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Original Studies |
Laboratory of Molecular Biology and Genetic Engineering (F.P., V.M.C., M.M., J.A.M.), University of Liège, B-4000 Sart Tilman, Liège, Belgium; Department of Obstetrics, Gynecology and Pediatrics (R.D.G.M., F.d.Z.), University of Leuven, B-3000, Leuven, Belgium; Department of Pediatrics (A.A.Z.A.A.), King Faisal Specialist Hospital and Research Center, 11211 Riyadh, Kingdom of Saudi Arabia
Address all correspondence and requests for reprints to: Joseph A. Martial, Laboratory of Molecular Biology and Genetic Engineering, University of Liège, B-4000 Sart Tilman, Liège, Belgium. E-mail: jmartial{at}ulg.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix of
the POU-homeodomain and is strictly conserved among all POU proteins.
The Pro239Ser mutant binds DNA normally but is unable to stimulate
transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abnormalities in the Pit-1 gene were first observed in Snell and Jackson dwarf mice. Their consequence was a combined pituitary hormone deficiency (CPHD) with hypoplasia of the anterior pituitary and no production of GH, PRL, or TSH (11). A number of CPHD patients are known to have a point mutation in the Pit-1 coding sequence (12, 13, 14, 15, 16, 17, 18, 19, 20). Some of these mutations affect the DNA-binding capacity (12, 20) of the Pit-1 protein, others alter its transactivation properties (13, 14, 15). From a comparison of their pedigrees, it seems that some mutations in the Pit-1 gene (Arg271Trp and Pro24Leu) lead to a dominant-negative phenotype (13, 15, 16, 17, 18, 19); others (Arg172stop, Glu250Stop, Ala158Pro, Arg143Gln), to a recessive phenotype (14, 15, 20).
Here, we report the identification of a novel homozygous Pro239Ser mutation in the Pit-1 gene of seven Middle Eastern hypopituitary children from three reportedly unrelated families. In addition, we present the properties of the mutant Pit-1 protein, with respect to transactivation, DNA binding, and interaction with its wild-type counterpart.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For DNA extraction from blood spots (5 mm2) on filter paper, we used the ChelexR 100 chelating resin, as described by Walsh and co-workers (21). PCR was used to amplify the six Pit-1 exons independently, using six pairs of oligonucleotides corresponding to the intron/exon boundaries (15). Reactions were carried out as follows: approximately 100 ng genomic DNA, 20 pmol of each primer, 200 µmol/L of each nucleotide, 1 U Dynazyme polymerase, and 1x Dynazyme reaction buffer were mixed in a final vol of 100 µL. Reactions were cycled 30 times for 1 min at 94 C, 1 min at 55 C, and 1 min at 72 C. The amplified products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide to evaluate their size, purity, and quantity. Sequencing was performed directly on the amplified fragments, thus allowing immediate evaluation of heterozygosity.
Site-directed mutagenesis
We used the CHAMELEON double-stranded, site-directed mutagenesis kit from Stratagene Cloning Systems (La Jolla, CA) to obtain the mutated Pit-1 complementary DNA (cDNA). A plasmid, containing nonmutated human Pit-1 cDNA cloned behind the RSV promoter (pRSVhPit-1) (22), was used as a template.
The oligonucleotide 5'CTCTTGAGAAGAAGATTTATTCTGTTC-TCC3', containing the specific T-to-C substitution in codon 239, on the antisense strand, was employed. Mutated clones were tested by DNA sequencing and restriction analysis.
In vitro transcription/translation reactions
For in vitro transcription and translation, both the wild-type and the mutant Pit-1 cDNA were inserted into the XbaI-XbaI sites of plasmid pSVK3 from Pharmacia LKB Biotechnology (Uppsala, Sweden), which contains a T7 promoter. The in vitro reactions were carried out using the coupled transcription/translation system: TnTa Coupled Reticulocyte Lysate Systems (from Promega, Madison, WI) and [35S]-labeled methionine. Translation products were checked by electrophoresis on 5% polyacrylamide gels, and two major species (31 and 33 kDa) were observed, as expected (data not shown).
Gel-retardation assays
For the binding assays, we used the same amount of in vitro translated proteins, as judged by protein gel autoradiography (overnight exposure). The assays were performed in a mixture containing 2 µL translated protein solution, 1 µg poly dIdC, 7 mmol/L HEPES (pH 7.9), 4% glycerol, 4% Ficoll, 100 mmol/L KCl, 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 2 mmol/L dithiothreitol, and 10,000 cpm labeled hPRLP1 oligonucleotide (8), in a final vol of 20 µL. Two microliters of anti-Pit-1 antibodies or control serum was added when necessary. Binding was carried out for 30 min at 4 C (1 h with antibodies), after which samples were loaded on a 5% polyacrylamide gel, in TBE 0.5X, for electrophoresis.
Production of anti-Pit-1 antibodies
A fragment of human Pit-1 cDNA, containing the coding regions for the N-terminus and a small part of the POU-domain (amino acids 1–193), was cloned in the pT7 bacterial expression vector and expressed in the bacterial strain BL21 after addition of 100 mmol/L IPTG and incubation at 37 C for 1 h. The approximately 23-kDa protein was subsequently purified for use as an antigen. The activity of the serum from injected rabbits was tested on Western blots (data not shown) with pituitary-derived cell extracts (GC, GH3), and the expected 31- to 33-kDa doublet bands corresponding to Pit-1 were obtained. We observed no extra bands corresponding to other POU family members, such as Oct-1, proving the high specificity of the antibodies against Pit-1. As expected, no bands appeared in the negative control performed with nonpituitary cell (HeLa) extracts.
Cotransfection assays
Human HeLa cells were plated on 60-mm plates at a density of 5 x 105 cells per plate and transfected with 5 µg of reporter plasmid p(GH1)6XTKCAT (23). The amount of effector plasmid was kept constant by addition of RSV-ßgal, where appropriate, as indicated in the figure legends. We used the calcium phosphate copreciptation method for transfection. Cells were harvested 48 h after transfection, and chloramphenicol-acetyl-transferase (CAT) assays were performed as previously described (23). The data shown are averages of at least three independent experiments, with error bars indicating the SEM.
Oligonucleotides and enzymes
The oligonucleotides used in this study were from Eurogentec (Seraing, Belgium) and the enzymes from Boehringer, Eurogentec, Gibco-BRL, New England Biolabs, and Pharmacia.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All described patients presented a clinical picture of congenital hypothyroidism and early growth failure and were referred to the King Faisal Specialist Hospital and Research Center. Studies were carried out with the informed consent of all parents.
The seven children were from three, reportedly independent families
from the Al-Baha region in the Southwest of Saudi Arabia. In each case,
the parents were second-degree relatives (see Fig. 1
) and of normal stature for this
community. The maternal height ranges from 149.5 to 159.2 cm and the
paternal height, from 162.6 to 166.4 cm.
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All children were treated with L-T4 from early infancy. They also received a GH therapy, which started between the ages of 4 months and 3 yr plus 4 months. The height deficit, after adjustment for midparental height SD increased with advancing age, amounting to -2.9 SD at early start and -6.0 SD at latest start of GH therapy.
Molecular studies
A new point mutation in the Pit-1 gene. The human Pit-1 gene is 17-kb long and composed of six coding exons (24). For each family member analyzed, all six exons of the Pit-1 gene were amplified by PCR and directly sequenced. Sequence analysis revealed only one alteration, a T->C transversion in codon 239, located in exon 6. This alteration leads to the substitution of serine for proline at codon 239. The affected children are all homozygous, the unaffected parents all heterozygous for this mutation.
Pro239Ser mutant is unable to transactivate. To analyze the
functional effects of the Pro239Ser mutation, we first used
site-directed mutagenesis to produce an identical substitution in human
Pit-1 cDNA, borne by the expression vector pRSVhPit-1. We shall refer
to the parental plasmid as pRSVPit-1WT and to the plasmid bearing
the Pro239Ser mutation as pRSVPit-1M. Cotransfection experiments in
HeLa cells were performed using one of the Pit-1 expression vectors
with a reporter plasmid, p(GH1)6XTKCAT, bearing six copies of the first
Pit-1 binding site of the human GH promoter (hGH1: from -71 to -91,
see Ref.8), inserted upstream from the TK promoter and the CAT
reporter gene. As shown in Fig. 2, the
mutant Pit-1 was unable to increase transcription of the (GH1)6XTKCAT
fusion-gene construct, whereas wild-type Pit-1 stimulates its
transcription 5-fold.
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, the
mutated and wild-type Pit-1 proteins bind with the same affinity to the
first Pit-1 DNA-binding site of the human PRL gene (hPRL1P: from -35
to -64, see Ref.8). The complexes are specific, because they
disappear with an excess of cold hPRL1P oligonucleotide but not with an
excess of cold, nonspecific DR4 oligonucleotide. The specificity is
further confirmed by the fact that the shifted bands disappear in the
presence of anti-Pit-1 antibodies but not in the presence of control
serum. A nonspecific lower band is observed with unprogrammed
reticulocyte lysate.
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, the CAT activity doubles when the
amount of pRSVPit-1WT is increased from 7 µg to 14 µg, whether or
not pRSVPit-1M is also present. We also observe 2-fold lesser
transactivation by wild-type Pit-1 at both vector concentrations when
the mutated form is expressed. The Pro239Ser form alone is unable to
stimulate transcription of the reporter plasmid.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proline 239 is strictly conserved among the Pit-1 proteins of several
species and among other related POU proteins (Fig. 5), implying that it must play an
important role in Pit-1 activity. This residue is located at the
beginning of the second -helix of the homeodomain, which was
originally described as the Pit-1 DNA binding domain (3). In gel
retardation experiments, we show that the Pro239Ser mutant form of
Pit-1 is still able to recognize the first Pit-1 binding site of the
human PRL proximal promoter and specifically binds to this sequence
with about the same affinity as the wild-type Pit-1. These results are
in agreement with the three-dimensional conformation of the POU domain
[recently established for Oct-1 (25) and for the Pit-1 POU domain (26)
cocrystallized with their respective binding sequence], which seems to
indicate that residue 239 is not in direct contact with DNA. In
addition, Proline 239 occupies the N-cap position of the helix 2 in
the Pit-1 homeodomain. Prolines are frequent at N-cap positions of
-helices, where they play a stabilizing role (27). In our mutant,
proline 239 is replaced by a serine, another residue known to have a
stabilizing effect on -helices when in the N-cap position. Thus, one
should not expect the stability of the second -helix of the Pit-1
homeodomain, and hence the conformation of the protein, to be seriously
affected by the Pro239Ser mutation. Furthermore, a genetic screen used
to define the important residues for Pit-1 DNA-binding, based on loss
of function of fusion proteins between the transactivation domain of
GCN4 and chemically mutated Pit-1 DNA binding domains, failed to detect
the Pro239Ser mutation described here (28). All these considerations
support our finding that this Pit-1 variant is capable of binding DNA
with the same affinity as the wild-type form.
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) clearly show
that the Pro239Ser mutation causes complete loss of transactivation
activity. The hypothesis of nonexpression of the mutated protein in
HeLa cells must be rejected, because activation by wild-type Pit-1 is
partially inhibited when the vector bearing the mutated cDNA is also
present (Fig. 4). Without expression of this cDNA, such an effect would
be hard to explain.
When pRSVPit-1WT and pRSVPit-1M are present in equal amount (7 or 14
µg each) in cotransfected cells, the transactivation potential of
wild-type Pit-1 is reduced by 50% (Fig. 4). These in vitro
results are in keeping with the clinically observed recessive phenotype
of this mutation if we assume that a 50%-reduced level of Pit-1
activity is sufficient to ensure a normal phenotype.
Because Pit-1 forms dimers on DNA through its POU-specific domain, two
mechanisms might account for the ability of the mutant to repress wild
type Pit-1 activity: competition between mutated and wild-type Pit-1
for DNA binding sites; or formation of inactive heterodimers. The first
possibility must be rejected because the reporter plasmid was in excess
in our experiments [as shown by the 2-fold increase of CAT activity
observed when the amount of vector expressing wild-type Pit-1 was
doubled, whether this vector was present alone or in combination with
the vector expressing the mutated form (Fig. 4)]. We therefore suggest
that the observed inhibitory effect of the Pro239Ser protein is caused
by formation of heterodimers that can bind to DNA but cannot stimulate
transcription, as is the case with mutant homodimers. This model
suggests that the mutation of Pro239 into a serine abolishes the
interaction of Pit-1 with another factor(s) required for
transcriptional activation.
In brief, our data show that a single point mutation in codon 239 of the Pit-1 gene, causing the substitution of a serine for a proline, leads to the phenotype of GH, PRL, and TSH deficiency and hypoplasia of the anterior pituitary, when present in both Pit-1 alleles. Because heterozygous individuals are seemingly unaffected, it further seems that a 50%-reduced level of Pit-1 activity is sufficient to ensure a normal phenotype.
The newly recognized, natural, recessive Pit-1 mutation that we describe has been found in only three Middle Eastern families. The relatively frequent and apparently exclusive occurrence of this mutation in a defined geographic region is striking. This occurrence may be based on a so-called founder effect, the three families having a single and common heterozygous ancestor in whom this mutation first arose. Alternatively, we may be in the presence of a hot spot for mutations in the Pit-1 gene within this ethnic community, distinct from the Arg271Trp hot spot that has been described in Caucasians and Mongolians (16).
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Acknowledgments |
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Footnotes |
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2 A fellow of the National Council for Scientific and Technological
Development, CNPq, Brazil. Current affiliation: Department of
Reproductive Medicine, University of California, La Jolla, San Diego,
California 92093-0674.
4 Current affiliation: Institute of Human Genetics, University of
Minnesota, Minneapolis, Minnesota 55455.
Received September 23, 1997.
Revised March 11, 1998.
Accepted March 18, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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