NF-E2 may be the mechanism, whereby tandem AP-1 binding sites confer erythroid specificity on the HS II enhancer.