©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Transcriptional Activator Originating from an Upstream Promoter in the Human Growth Hormone Gene (*)

(Received for publication, March 2, 1995; and in revised form, June 15, 1995)

Nathalie Labarrière (1)(§) Philippe L. Selvais (2)(¶) Frédéric P. Lemaigre (1)(**) Alain Michel (3) Dominique M. Maiter (2) Guy G. Rousseau (1)(§§)

From the  (1)Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology and University of Louvain Medical School, 75 Avenue Hippocrate, B-1200 Brussels, Belgium, the (2)Diabetes and Nutrition Unit, University of Louvain Medical School, 54 Avenue Hippocrate, B-1200 Brussels, Belgium, and the (3)Laboratory of Biological Chemistry, University of Mons, 15 Avenue Maistriau, B-7000 Mons, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transcription of the human growth hormone gene can start in vitro and in vivo 197 base pairs upstream from the cap site of growth hormone mRNA (Courtois, S. J., Lafontaine, D., and Rousseau, G. G.(1992) J. Biol. Chem. 267, 19736-19743). We have now characterized the mRNA that originates from this optional promoter and have found that it occurs in human hypophysis and placenta but not in 10 other tissues. This mRNA contains an open reading frame for a protein of 107 residues that shares sequence similarity with three domains of hepatic nuclear factor-1alpha. With antibodies directed against a peptide corresponding to the C terminus of this protein, immunoreactive material was detected in a subset of cells of the adenohypophysis. When fused to the DNA-binding domain of the yeast transcription factor GAL4, the protein stimulated transcription from a GAL4-sensitive reporter gene in transiently transfected pituitary and placental cells.


INTRODUCTION

The sequence between -294 and -177 in the human growth hormone (hGH-N or hGH-1) (^1)gene resembles the adenovirus-2 major late promoter in that it contains a TATA box (-229 to -223) 30 base pairs downstream from a binding site (-266 to -253) for upstream stimulatory factor(1) . This sequence functions as a promoter in cell-free assays, and it drives upstream stimulatory factor-dependent transcription from a promoterless reporter gene in transfected cells(2) . Transcripts starting at -197 were detected by RNase mapping in human pituitary tissue but not in HeLa cells(2) . If translated, such transcripts are not expected to code for GH. Indeed, there are three ATG codons in a Kozak's consensus (3) between -197 and -63 and a potential splice site at -63. The first and third ATG are followed by an open reading frame (ORF) not longer than 6 residues. The second ATG (at -151) is in a better Kozak's consensus than the first one and is followed by an ORF that reaches position -63 without being interrupted by a stop codon. GH mRNA contains five (I-V) coding exons. Whether the 3` end of the exon starting at -197 is at the splice site -63 or coincides with that of exon I, none of the possible splicing combinations with exons II-V of GH mRNA restitutes an ORF corresponding to that of GH. We therefore decided to clone and sequence the mRNA actually spliced from the primary transcript originating from the upstream promoter and to study its translation product. We show here that this product behaves as a GH gene-derived transcriptional activator (GHDTA).


EXPERIMENTAL PROCEDURES

Cloning and Detection of GHDTA mRNA

A human pituitary cDNA expression library in gt11 (Clontech) was screened (5.10^5 plaques) with the cDNA probe (c in Fig. 1A) corresponding to the hGH-1 gene fragment from -294 to -58. The positive clones, which also scored positive with the anti-AMR544 antibodies (see text), were isolated from phage DNA by EcoRI digestion, subcloned in pBluescript, purified with a Qiagen kit, and sequenced by polymerase chain reaction (PCR), using an U. S. Biochemical Corp. kit, with a LI-COR DNA sequencer model 4000L and dye-labeled T3 and T7 primers. Ambiguous sequences were verified by sequencing with a T7 QuickPrime kit (Promega) using nested primers. For in situ hybridization (4) pPSP72phGH was obtained by cloning the -294/-58 hGH gene fragment in pSP72 digested with EcoRI and HindIII. S-Labeled (Boehringer kit) sense and antisense RNA probes were prepared by transcribing the linearized vector with T7 and SP6 RNA polymerase, respectively. Pituitary tissue obtained 6 h postmortem was fixed with 4% paraformaldehyde and embedded in paraffin. Sections (5 µm) were hybridized overnight at 56 °C with the antisense or sense riboprobes. To detect GHDTA mRNA in tissues, total RNA was reverse transcribed with oligo(dT), and the cDNA (corresponding to 50 ng of RNA) was amplified by PCR with Taq polymerase (1 min at 95 °C, 1 min at 57 °C, 1 min at 72 °C, 30 cycles) using the sense (5`-TCCATTAGCACAAGCCCGTC) and antisense (5`-GAATGGTTGGGAAGGCACTG) primers indicated in Fig. 1A by a and b, respectively.


Figure 1: Structure of GHDTA mRNA (A) and its detection by in situ hybridization in human pituitary (B). GHDTA mRNA is identical to GH mRNA (from exon I, starting at +1, to exon V) except that exon I has a 0.2-kb 5` extension (shadedarea). The ORFs for GH (thick dashedline), presumably not translated from this mRNA (see text), and for GHDTA (thickline) are in a different frame. The double-headed arrow refers to the location of the cDNA probe used for cloning the mRNA (A) and of the antisense riboprobe used in B. The arrows refer to the PCR primers used to detect GHDTA mRNA in tissues.



Detection of the GHDTA Protein

Peptides AMR544 (see Fig. 2A), with or without an additional tyrosine residue at the N- or C-terminal end to facilitate labeling, were synthesized by solid-phase synthesis using 9-fluorenylmethyloxycarbonyl chemistry on an Applied Biosystems 431A instrument (5) and were purified by reverse phase high performance liquid chromatography (Vydac C18) in the 0.1% trifluoroacetic acid/acetonitrile system. Antibodies were obtained from rabbits injected with AMR544 (15-mer) coupled to keyhole limpet hemocyanin. Immunochemistry was performed on paraffin sections(6) , using antisera directed against AMR544 (1:10,000), human corticotropin (ACTH), GH, prolactin, beta-thyrotropin, beta-luteinizing hormone and beta-follicle-stimulating hormone (UCB, 1:2,000), and porcine galanin (Peninsula, 1:3,000). The specificity of AMR544 labeling was demonstrated by serial dilution of the antiserum and by the disappearance of the signal after omission of the antiserum, its replacement by a nonimmune rabbit serum, or its preadsorption with synthetic AMR544 peptide (10 µM). In contrast, the signal persisted after preadsorption of the anti-AMR544 antiserum with human ACTH or porcine galanin. It was also shown by radioimmunoassay that neither ACTH nor galanin was recognized by the antiserum.


Figure 2: Structure and detection of the GHDTA protein. A, predicted amino acid sequence of GHDTA and comparison with the dimerization domain (residues 20-54), activation domain II (residues 291-306), and activation domain I (residues 614-621) of human HNF-1alpha. The peptide (AMR544) synthesized to raise antibodies is underlined. The amino acid sequence shown is the ORF starting at the first ATG (at -151 in the hGH gene, see Fig. 1A) common to the GHDTA cDNA clones and ending, because of a stop codon, at the 98th nucleotide of exon II. Discrepancies in published sequences (7, 24, 25) suggest a polymorphism in the hGH gene. As a consequence, some GHDTA mRNAs would yield a peptide ending with a valine after glutamine 40. B, immunostaining for AMR544 (a, c) and ACTH (b, d) in the normal human pituitary gland. On a sagittal section (a, 40) AMR544-immunoreactive cells are observed in a restricted area of the anterior pituitary (AP) and in the intermediate lobe (IL) and are spreading into the posterior pituitary (PP). This distribution is similar to that of the corticotrophs (b), although some of the latter do not show AMR544 immunoreactivity in the anterior pituitary. Colocalization of immunoreactivities of AMR544 (c) and ACTH (d) within adenohypophysial cells is apparent on semi-serial sections (100), as indicated by arrows.



Transactivation Assays

pGAL4, pGHDTA, or pGAL4-GHDTA expression vectors were constructed by inserting into pCMV-NH a cDNA coding for residues 1-95 of the yeast transcription factor GAL4, for the 107 residues of GHDTA, or for an in-frame fusion of the two proteins, respectively. The pGAL4-TFIIB expression vector was constructed by in-frame fusion of amino acids 1-100 of the human basal transcription factor TFIIB downstream from amino acids 1-95 of GAL4 in pGAL4. The reporter plasmid was pE1B-CAT, which contains five consensus 17-mer GAL4-binding sites upstream from the minimal promoter of the adenovirus E1b TATA box fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Cells were cotransfected, using the calcium phosphate precipitation (2) (GC cells) or Lipofectin (Life Technologies, Inc.) (JEG-3 cells) method, with 1 µg of expression vector and 5 µg of reporter plasmid. After 38 h, CAT activity and protein concentration were determined in cell lysates with the Quan-T-CAT assay system (Amersham Corp.) and with the Bio-Rad reagent, respectively. Phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) or the vehicle dimethyl sulfoxide (0.01%) was added 20 h prior to cell harvest. Background activity (CAT/mg of protein) measured in nontransfected cells was subtracted.


RESULTS AND DISCUSSION

To clone the mRNA transcribed from the upstream promoter of the hGH gene, a human pituitary cDNA library was screened with a cDNA probe corresponding to the hGH gene fragment from -294 to -58. The tertiary screening yielded five clones, all originating from the hGH gene and ending with a poly(A) tail. Two clones (2 and 1.6 kb) corresponded to immature transcripts. Clones S4 (1 kb) and S6 (1 kb) corresponded to mRNAs spliced in the same way as the major GH mRNA coding for native (22 kDa) GH, but their 5` end extended to -174 for S4 and to -164 for S6. Clone S7 (0.95 kb) corresponded to a mRNA that extended to -167 but was spliced in the same way as the GH mRNA coding for the 20-kDa GH variant, which results from splicing of exon II with a splice site inside exon III ((7) ). Thus, mRNAs originating from the upstream promoter of the hGH gene are polyadenylated and are identical to GH mRNAs except that their exon I has a 5` extension of about 0.2 kb (Fig. 1A).

Transcription of the hGH gene to give GH mRNA occurs only in a subset of anterior pituitary cells, the somatotrophs. This results mainly from the presence in these cells of a cell type-specific, POU homeodomain transcriptional activator called Pit-1 (reviewed in (8) ), which binds from -65 to -92 and from -105 to -130 in the hGH gene. To determine whether transcription from the upstream promoter was similarly cell-restricted, we performed in situ hybridization on human pituitary tissue with a specific riboprobe. As shown in Fig. 1B, positive signals were detected with the antisense probe only in a limited number of anterior pituitary cells. No signal was seen with the sense riboprobe (not shown). The corresponding mRNA was searched for in extrapituitary human tissues by reverse transcription-PCR amplification using intron-spanning specific primers (Fig. 1A). While a positive signal of the expected length (268 base pairs) was seen with placental RNA, none was found with RNA from umbilical cord, uterus, liver, muscle, bone marrow, kidney, adrenal, skin, brain, and cerebellum, under conditions where actin mRNA was detectable.

The sequence of the mRNAs derived from the upstream promoter predicts that their translation yields a 11,421-Da protein of 107 residues (including the initial methionine) whose sequence bears no relationship with that of GH (Fig. 2A). This protein, which we call GHDTA, starts at the AUG corresponding to position -151 in the gene, and it ends at a stop codon in exon II because of a frameshift encompassing the entire sequence that GHDTA mRNA shares with GH mRNA (Fig. 1A). To detect the GHDTA protein in tissues, we synthesized a peptide (AMR544) corresponding to residues 92-106 of GHDTA and raised antibodies in rabbits. Using anti-AMR544 antiserum (1:10^4) and I-labeled C-Tyr-AMR544, immunoreactive material was detected by radioimmunoassay in extracts from a normal human pituitary and from a corticotroph adenoma. Consistent with the in situ hybridization data, immunohistochemistry with anti-AMR544 antiserum showed specific labeling of a subpopulation of cells located in the anterior and intermediate lobes and spreading into the posterior lobe of the pituitary (Fig. 2B). To identify these cells, semi-serial sections were specifically labeled with antibodies against several human pituitary hormones. Anti-AMR544 immunoreactivity did not colocalize with GH, prolactin, beta-follicle-stimulating hormone, beta-luteinizing hormone, and beta-thyrotropin. In contrast, the labeling did colocalize with ACTH and galanin, a hormonal pattern typical of corticotrophs in humans(9) . The colocalization of immunoreactivity against AMR544 and ACTH was confirmed by double-labeling experiments on the same tissue sections. All these data strongly argue for the pituitary-specific transcription and translation of the GHDTA mRNA.

Data bank searches showed no identity of the GHDTA protein with known sequences. However, there was a striking similarity (45, 68, and 87%) with three regions (Fig. 2A) of the homeodomain protein called hepatocyte nuclear factor-1alpha (HNF-1alpha) (10) or liver-specific transcription factor B-1 (LFB-1)(11) . One of these regions of HNF-1alpha overlaps with the dimerization domain; the two other regions are in the activation domains(12) . This suggested that GHDTA might be a transcription factor. To address this question, we constructed a chimeric protein in which GHDTA replaces the transcription activating domain of the yeast zinc-finger protein GAL4. To do so, a cDNA corresponding to the coding sequence of GHDTA mRNA was inserted into an eukaryotic expression vector downstream from, and in-frame with, the cDNA fragment coding for the otherwise transcriptionally inactive DNA-binding domain of GAL4. The latter was chosen because it functions with a variety of heterologous transcription activation domains but not with proteins devoid of such domains(13, 14, 15) . The activity of the resulting GAL4-GHDTA chimeric protein was tested by cotransfecting this vector with a plasmid containing a CAT reporter gene driven by a GAL4-sensitive promoter. In rat pituitary GC cells, some CAT activity was detected after cotransfection with a vector expressing only the GAL4 DNA-binding domain. This presumably reflected the basal activity of the reporter gene. The same basal CAT activity was seen after cotransfection with a vector expressing only the GHDTA protein. In contrast, CAT activity was reproducibly increased after cotransfection with a vector expressing the GAL4-GHDTA chimeric protein but not after cotransfection with a control vector expressing a GAL4-TFIIB chimeric protein (Fig. 3). This transcriptional activity of the GAL4-GHDTA construct was also demonstrable in transfected human placental JEG-3 cells (Fig. 3). Residues 10, 57, and 93 of GHDTA are putative protein kinase C (PKC)-dependent phosphorylation sites(16) . PKC-dependent phosphorylation of transcription factors can affect their nuclear translocation, DNA binding, or transactivation potential(17) . We therefore determined whether stimulation of PKC by PMA would affect the activity of the chimeric protein in transfected cells. The chimeric protein was indeed three times more active in JEG-3 cells treated with PMA than in nontreated cells. PMA had no effect in GC cells (Fig. 3). This suggests that in the placenta, but not in the pituitary, GHDTA activity is liable to PKC-dependent control.


Figure 3: Transactivation reporter assays using the GHDTA protein fused to the GAL4 DNA-binding domain. CAT activity was measured in cultured rat pituitary GC and human choriocarcinoma JEG-3 cells transiently transfected with a CAT reporter plasmid and an expression vector for GAL4 alone, for GHDTA alone, or for the chimeric proteins GAL4-GHDTA and GAL4-TFIIB, as indicated below the histograms. The data, obtained with two different preparations of expression vectors, are the means ± S.E. for three to six (no PMA) and one or two (PMA, 100 ng/ml) independent experiments.



Transcription activation domains of proteins have been classified as acidic, glutamine-rich, and proline-rich(13) . Proline-rich domains occur in HNF-1(18) , CAAT box transcription factor/nuclear factor-I (CTF/NF-I)(19) , and myocyte nuclear factor (MNF)(20) . In GHDTA 13% of the residues are prolines, 10 being clustered within a 40-mer (49-89) segment. GHDTA has a net positive charge of 10.5, 10 basic amino acids being clustered within a 22-mer (27-48) segment as in the DNA-binding domain of Jun(13) . Thus, despite its small size, GHDTA could be a DNA binding transcription factor like the ICER (inducible cAMP early repressor) basic leucine zipper proteins (108 and 120 residues), which originate from an optional promoter of the CREM (cAMP-responsive element modulator) gene(21) . Alternatively, GHDTA could act as a nonacidic activator of a DNA binding factor(s)(22) , such as the 11-kDa DCoH protein (104 residues), which acts as a cofactor of HNF-1(15) . As translation of GHDTA mRNA in the pituitary appears to be restricted to corticotrophs, GHDTA might help activate genes whose expression is corticotroph-specific, such as the proopiomelanocortin gene(23) , or it might repress in these cells the GH gene by virtue of a negative autoregulatory loop similar to the one exerted on the cAMP-responsive element modulator gene by the inducible cAMP early repressor proteins(21) .


FOOTNOTES

*
This work was supported by grants from the Fonds de la Recherche Scientifique Médicale (Belgium) and from the Interuniversity Poles of Attraction of the Services Fédéraux des Affaires Scientifiques, Techniques et Culturelles (Belgium). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institut de Biologie, U419-INSERM, 9 Quai Moncousu, F-44035 Nantes Cedex, France.

Research assistant of the Fonds National de la Recherche Scientifique (Belgium). Present address: NICHD-ERRB, NIH, Bethesda, MD 20892.

**
Research Associate of the Fonds National de la Recherche Scientifique (Belgium).

§§
To whom correspondence should be addressed: ICP-UCL, Box 7529, 75 Avenue Hippocrate, B-1200 Brussels, Belgium. Tel.: 32-2-7647530; Fax: 32-2-7627455.

(^1)
The abbreviations used are: (h)GH, (human) growth hormone; ORF, open reading frame; GHDTA, GH gene-derived transcriptional activator; PCR, polymerase chain reaction; ACTH, corticotropin; HNF-1, hepatocyte nuclear factor-1; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C; kb, kilobase(s); PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We thank J. Martial for providing the pituitary cDNA library and the cell lines, F. Brasseur for providing human cDNAs, C. Southgate for providing expression vectors, J. Lejeune and F. Opperdoes for help with data bank searches, M. De Cloedt for preparing antibodies, I. Kokorine and C. Landry for help with in situ hybridization, J. M. Brucher, J. F. Denef, and J. Rahier for providing histological material, J. M. Ketelslegers for advice on radioimmunoassay, and N. Aidant for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.