Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.     

Differential binding of rat pituitary-specific nuclear factors to the 5′-flanking region of pituitary and placental members of the human growth hormone gene family

Summary Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (–140/–107) and proximal site (–97/–66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (–140/–116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer. However, the data also indicate that rat Pit 1 binds to human and rat pituitary growth hormone in a similar manner (two sites of interaction) and that the pattern of binding is distinct from the placental members of the hGH gene family. These data indicate that human Pit 1, unlike the rat equivalent, might distinguish these genes functionally (tissue-specifically) as well as structurally.     

Tissue-specific activity of the pro-opiomelanocortin gene promoter.

The pro-opiomelanocortin (POMC) gene is specifically expressed in corticotroph cells of the anterior pituitary. To define the POMC promoter sequences responsible for tissue-specific expression, we assessed POMC promoter activity by gene transfer into POMC-expressing pituitary tumor cells (AtT-20) and fibroblast L cells. The rat POMC promoter was only efficiently utilized and correctly transcribed in AtT-20 cells. 5'-End deletion analysis revealed two promoter regions required for activity in AtT-20 cells. When tested by fusion to a heterologous promoter, DNA fragments corresponding to both regions exhibited tissue-specific activity, suggesting the presence of at least two tissue-specific DNA sequence elements within the promoter. In summary, POMC promoter sequences from -480 to -34 base pairs appear sufficient to mimic the specificity of anterior pituitary expression.     

Human chorionic somatomammotropin and growth hormone gene expression in rat pituitary tumour cells is dependent on proximal promoter sequences.

Human placental chorionic somatomammotropin (hCS-A or hCS-B) and pituitary growth hormone (hGH-N) are related by structure and function. The hCS-A gene is expressed in rat pituitary tumour (GC) cells after gene transfer. Deletion of hCS-A 5'-flanking DNA reveals repressor activity upstream of nucleotide -132, and a region essential for expression in GC cells between nucleotides -94 and -61. The sequences in this region differ from the equivalent hGH-N gene DNA by one nucleotide, and include the binding site (-92 to -65) for a pituitary-specific factor (GHF-1), required for hGH-N expression in GC cells. Exchange of hGH-N with hCS-A gene DNA in this region maintains expression in GC cells. By contrast, modification of these sequences blocks expression. These data indicate that proximal promoter sequences, equivalent to those bound by GHF-1 on the hGH-N gene, are required for hCS-A expression in GC cells.     

Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.     

Tissue specific trans-acting factor interaction with proximal rat prolactin gene promoter sequences.

Using an exonuclease III protection assay, strong, reversible and tissue-specific binding of GH3 cell nuclear factors to proximal regions of the rat prolactin (rPrl) promoter (-31 to -77) has been detected. A second less prominent region of factor binding, that may have a correlate in HeLa cell extracts, was detected in the region (-155 to -180). The binding is eliminated in the presence of excess unlabelled rPrl promoter sequences (-423 to +38), excess unlabelled distal rPrl 5'-flanking sequences (-1960 to -1260) and SV40-enhancer/promoter sequences; it is largely unaffected by growth hormone (rGH) promoter and RSV-LTR sequences. A plasmid containing the proximal rPrl promoter sequences (-75 to +38) was also shown to be an avid inhibitor, at low concentrations, of rPrl promoter driven chloramphenicol acetyl transferase (CAT) gene expression in transient cotransfection competition studies; under these assay conditions distal rPrl 5'-flanking sequences and RSV and rGH promoter plasmids do not compete. The results emphasize the critical importance of proximal rPrl promoter sequences for prolactin gene expression in GH3 cells but recognize the related functional potential of more distal sequences.     

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)     

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.     

Sequences required for cell-type specific thyroid hormone regulation of rat growth hormone promoter activity

We have located sequences within the rat growth hormone (rGH) promoter region which are required for pituitary cell-type specific responsiveness to T3 (thyroid hormone, 3,5,3'-L-triiodothyronine). Transient transfections with a series of plasmids containing as few as 202 nucleotides upstream of the start site of the rat growth hormone mRNA showed specific induction by T3 in rat pituitary cell lines. Both the magnitude and the kinetics of this response were similar to those of the endogenous rGH gene, showing a strong early induction followed by a decline in T3 effect. Deletion of an additional 19 base pairs (to -183 relative to the start site) eliminated this induction. Plasmids containing sequences up to -237 or -202 showed significant promoter activity but no T3 responsiveness in transfections of mouse fibroblasts or monkey kidney cells. The presence of high affinity nuclear T3 binding proteins was demonstrated in both cell types. These results show that sequences between -183 and -202 are required for pituitary cell specific T3 regulation of the rGH promoter. The lack of T3 responsiveness in non-pituitary cells suggests that such regulation may be mediated by factors present in pituitary cells and absent in other cells.     

Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.