Combined Pituitary Hormone Deficiency due to the F135C Human Pit-1 (Pituitary-Specific Factor 1) Gene Mutation: Functional and Structural Correlates
Sophie Vallette-Kasic,
Isabelle Pellegrini-Bouiller,
François Sampieri,
Ginette Gunz,
Adriana Diaz,
Sally Radovick,
Alain Enjalbert and
Thierry Brue
Laboratory ICNE UMR 6544 (S.V.-K., I.P.-B., G.G., A.D., A.E.,
T.B.) Ingéniérie des Protéines, UMR 6560
(F.S.) IFR Jean-Roche Faculté de Médecine Nord
13916 Marseille Cedex 20, France
Division of
Endocrinology Childrens Hospital (S.R.) Harvard Medical
School Boston, Massachusetts 02115
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ABSTRACT
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The pituitary-specific transcription factor Pit-1
(pituitary-specific factor 1) is known to play a key role in the
differentiation of PRL-, GH-, and TSH-secreting cells, and in the
regulation of expression of the corresponding genes. In recent years,
12 distinct mutations of the Pit-1 gene have been shown to
be responsible for a phenotype of multiple congenital pituitary hormone
deficiency involving PRL, GH, and TSH. We had previously identified, in
four siblings with GH, PRL, and TSH deficiencies, a mutation (F135C)
resulting in a single amino acid change within the POU-specific binding
domain of the Pit-1 molecule. In the present report, we have explored
the functional effect of the F135C mutation. In vitro
activity tests performed by transfection in human HeLa cells showed
decreased transactivation capacity on the PRL, GH, and
Pit-1 genes. The DNA binding experiments performed by gel
shift showed that the F135C mutation generated a protein capable of
binding to DNA response elements. To analyze how the F135C mutation
might affect functionality of the transcription factor despite a normal
DNA binding, we used a structure modelization approach and also
analyzed two other Pit-1 mutant proteins (F135A and F135Y). The loss of
functionality in these two mutants was similar to that of F135C.
This finding was in keeping with our molecular modeling studies.
According to structural data derived from the crystallographic analysis
of the DNA/Pit-1 POU domain complex, the conformation of the first
helix of the F135C-mutated POU-specific domain could be perturbed
to such an extent that any interaction with other transcription
cofactors might be definitively prevented.
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INTRODUCTION
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Pit-1 (pituitary-specific factor 1) as most commonly termed, or
POU1F1 according to the official nomenclature, is a pituitary-specific
transcription factor responsible for pituitary development and hormone
expression in mammals. It is required for the expression of pituitary
hormones through transactivation of the GH, PRL, and TSH-ß genes, for
the differentiation and proliferation of somatotrophs, lactotrophs, and
thyrotrophs, and for the regulation of the corresponding hormones (1, 2). As a member of the family of POU domain transcription factors,
Pit-1 contains a C-terminal transactivation domain and a DNA-binding
region. The DNA-binding domain consists of a POU homeodomain (POU-HD)
essential for DNA binding and a POU-specific domain (POU-S), which is
required for high-affinity and high-specificity DNA binding on the GH
and PRL genes, and for protein-protein interactions (2). Pit-1 can bind
to and transactivate the TSH-ß promoter and is also expressed in the
thyrotrophs (3). Pit-1 is monomeric in solution but associates as a
dimer on most of its DNA response elements (2, 4). Pit-1 transcription
is maintained by autoregulation through two Pit-1-binding elements and
additional transcriptional regulation (5). Naturally occurring
mutations in the Pit-1 gene were first recognized in two
murine models. The Jackson dwarf mouse has an inversion or insertion of
a more than 4 kb segment of the Pit-1 gene DNA. The gross
structural alteration of this gene is responsible for a phenotype of
combined pituitary hormone deficiency (CPHD) of GH, PRL, and TSH, a
hypoplastic anterior pituitary, and absence of Pit-1 gene
expression (6). Snell dwarf mice also have hypoplastic anterior
pituitaries and CPHD, but they have a low level of Pit-1
gene expression. In these animals, a G-to-T mutation in both alleles of
the Pit-1 gene alters a tryptophan to a cysteine in codon
261 (W261C) in the DNA recognition helix of the POU-HD. The loss of
functionality of the mutant protein was clearly related to a defective
DNA binding to Pit-1 response elements. In recent years, 12 distinct
mutations of the human Pit-1 gene have been shown to be
responsible for a phenotype of multiple congenital pituitary hormone
deficiency involving PRL, GH, and TSH (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In contrast to the Snell
mouse model, functional in vitro experiments performed on
four of these mutations have shown that mutant proteins had defective
transcription activation properties despite a normal DNA binding.
Inhibition of transactivation may be explained by several different
mechanisms, which can be examined in view of the recent findings on
Pit-1 structure (4, 20) and protein-protein interactions (21, 22, 23). We
had previously identified, in four siblings with GH, PRL, and TSH
deficiencies, a homozygous mutation resulting in a single amino acid
(aa) change (a phenylalanine (Phe) to a cysteine (Cys) in codon 135:
F135C) within the POU-S binding domain of the Pit-1 molecule (8). In
the present report, we have explored the functional effect of the F135C
mutation. This mutation inhibits activation of the PRL and GH target
genes, despite a conserved DNA binding capacity. Taking advantage of
crystal structure data available for the Pit-1 POU domain-cognate DNA
complex, we also modelized the structural consequences of this
mutation and applied these findings to our experimental data. We
also analyzed two other mutant proteins (F135A and F135Y) to confirm
the critical role of the Phe residue at position 135.
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RESULTS
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The F135C Mutant Has Decreased Transactivation Capacity on the GH,
PRL, and Pit-1 Genes
In HeLa cells, wild-type (wt) Pit-1 induced a 22 ± 3.3-fold
increase in the activity of the luciferase gene fused to the PRL-250
promoter (Fig. 1A
). A similar 21.5
± 5.1-fold increase was obtained using the PRL-164 promoter (not
shown). The F135C mutant Pit-1 protein had a decreased transactivation
capacity on this reporter gene (6 ± 2.1-fold), representing 27%
of the wild type (Fig. 1
). The mutant Pit-1 had also a decreased
transactivation capacity on the GH gene and on its own gene, with a
mean stimulation of 2.2 ± 0.35- and 1.6 ± 0.4-fold,
respectively, while the transcription activation of GH and
Pit-1 genes by the wt Pit-1 protein was 3.9 ± 0.8-fold
and 6.1 ± 1.4-fold, respectively (Fig. 1
, B and C). Similar
results were obtained in the CV-1 cells (not shown). Nuclear expression
of both wt and mutant Pit-1 vectors was ascertained by Western blot
analysis (Fig. 1D
).

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Figure 1. Activation Capacity of the PRL (A), GH (B), and
Pit-1 (C) Promoters in Pit-1-Deficient Cells
HeLa cells were transiently transfected by electroporation with 5
µg of an effector plasmid [pcDNA3 EV, wild type Pit-1 (wt), F135C,
F135A, or F135Y Pit-1 mutants] and 5 µg of a reporter plasmid
[PRL-250 (A), GH (B), or Pit-1 (C) promoter constructs], as
indicated. Electroporations were performed in triplicate for each
condition within a single experiment, and experiments were repeated
five times using different plasmid preparations of each construct.
Results are expressed as fold activation over control and represent the
mean ± SD of triplicates in a representative
experiment. D, Western blot analysis of the cellular extract.
Equivalent amounts of wt and F135C, F135A, or F135Y Pit-1 mutant
proteins of the expected 31- and 33-kDa size isoforms were detected
with a Pit-1 mAb.
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Because of the short stature (149 cm) of the mother who is heterozygous
for the F135C mutation (8), we tested the ability of the mutant form to
interfere with transactivation of PRL promoter construct by wt Pit-1.
We cotransfected HeLa cells with the PRL-250 promoter construct and
equal or double amounts of wt and F135C vectors. As shown in Fig. 2
, the coexpression of the F135C mutant
did not interfere with wt Pit-1 activity.

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Figure 2. Coexpression of the F135C Mutant Did Not Interfere
with the wt Pit-1 Activity
HeLa cells were transiently cotransfected by electroporation with
various concentrations of wt Pit-1 (wt) and F135C Pit-1 mutant as
indicated (1 = 5 µg, 2 = 10 µg) and 5 µg of the PRL-250
reporter plasmid. Electroporations were performed in triplicate for
each condition within a single experiment, and experiments were
repeated three times using different plasmid preparations of each
construct. Results are expressed as fold activation over control and
represent the mean ± SD of triplicates in a
representative experiment. Similar results were obtained on GH and
Pit-1 vectors.
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The F135C Mutant Protein Displays Normal DNA Binding Properties
Electrophoresis mobility shift assay (EMSA) analysis revealed that
both wt and F135C mutant Pit-1 proteins were able to bind a
high-affinity Pit-1 binding site as shown in Fig. 3
. Such a binding was specific for Pit-1
as it was displaced (supershift) by addition of a Pit-1 monoclonal
antibody (Pit-1 mAb), and the binding was cancelled by using a Pit-1
polyclonal antibody (Pit-1 pAb) or by addition of the nonlabeled
oligonucleotide.

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Figure 3. Gel Shift Analysis of the Human PRL Response
Element Using the wt and Mutant Pit-1 Proteins
DNA fragment used: human PRL-1P,
-66-AATGCCTGAATCATTATATTCATGA AGATATC-34. Approximately
equal amounts of the pcDNA3 EV, wt, and mutant proteins (C, A, and Y)
were used. The longer arrow denotes specific Pit-1-DNA
complexes. The shorter arrow denotes the supershifted
complex with the addition of the monoclonal Pit-1 antibody. The binding
was cancelled by addition of the unlabeled oligonucleotide (U.O.).
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Molecular Modeling of the F135C Mutant Suggests Structure
Alterations
The crystallographic analysis of the DNA/Pit-1 POU domains complex
reported by Jacobson and co-workers (4) showed three-dimensional
structure of the Pit-1 dimer associated with the double-stranded DNA
helix. The POU-S domain (residues 128199) and the POU-HD (residues
226293) of each monomer are linked together by a 26-residue extended
arm that is not resolved in the structure. The POU-S domain is made of
four tightly associated
-helices (h1 to h4), while there are three
helices in the POU-HD. The sequence alignment of the POU-S domains in
POU transcription factors and in Pit-1 from distinct species showed a
highly conserved Phe residue in position 135, similar to the six
residues with which it establishes direct contacts (Leu132, Phe139,
Phe173, Lys187, Leu190, and Leu194), as shown in Fig. 4
. Only the POU-S domain was taken for
molecular mechanics computations. The replacement of Phe135 by Cys
introduced no constraint but left a cavity (Fig. 5
). According to the PROCHECK
test, all models obtained either with INSIGHT II or with MODELLER were
geometry compliant, i.e did not comprise distorted
conformations. The PROSA pseudoenergy profiles of the F135C mutant and
of the wt molecule were compared (not shown). They showed a shift
toward higher values for the residues close to the mutated residue in
the first helix of the POU-S domain, and for the mutated residue
itself, indicating a lower stability of the mutated region. Moreover,
when compared with the wt models, all mutant models obtained with
MODELLER showed a significant increase of the computed solvation free
energy per residue calculated with ENVIRON (not shown) for the mutated
residue in position 135 and for the six residues with which it showed
direct contacts (Leu132, Phe139, Phe173, Lys187, Leu190, and Leu194;
Fig. 5
). Both results suggested that the mutated molecules would be
less stable if the mutated residues were adopting the same conformation
and environment as in the wt molecule. We also tested the likelihood of
the given mutations to be observed in natural structures. Several
empirical methods provide such propensities. Table 1A
shows the probability for a given F to X (X = C, A, or Y) mutation
to be observed in homologous proteins (24). These values are derived
from statistics of the observed mutations in experimental
three-dimensional structures of homologous proteins. Since the F135
residue was half-buried in the h4 helix, the table shows that the
substitutions found in our studied mutants have been rarely (F to A or
F to Y) or never (F to C) observed in helices, which indicates that
there is a very low probability that any of the mutated residues kept
the same conformation as that in the wt molecule, i.e
half-buried and/or in an
-helix. Another way to evaluate the
likelihood of a residue substitution to occur was by computing a
pseudoenergy change, taking into account the mutated residue and its
most often observed direct neighbors (25). These changes are shown in
Table 1B
for the same mutations and give rather high positive values in
accordance with the low substitution probabilities given in Table 1A
and indicate that the F135 changes were very unfavorable in the
mutants.

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Figure 4. Sequence Alignment of the POU-S Domain of the Pit-1
Family Proteins, Obtained with the CLUSTAL-X Program
The gray boxes and the row of asterisks
under the sequences show the residues that are identical in all
sequences. The horizontal bar indicates the POU-S domain
in the crystallographic structure of Jacobson et al.
(4 ), with the four -helices h1h4 delimited with "h" letters.
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Figure 5. Models of the F135C pit-1 Mutant and of the
Crystallographic Structure of the Pit-1 POU-S Domain, Both Associated
with the DNA
a, Model of the dimer mutated protein, in the same quaternary structure
arrangement as in the crystallographic structure of the rat pit POU
domain (4 ). The first monomer appears in green ribbon
with a representation of -helixes with red
semitransparent cylinders. The second monomer is in
purple. The Cys135, Phe135 residues and their direct
neighbors are represented with space-filling atoms. b, Model of the
dimer wt protein with the same orientation and colors as in panel a. c,
Close view of the first and fourth helix contact region of the POU-S
domain in the F135C mutant. Some of the residues in contact with the
Cys135 are labeled. The four h1h4 domains are labeled in green
italics. The broken h1 helix and the cavity around the mutated
residue can be seen. d, Close view of the same domain of the wt protein
structure.
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Because of the presence of a new Cys residue in the mutant protein, it
was first considered whether this residue might create a new disulfide
bond between C135 and C184. Since we found in an early model that a
C135C184 disulfide closure could occur at a low energy expense, the
mutated protein could also be trapped in a disulfide-stabilized
conformation, differing from that of the wt mainly where the first and
fourth helices are in contact (not shown). Moreover, the relaxed
disulfide-bonded model had a total potential energy similar to that of
the wt Pit-1 monomer, but a PROSA profile less favorable than the wt.
The solvation free energy, however, was slightly lower for the
disulfide-bonded model. Alternatively, to investigate whether the F135C
residue by itself was critical for protein function, we examined amino
acid conservation among homologous factors and created two other
mutations at the F135 residue, changing Phe into structurally similar
or distinct residues [tyrosine (Tyr) or alanine (Ala),
respectively].
Functional and Structural Properties of the F135A and F135Y Mutant
Pit-1 Proteins
Like F135C, F135A and F135Y mutant proteins also had a decreased
transactivation capacity: 8.4 ± 1.7-fold and 8.5 ± 1.0-fold
increase in PRL reporter gene activity, representing 38% of the
stimulation induced by wt Pit-1 (Fig. 1A
). These two mutants also had
markedly decreased transcription activity on the GH gene (Fig. 1B
):
2.1 ± 0.2- fold (F135A) and 1.4 ± 0.1-fold (F135Y)
representing 54% and 36% of the stimulation induced by the wt,
respectively, and on Pit-1 promoter construct (Fig. 1C
):
2.2 ± 0.3-fold (F135A) and 1.3 ± 0.2-fold (F135Y),
representing 36% and 21% of the stimulation induced by the wt,
respectively. The decreased transactivation properties of the three
proteins mutated at residue 135 were not accounted for by differences
in protein expression, since Western blot analysis of nuclear extracts
from transfected HeLa cells showed expression of the wt and the three
mutant Pit-1 proteins (Fig. 1D
). EMSA analysis revealed that F135A and
F135Y mutant Pit-1 proteins expressed in an in vitro
reticulocyte lysate system were able to bind to a high-affinity Pit-1
binding site as did wt Pit-1 and F135C proteins, as shown on
Fig. 3
.
The replacement of Phe135 by Tyr induced a strong steric hindrance,
particularly with the Ser 191 residue. The mutated tyrosine side chain
appeared trapped in a narrow niche from which it could not escape
without breaking the h1/h4 helix association. By contrast, the
replacement of F135 with less bulky cysteine or alanine residues
introduced no constraint, but left a cavity (Fig. 5
). Comparison of the
PROSA pseudoenergy profiles of the three mutants and the wt molecule
showed a positive shift for the residues close to the mutated residue
in the first helix of the POU-S domain, and for the mutated residue
itself. All three studied mutations were unfavorable in the Pit-1
context (Table 1).
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DISCUSSION
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Several patients with CPHD and Pit-1 gene mutations
have been described since 1992 (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). However, in only a minority of
these mutations, functional studies have been able to ascribe a
particular genotypic alteration to the observed phenotype. We had
previously found the F135C mutation to be present in both alleles of
the Pit-1 gene in four siblings with CPHD (8). Among 11
normal siblings of patients presenting with CPHD phenotype, we did not
find any polymorphism at codon 135 (data not shown). Moreover, the F135
residue was invariant among POU-S domains of POU transcription factors
and Pit-1 from different species (Fig. 4
). To ascertain the link
between such a genetic alteration and the CPHD phenotype, we performed
transcriptional activation and DNA binding studies on the F135C mutant.
Our data clearly showed that the F135C mutant had markedly decreased
transactivation capacity on the three target genes studied, confirming
the causative role of this mutation in the phenotype of our patients.
Indeed, the F135C mutant had a decreased transactivation ability on two
different PRL promoter constructs as well as on the GH promoter and on
the Pit-1 gene itself. This latter finding is of particular
functional significance in view of the demonstration of the critical
importance of Pit-1 gene autoregulation as illustrated by
the low levels of Pit-1 expression in W261C Snell dwarf mouse mutants,
and by the recently reported study on a CPHD associated with the K216E
Pit-1 gene mutation (7). The mother of our index case for
the F135C mutation was heterozygous for the same mutation and, although
of short stature, had normal pituitary function (8). As the only
heterozygous subject that was available for study in this family had a
short stature (149 cm), we addressed the hypothesis that the presence
of a single mutant allele might exert a dominant negative effect on the
wt protein. This possibility was ruled out by the absence of inhibition
induced by the addition of the mutant to the wt form in cotransfection
experiments. Moreover, the four unaffected siblings, unfortunately
unavailable for genotyping, were all of normal height.
The mechanism by which the F135C mutation decreases the transcriptional
activity was not due to a DNA binding defect, as shown in gel
retardation experiments. In the literature, the W261C murine mutation
and only 5 of 12 human mutations of Pit-1 gene have been
functionally studied. The W261C mutant was unable to bind to a high
affinity binding Pit-1 site. In addition, in vitro
mutagenesis and functional studies were performed to examine the
contributions of individual amino acids to the function of the DNA
binding domains of Pit-1 protein (26). One of these mutations was the
same as a spontaneously occurring mutation responsible for a human CPHD
phenotype, R143Q, that was found to be defective in its binding
activity (13). Crystallization and x-ray analysis of the Pit-1 POU
domains confirmed that this arginine 143 residue was necessary to
protein-DNA contacts, forming hydrogen bonds to the guanine of the
first base pair (4). The four other mutations had decreased
transactivation but had normal binding capacity. Two of these mutations
are dominant: K216E and R271W. The latter mutation represents the most
frequently reported genetic alteration among human Pit-1
gene defects (12). It generates a protein that acts as a dominant
inhibitor of Pit-1 action in the pituitary (10). Although the K216E
mutant was able to transactivate GH and PRL genes, it was defective in
its ability to activate transcription of the Pit-1 gene in
synergy with the retinoic acid receptor (7, 27). As observed with our
mutant, the A158P and the P239S are recessive mutations that generated
proteins capable of DNA binding to Pit-1 response elements but unable
to effectively transactivate its target genes (9, 18). The mechanism
suggested for the P239S mutation is an abolishment of the interaction
of Pit-1 with other factors required for transcriptional activation
(18). A Pit-1 variant mutated on two sites of the
-1 helix of the
POU-S domain around residue 135 (E133P, A136P), recently found to have
decreased transactivation properties, was unable to bind to
Pit-1-specific DNA and partitioned only to the insoluble nuclear
compartment (28). In contrast, the three F135 mutants tested in our
study retained DNA binding capacity. As the POU-S domain has been shown
to be a necessary and sufficient nuclear matrix targeting signal, an
altered subnuclear targeting may contribute to the decreased activity
of these mutants, which have been shown by Western blot analysis to be
expressed in nuclear extracts of transfected cells.
To understand how the F135C mutation might affect functionality of the
transcription factor, we used a structure modelization approach and
also analyzed F135A and F135Y mutants. These mutants were engineered
because we presumed that a disulfide bridge might occur between C135
and C184 in the F135C mutant, which could explain the loss of function.
Accordingly, like F135C, F135A and F135Y mutants had a decreased
transactivation capacity despite a normal DNA binding. This finding was
in keeping with our molecular mechanics studies. Indeed, Phe 135 was
involved neither in POU-S domain-DNA contacts nor in the dimer
interface formed between the POU-S domain of one monomer and the POU-HD
of the symmetry-related monomer. As seen in Table 1, the amino acid
substitution tables of Overington et al. (24) gave
unfavorable scores for the F to A, C, or Y substitutions in any
environment. These values only represented a measure of the relative
propensity in change because they were statistically derived, but in
addition, the average contact energy change in substitution (25)
clearly showed a high positive change in contact energy due to any of
the studied mutations. Thus, before obtaining any model of the mutants,
these preliminary observations suggested a low probability of the
mutants to maintain their native conformation.
The three-dimensional structure established by Jacobson and co-workers
showed the POU domains of the rat Pit-1 transcription factor bound as a
homodimer to a 24-bp DNA element (4). The very strong similarities
between the rat and human Pit-1 POU domain sequences allowed the human
Pit-1 structure to be modeled from that of the rat species. The POU-S
domain consists of four
-helices tightly bound by interactions
between residues mostly hydrophobic at conserved positions, as shown by
the alignment of the POU family sequences in Fig. 4
. The Phe135 is one
of such invariant residues. In the crystallographic structure, it was
buried in a cleft made of the side chains of six highly conserved
residues at the interface of the
-helices: Leu 132, Phe 139, Phe
173, Lys 187, Leu 190, and Leu 194, and of the backbone of the more
variable S191. The simple replacement of F135 in the rat Pit-1 POU-S
domain, followed by energy minimization, gave unrealistic structures,
for which the PROSA and ENVIRON scores indicated that the mutation
could not occur without some expense in packing or conformational
energy (not shown). In the models, a closer view of the contacts
between the first and fourth helices showed a cavity left by the less
bulky Cys or Ala 135 residue which, when comparing the F135C to the wt,
had looser contacts with the seven residues mentioned above. Such
perturbations may well be revealed by computed energy profiles. All 20
models of each of the human wt and mutant molecules were screened with
the program ENVIRON (29). All mutant models showed an increase in the
pseudosolvation energy of the mutated residues and of the
aforementioned neighbor hydrophobic residues. The F135Y mutant
was found to be less markedly affected than the others, but the
solvation energy penalty was more diffuse in several parts of the
sequence.
The very tight association between the h1, h3, and h4 helices in the wt
molecule proceeds from interactions of hydrophobic residues forming a
buried hydrophobic cluster. As seen in Fig. 4
, most of the hydrophobic
buried residues participating in the h1h4 helices association are
strictly conserved in the sequence alignment of the Pit-1 family.
Remarkably, both h1 and h4 helices are curved and associated by their
concave faces, which reveals the shortening of the hydrogen bonds of
the buried sides of the helices. This kind of curved parallel
-helices associated by hydrophobic residues was found very
stabilizing in the leucine-zipper helix dimer (30). By analogy, this
strongly suggested for the Pit-1 POU-S domain that the conserved
hydrophobic residues play a very important role in the stability of the
helix association. Moreover, the F135 residue showed a close
interaction with the side chain of the F173 residue of the h3 helix.
Thus the F135 residue acted as an actual key in the assembling and
stability of the h1, h3, and h4 helices. All data obtained from the
propensities tables or from pseudoenergy calculations on the models
strongly suggested that the F135C mutation induced a change in the
association between the h1, h3, and h4 helices of Pit-1. The mutated
POU-S domain could be perturbed in such a way that any interaction with
other transcription cofactors could be definitively prevented,
particularly in the mutated region. By contrast, the third helix, which
is the domain of the specific interaction with the DNA, did not appear
to be affected by the mutation in any of the models, which was in
accordance with the experimentally observed ability of the mutant
protein to bind to the DNA.
Several studies have underlined the critical role of
protein-protein interactions in Pit-1 function. For example, Dasen
et al. (31) have evidenced a DNA binding-independent
function of Pit-1 whereby Pit-1 suppresses the ventral gonadotrope
program by direct interaction with GATA-2. The mechanism by which the
F135C mutation decreased the transcriptional activity despite a
conserved DNA binding was likely due to altered interactions with
cofactors of the transcriptional complex. Functional and physical
interactions of Pit-1 with several other transcription factors have
been described in the regulation of several pituitary-specific genes,
and protein-protein interactions have been mapped to distinct regions
of Pit-1 (23). The majority of Pit-1 interacting partners bind to the
DNA-binding region of Pit-1, involving either the POU-S domain and/or
the POU-HD. These include coactivators such as the CREB binding
protein, CBP, and corepressors such as N-CoR (21, 22, 23). Interestingly, a
Pit-1 mutant (E254A) obtained by systematically mutating surface
residues in the POU-HD was found to inhibit Pit-1 activity without
disrupting its ability to bind to DNA or to CBP, while exhibiting
increased affinity for N-CoR (21). The POU-S and POU-HD are required
for interactions with N-CoR corepressor and CBP co-activator complexes
(21, 32). Similarly, a pituitary LIM-homeodomain protein, P-LIM (or
Lhx3), interacts with Pit-1 via both the POU-S and POU-HD domains (33, 34). Moreover, we found that decreased CBP-Pit-1 interactions might
help to explain the defective functionality of the K216E Pit-1 mutant
(35, 36).
In conclusion, despite preserved DNA binding properties to a
specific Pit-1 response element, the F135C mutant was unable to
transactivate Pit-1 target genes. These findings confirmed that this
mutation was responsible for the CPHD phenotype observed in the
subjects homozygous for this mutation. Functional studies and molecular
modelization data based on the crystallization of the Pit-1 DNA complex
support the hypothesis that this mutant is functionally deficient in
its ability to interact with other proteins. Recent data have
underlined the key role of combinatorial interactions of Pit-1 with
other transcription factors. Further studies will try to decipher more
precisely the mechanisms whereby this and other mutants alter the
transactivation complex mediating Pit-1 function.
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MATERIALS AND METHODS
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Plasmids
wt Human Pit-1 cDNA was inserted into the effector plasmid
pcDNA3. The various reporter constructs that contained Pit-1 binding
sites within the context of different gene-regulatory regions were
fused to a firefly luciferase gene. We used two PRL reporter constructs
from the proximal promoter regions of the human PRL gene: 164; +34 bp
(PRL 164) and 250; +34 bp (PRL 250) containing two or three Pit-1
binding sites, respectively (gifts of J. A. Martial, Liège,
Belgium). The proximal promoter of the human GH gene (Pa3-Ghp-Luc)
contained two Pit-1 response elements (gift of N. L. Eberhardt,
Rochester, MN). A reporter construct containing the positive
autoregulatory site of the human Pit-1 promoter gene was used (gift of
M. Delhase, San Diego, CA).
Site-Directed Mutagenesis
We made an identical substitution from a thymine to a guanine in
the normal human Pit-1 cDNA by in vitro
site-directed mutagenesis using the Quickchange kit
(Stratagene Cloning Systems, La Jolla, CA). This reaction
deriving from the PCR technique was performed with a double-stranded
template generated from the wt human Pit-1 cDNA into the
plasmidic vector pcDNA3, and two complementary mutagenetic primers
(sense primer: C AGA GAA CTT GAA AAG TGT GCC AAT
GAA TTT AAA G; antisense primer: G TCT CTT GAA CTT TTC
ACA CGG TTA CTT AAA TTT C). To extend the
functional study of the F135C point mutation, two other mutations were
performed in the wt human Pit-1 cDNA by in vitro
site-directed mutagenesis: F135A (substitution from two thymines to
a guanine and a cystine) and F135Y (substitution from a thymine to an
adenine).
After DNA purification and amplification (QIAGEN
maxi kit. Chatsworth, CA), the sequence of plasmid containing F135C,
F135A, and F135Y mutations was confirmed by the ABI Prism 310 sequencer
(ABI Advanced Biotechnologies, Inc., Columbia, MD) with specific
forward and reverse primers.
Cotransfection in Eukaryotic Cells
Cotransfection experiments were carried out in the human HeLa
cell line, using the various reporter constructs and effector plasmids.
HeLa cells were maintained in DMEM (4.5 g, Life Technologies, Inc., Gaithersburg, MD) supplemented with 2 mM
glutamine and 10% FCS. Cells were grown at 37 C in 5%
CO2 and were harvested at 5070% confluency.
Aliquots of 1.5 x 106 cells per well in 400
µl of medium were added to plasmid DNA (5 µg of effector plasmid
and 5 µg of reporter plasmid), transfected by electroporation at 240
V (EASYJECT), and plated in six-well tissue culture plates.
Total DNA was kept constant, and nonspecific effects of viral promoters
were controlled by using the pcDNA3 empty vector (EV). Luciferase
activity in relative light units was measured at 48 h.
Cotransfections were also performed in CV-1 cells using a
calcium-phosphate precipitation technique (Transfection MBS
transfection kit, Stratagene Cloning Systems, La Jolla,
CA) in six-well tissue culture plates. We transfected 0.2 µg
per well of effector plasmid (EV, wt, or mutants Pit-1) with
2 µg per well of luciferase reporter construct (PRL-250, GH, or
Pit-1 promoter) and with 0.1 µg pCMVß (CLONTECH Laboratories, Inc., Palo Alto, CA) as an internal control for
transfection efficiency. Luciferase assay in relative light units and
ß-galactosidase activity determined spectrophotometrically using the
chromogenic substrate
o-nitro-phenyl-ß-D-galactopyranoside were
measured at 48 h. Total luciferase light units were normalized to
total ß-galactosidase activity.
Transfections were performed in triplicate for each condition
within a single experiment, and experiments were repeated five times
using different plasmid preparations of each construct. The relative
luciferase activity for each control was set to 1, and results were
expressed as fold promoters activation and represented the mean ±
SD of a representative experiment.
Western Blot Analysis
After transient transfection in HeLa cells as above, nuclear
proteins were extracted and Western blot analysis was performed
according to published protocols (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and Amersham Pharmacia Biotech, Arlington Heights, IL, respectively). A Pit-1 mAb
(Transduction Laboratories, Inc. Lexington, KY) was used
for detection.
EMSA
EMSA was performed with Pit-1 recombinant proteins and
32P-labeled DNA fragments. Pit-1 and mutant
proteins were synthesized using the TNT coupled
transcription-translation reticulocyte lysate system with T7
polymerase, according to the manufacturers protocol (Promega Corp., Madison, WI). For testing Pit-1 binding, we used a
high-affinity Pit-1 DNA binding site from the human PRL gene (PRL-P1):
5'-AATGCCTGAATCAT TATATTCATGAAGATATC-3'. This oligonucleotide was
P32-labeled with Klenow polymerase. The Pit-1
complexes were supershifted with a Pit-1 mAb (Transduction Laboratories, Inc.). The binding was cancelled by addition of
the unlabeled oligonucleotide in excess.
Molecular Modeling
Automatic Sequence Alignment.
Seventeen amino acid sequences of the Oct, Pit-1, and Unc
family were picked up from the Swiss-Prot database (25) and aligned
using the newly released version (CLUSTAL-X) of the CLUSTAL-W
program (37).
Molecular Model Building.
Three-dimensional models of the F135C, F135A, and F135Y mutants
of the Pit-1 POU-S domain were derived from the crystallographic
structure of the rat Pit-1 POU domains dimer bound to a 24-bp DNA
element (4). All computations were achieved on a Indigo R 3000
workstation (Silicon Graphics, Inc., Mountain View, CA). The
Pit-1 POU-S domain of the A monomer was taken as template (72 residues
from G5 to Q76 in the crystallographic structure). Two methods were
used. In the first method, the mutant structures were derived from
the replacement of the Phe 135 residue of the template by the mutated
residue, followed by energy minimization using the program INSIGHT
II/DISCOVER (Molecular Simulations, Inc., San Diego, CA). This method
gave the actual corresponding mutants of the rat Pit-1 transcription
factor. After the replacement of the Phe 135 residue into either Cys,
Ala, or Tyr residues, the three models obtained were relaxed by 50
steps of steepest descent molecular energy minimization, followed by
500 steps of conjugate gradient minimization. The same molecular
relaxation was applied to the POU-S domain of the experimental
structure. The second method used the freely distributed program
MODELLER (38). The modeling process used the same rat Pit-1
POU-S domain template for deriving distance constraints to be used to
drive the human Pit-1 model building, according to the sequence
HMP1-HUMAN in Fig. 4
. Twenty different conformers of the human wt, and
of each of the F to C, F to A, and F to Y mutants, were eventually
obtained.
Model Evaluation.
The models were tested using the free PROSA (39), ENVIRON (29),
and PROCHECK programs (40). A pseudoenergy profile was calculated with
PROSA for the POU-S domain of the rat wt Pit-1 molecule and of the
three mutants obtained with INSIGHT II. The program ENVIRON was used to
compute an approximation of the free solvation energy per
residue of the human POU-S domain for the wt and the mutants obtained
with INSIGHT II and MODELLER.
The effect of the punctual mutations was also assessed from
tables of propensity changes computed by Overington et al.
(24) and of average contact energy changes computed by Miyazawa and
Jernigan (25).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. P. Koehl and Dr. M. Delarue for providing the
program ENVIRON, Dr. M. Sippl for the program PROSA, and Dr. J. D.
Thompson and his co-workers for the program CLUSTAL-X. We also thank
Dr. E. M. Jacobson and his co-workers who deposited the
coordinates of the Pit-1 dimer-DNA complex in the PDB. We are
indebted to Professor J. A. Martial and Dr. M. Müller,
Liege, Belgium) for providing us not only with the gift of several
vectors but also for helpful discussions and technical support.
 |
FOOTNOTES
|
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Address requests for reprints to: Thierry Brue, M.D., Ph.D., Hopital de la Timone, 264 rue St. Pierre, 13385 Marseille Cedex 5, France. E-mail: tbrue{at}ap-hm.fr
This work was supported by funding from the Programme
Hospitalier de Recherche Clinique 1996 (T. B.), by a grant from
the Association pour la Recherche sur le Cancer (ARC 1996 No. 5069),
and by the Association pour le Développement des Recherches
Biologiques et Médicales au Centre Hospitalo-Universitaire de
Marseille (ADEREM).
Received for publication July 7, 2000.
Revision received October 3, 2000.
Accepted for publication October 9, 2000.
 |
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