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Transcription of the human growth hormone (hGH) gene depends on cis-acting elements contained within 300 base pairs of its 5'-flanking sequence. An earlier in vitro study of the transcriptional activity of this 5'-flanking region suggested that transcription can start upstream from position +1. We have investigated this phenomenon by cell-free transcription and transient transfection of chimeric constructs in cultured pituitary cells and in HeLa cells and by analysis of RNA from human pituitary glands and HeLa cells. Transcription initiation sites were identified at positions -54 and -197 by cell-free transcription assays and by RNAse mapping of human pituitary RNA. In transfection assays, the hGH gene 5'-flanking sequence upstream from position -197 displayed transcriptional activity, which critically depended on the upstream stimulatory factor-binding site located between positions -253 and -266. Transcripts initiated upstream from position +1 were detected in human pituitary RNA by polymerase chain reaction amplification and Northern blotting. These transcripts were longer than the mRNA encoding hGH. They might control initiation at position +1 or code for a novel peptide.

DNase I footprinting experiments showed that binding activities of Sp1 and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Sp1 site did not affect promoter activity in cell-free transcription. Still, Sp1 could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.

Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene.

The expression of the human growth hormone (hGH) gene is regulated by several transcription factors. Basal level transcription factors include TATA box-binding proteins, Sp1, USF and CTF/NF-1. The hGH gene is expressed only in pituitary somatotrophs, and the pituitary-specific GHF-1/Pit-1 protein is a potent transcriptional stimulator. Glucocorticoid and thyroid hormones, insulin, and GHRH which acts via cAMP, also control hGH gene transcription via trans-acting factors some of which are the hormone receptors themselves. Three transcription initiation sites were detected when hGH gene promoter activity was studied in a cell-free system. This system enabled us to delineate the respective role of some transcription factors and to propose a model that accounts for the basal, pituitary-specific, and hormonal control of hGH gene expression.

Glucocorticoid and thyroid hormones modulate the expression of the growth hormone gene. To investigate this control mechanism, we have determined whether this gene contains sites that bind the human glucocorticoid and thyroid hormone receptors in vitro. To do so, we have designed a novel assay for studying binding of the purified glucocorticoid receptor to cloned fragments of the human growth hormone (hGH) gene, and have adapted a DNA-competition assay for the thyroid receptor in nuclear cell extracts. Two glucocorticoid receptor binding regions were found in the hGH gene, one of high affinity in a fragment of the gene containing the first intron, and one of low affinity located within a 290 bp-fragment of 5'-flanking DNA. In contrast, the thyroid receptor bound with high affinity to the 5'-flanking fragment. Homologous binding regions for the two types of receptor were found in the human placental lactogen (chronic somatomammotropin) gene. DNA binding of the two receptor types appeared to depend on the presence of the hormone, yet antagonist-bound glucocorticoid receptor was still capable of interacting specifically with DNA. There was no evidence for synergism or antagonism of the two receptor types in binding to their respective sites on the hGH gene. The data also make it unlikely that the thyroid receptor negatively controls gene transcription and that the stimulatory effect of thyroid hormone results from a derepression mechanism.

The human genes coding for growth hormone (GH), chorionic somatomammotropin (placental lactogen, CS), and prolactin (Prl) are related evolutionarily but are expressed in phenotypically distinct cell types despite their nucleotide sequence homology. We show here that the promoters of the human Prl and CS genes contain cis-acting sequences that confer pituitary-specific expression in a cell-free transcription assay. Similar data are obtained with the human GH gene, consistent with earlier work by others. Footprinting analysis shows that neighboring sequences in each of these three promoters are protected from deoxyribonuclease I digestion by rat pituitary cell extracts. Footprinting competition experiments and gel retardation assays with synthetic oligonucleotides suggest that a single factor is responsible for the pituitary-specific footprints seen on the human Prl, CS, and GH genes. They also suggest that this factor is identical or closely related to the trans-acting factor GHF-1/Pit-1. Similarities with and differences from the rat GH and Prl genes are discussed.

Expression of the human-growth-hormone gene is restricted to pituitary somatotrophs. Two protein-DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary-specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease-I protection and gel-retardation experiments. The first footprint, located between -257 and -290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides -257 and -267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides -263 and -290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I. The second footprint, located between nucleotides -116 and -140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human-growth-hormone gene is therefore a candidate for regulation by these factors in vivo.