Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.

In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcriptional induction of the human prolactin gene by cAMP requires two cis-acting elements and at least the pituitary-specific factor Pit-1.

To identify the cis-acting elements responsible for cAMP stimulation of human prolactin (hPRL) promoter activity, pituitary GC cells were transfected with 5'-deleted hPRL promoters fused to the chloramphenicol acetyltransferase reporter gene. The proximal regulatory region (coordinates -250 to -42) was sufficient to confer strong cAMP stimulation (+/- 25 fold). Further 5' and 3' deletions performed within this proximal region demonstrated that two types of cis-acting elements are involved in the cAMP regulation: (i) the binding sites of the pituitary-specific factor Pit-1, and (ii) the sequence between coordinates -115 and -85 (named fragment A), which contains a TGACG motif. We show by gel-shift and Southwestern experiments that fragment A binds Pit-1 monomer and also a ubiquitous factor that is neither cAMP-responsive element-binding protein nor activator protein-1. Strong cAMP induction was observed when fragment A was juxtaposed to a Pit-1 binding site. That Pit-1 plays an important role was supported further by the finding that the hPRL proximal region conferred cAMP regulation when linked to the herpes simplex virus thymidine kinase promoter only in pituitary GC cells and not in other heterologous cells, which do not express Pit-1. Furthermore, we observed that concatenated Pit-1 binding sites were able to confer cAMP responsiveness to the thymidine kinase promoter in GC cells.     

Analysis of the promoter region of the human FcRn gene

The 5'-flanking region of the human FcRn alpha-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region -660 to +300 of the 5'-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region.     

Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta.

Regulation of the human type 1 plasminogen activator inhibitor (PAI-1) promoter by transforming growth factor-beta (TGF beta) was studied. An 800-base pair fragment from the PAI-1 promoter and 5'-flanking region was fused to the firefly luciferase reporter gene and transfected into Hep3B human hepatoma cells. Treatment of the cells with TGF beta induced luciferase activity by more than 50-fold. Transfection studies using constructs with 5' or 3' deletions through this region revealed that two sequences were important in the TGF beta response. The first sequence was located in the proximal promoter (-49 to -87) and mediated an 11-fold induction with TGF beta, while the second more distal region (-636 to -740) contained two sequences which together mediated a 50-fold or greater response. Sequence comparison indicated that both of the responsive regions contained sequences with high homology to the AP-1 consensus binding site. Moreover, gel retardation analysis experiments demonstrated that both sequences bound a common nuclear protein, and that an oligonucleotide containing a consensus AP-1 sequence was able to compete for the binding of this common protein. Thus, the response of the PAI-1 gene to TGF beta is mediated by at least two separate regions, and both of these regions contain DNA sequences homologous to the AP-1 binding site.     

Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.     

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RII beta of type II cAMP-dependent protein kinase

The promoter and exon 1 of the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5'-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RII beta 5'-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp -291/-121. An enhancer element was located between bp -1426/-1018.     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D(3). Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides -1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.