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 Children’s Hospital (S.R.) Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1Go). 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. 1Go, 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. 1DGo).



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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.

 
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. 2Go, 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.

 
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. 3Go. 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.).

 
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 128–199) and the POU-HD (residues 226–293) 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 {alpha}-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. 4Go. 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. 5Go). 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. 5Go). 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 1AGo 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 {alpha}-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 1BGo for the same mutations and give rather high positive values in accordance with the low substitution probabilities given in Table 1AGo 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 {alpha}-helices h1–h4 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 {alpha}-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 h1–h4 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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Table 1A. Probability of Mutation in Homologous Proteins

 

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Table 1B. Contact Energy Change in RT Units

 
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 C135–C184 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. 1AGo). These two mutants also had markedly decreased transcription activity on the GH gene (Fig. 1BGo): 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. 1CGo): 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. 1DGo). 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. 3Go.

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. 5Go). 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 4Go). 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 {alpha}-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 {alpha}-helices tightly bound by interactions between residues mostly hydrophobic at conserved positions, as shown by the alignment of the POU family sequences in Fig. 4Go. 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 {alpha}-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. 4Go, most of the hydrophobic buried residues participating in the h1–h4 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 {alpha}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 50–70% 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 manufacturer’s 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 {gamma}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. 4Go. 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
 
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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