(Received for publication, March 2, 1995; and in revised form, June 15, 1995)
From the
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-1. 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.
The sequence between -294 and -177 in the human
growth hormone (hGH-N or hGH-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).
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.
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-1. 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.
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) 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,
-follicle-stimulating hormone,
-luteinizing hormone, and
-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-1 (HNF-1
) (10) or liver-specific
transcription factor B-1 (LFB-1)(11) . One of these regions of
HNF-1
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) .