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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2289
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 4981-4987
Copyright © 2006 by The Endocrine Society

Identification and Functional Analysis of the Novel S179R POU1F1 Mutation Associated with Combined Pituitary Hormone Deficiency

Ichiro Miyata1, Sophie Vallette-Kasic1, Alexandru Saveanu, Mizuho Takeuchi, Hideki Yoshikawa, Asako Tajima, Katsuyoshi Tojo, Rachel Reynaud, Magali Gueydan, Alain Enjalbert, Naoko Tajima, Yoshikatsu Eto and Thierry Brue

Departments of Pediatrics (I.M., H.Y., A.T., Y.E.) and Diabetes, Metabolism, and Endocrinology (M.T., K.T., N.T.), Jikei University School of Medicine, Tokyo 105-8461, Japan; and Laboratoire des Interactions Cellulaires Neuro-Endocriniennes (S.V.-K., A.S., R.R., M.G., A.E., T.B.), Unité Mixte de Recherche 6544 Centre National de la Recherche Scientifique, Université de la Méditerranée, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, 13926 Marseille, France

Address all correspondence and requests for reprints to: Ichiro Miyata, M.D., Ph.D., Department of Pediatrics, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan. E-mail: i-miyata{at}jikei.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patient and Methods
 Results
 Discussion
 References
 
Context: The pituitary-specific transcription factor 1 plays a key role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Several mutations of the human gene (called POU1F1) have been shown to be responsible for a phenotype of combined pituitary hormone deficiency involving GH, prolactin (PRL), and TSH.

Objective: We have identified a novel homozygous C to G mutation in exon 4 of the POU1F1 gene (S179R) in a patient with this rare phenotype. We analyzed the functional consequences of this S179R mutation associated with a single-amino acid change in the POU-specific domain.

Methods: Consequences of this mutation on transcriptional activities by transfection studies in {alpha}T3 cells, DNA binding ability by EMSA, structural properties, and nuclear accumulation of POU1F1 were investigated.

Results: The transactivation capacity of this mutant was markedly decreased on the GH1, PRL, TSHß, and POU1F1 genes. Interestingly, this mutation abolished the functional interaction of POU1F1 on the PRL promoter with the coactivator cAMP response element-binding protein-binding protein but not with the transcription factor LIM homeodomain transcription factor 3. The S179R mutant displayed normal nuclear accumulation but a markedly decreased binding to a DNA response element in keeping with crystallographic data, suggesting that the S179R mutation might interfere with DNA binding.

Conclusions: Together with previous data, our study indicates that both DNA binding and interaction with cofactors like cAMP response element-binding protein-binding protein are critical for POU1F1 function and that functional and structural properties of abnormal POU1F1 proteins are variously influenced by the type of mutations.


   Introduction
Top
 Abstract
 Introduction
Patient and Methods
 Results
 Discussion
 References
 
PITUITARY-SPECIFIC TRANSCRIPTION factor 1 (PIT-1) (murine ortholog of human POU1F1) is a pituitary-specific nuclear transcription factor that plays a pivotal role in pituitary development and expression of the GH, prolactin (Prl), and Tshß genes. Pit-1 is required for the differentiation and proliferation of somatotrophs, lactotrophs, and thyrotrophs (1, 2). Therefore, abnormalities of the Pit-1 gene result in GH, PRL, and TSH deficiencies. As a member of the homeobox family of POU domain transcription factors, Pit-1 contains an N-terminal transactivation domain and a DNA-binding region. The DNA binding domain consists of a POU-homeodomain essential for DNA binding and a POU-specific domain, which is required for high-affinity and high-specificity DNA binding on the GH and Prl genes (2). Pit-1 can also bind to and transactivate the Tshß promoter (3). Furthermore, Pit-1 transcription is maintained by autoregulation through two Pit-1-binding elements on the Pit-1 promoter region (4). Pit-1 is reported to regulate its target genes by binding to response elements of their promoter regions and recruiting coactivator proteins, such as the cAMP response element-binding protein-binding protein (CBP) (5) and other transcription factors like LIM homeodomain transcription factor 3 (LHX3) to the transcriptional complex (6).

Naturally occurring mutations in the Pit-1 gene were first reported in the Jackson and Snell dwarf mice. In Snell mice, the loss of functionality of the Pit-1 mutant protein was clearly related to a defective DNA binding to Pit-1 response elements (7). To date, more than 20 different mutations of the human POU1F1 gene related to combined pituitary hormone deficiency (CPHD) involving GH, PRL, and TSH have been reported (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Most mutations are recognized in the POU domain of POU1F1, which is important for its ability to dimerize and bind to DNA. In addition, two mutations located in the transactivation domain of the POU1F1 gene have also been reported in patients with CPHD (18, 19). Relations between clinical symptoms and functions of abnormal POU1F1 proteins have however not been well clarified because functional studies of observed human POU1F1 mutations have only been carried out in a limited number of the mutants identified.

We have recently identified a novel C to G homozygous missense mutation at position 537 in exon 4 of the POU1F1 gene, resulting in an amino acid change at codon 179 from serine (S) to arginine (R) (S179R). In the present report, we describe the functional effects of the S179R POU1F1 mutation, which was found to have deleterious functional effects on the mutant transcription factor by selectively affecting protein-protein interactions and DNA binding properties. We also analyzed the structural consequences of this mutation with reference to crystallographic structure data available for the POU1F1 POU-domain-cognate DNA complex (24).


   Patient and Methods
Top
 Abstract
 Introduction
 Patient and Methods
Results
 Discussion
 References
 
Clinical studies

The index case was referred at the age of 20 yr to our university hospital in Tokyo, Japan for evaluation of hypopituitarism. Informed consent was obtained from the patient and his mother for all studies performed. A complete clinical, biological, and neuroradiological workup was carried out. GH response was studied by a GHRH infusion test (normal peak value > 15 ng/ml). Basal plasma ACTH and cortisol were measured at 0800 h (10–50 pg/ml and 210–560 nmol/liter, normal range for ACTH and cortisol, respectively). A CRH test was also carried out with measurements of ACTH and cortisol at 0, 15, 30, 60, 90, and 120 min after a 1.5 µg/kg iv bolus. The gonadotropin axis was investigated by measuring LH and FSH levels at baseline and at 15, 30, 60, 90, and 120 min after a GnRH provocative test (100 µg iv) and by determining the basal level of testosterone. Investigation of the thyrotroph axis included determination of basal thyroid hormone levels (normal range of free T3 and free T4, 3.0–9.0 and 10–30 pmol/liter, respectively) and TSH concentration (normal range, 0.2–6.0 mIU/liter). Four weeks after discontinuation of thyroid hormone replacement, a TRH test was carried out with measurements of TSH and PRL at 0, 15, 30, 60, 90, and 120 min after a 500-µg iv bolus. Pituitary magnetic resonance imaging was performed using precontrast sagittal and axial spin echo T1-weighted images, followed by T2-weighted imaging. Because a POU1F1 abnormality was suspected in this patient, we performed the POU1F1 gene analysis.

Genomic analysis of the POU1F1 gene (NM000306)

All six exons and exon-intron boundaries of the POU1F1 gene were PCR amplified from genomic DNA by primers flanking the exons for direct sequencing. DNA was extracted from peripheral lymphocytes by the QIAamp Blood kit (QIAGEN Sciences, Germantown, MD). Exons were amplified by PCR using six sets of flanking intronic primers: F1, 5'-ATCGGCCCTTTGAGACAGTAA-3'; R1, 5'-CCCGGTCATATGTAAACTG-3'; F2, 5'-TTTCTCGGTGACAACGTTG-3'; R2, 5'-GTGTCCCCAAATTCAATAAC-3'; F3, 5'-AAGGAGAATGACAAATGGTC-3'; R3, 5'-AAGTTCTTTTTCCTGTTGCC-3'; F4, 5'-AAAGTTGGAGCTGATGGTC-3'; R4, 5'-CACAGCCTTCAGAGACAC-3'; F5, 5'-TTTGTAATTATCTCTCTTTTCC-3'; R5, 5'-TACACTCAAATGCTCATTCC-3'; F6, 5'-AATTTCACCCCCTATGTCC-3'; and R6, 5'-GAAACGGGAGAAAAAGGCT-3'. Amplification was carried out in a 50-µl reaction, using 200 ng genomic DNA, 0.25 nmol/liter of each deoxy-NTP, 25 pmol of each primer, and 1.5 U Pfu DNA polymerase (Promega, Madison, WI). The reaction consisted of 3 min at 95 C, followed by 30 cycles of 30 sec at 95 C, 30 sec at 55 C (exons 1 and 3) or 60 C (exon 2), 2 min at 72 C, and 5 min at 72 C. PCR products were purified using the Qiaquick PCR purification kit (QIAGEN). Direct sequencing of the double-stranded PCR fragments was carried out according to the thermal cycle sequencing big dye terminator protocol (ABI Prism 310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster City, CA) using the same PCR primers. Mutation was confirmed by repeat PCR and subsequent sequencing of PCR products.

Functional analysis

Transcriptional activities. The POU1F1 expression vector consisted of the human POU1F1 cDNA subcloned into a mammalian expression vector (pcDNA3.1) as previously described (25). The mutant form of the POU1F1 (S179R) expression vector was constructed using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Briefly, Pfu DNA polymerase was used to react 50 ng template DNA [pcDNA3.1-wild-type (WT) POU1F1] with the mutant sense primer (5'-CGATTTGAAAATCTGCAGCTCAGGTTTAAAAAT-GCATGCAAACTGAA-3') and mutant antisense primer (5'-TTCAGTTTGCATGCATTTTTAAACCTGAGCTGCAGATTTTCAAATCG-3'). This reaction involved 30 sec of denaturation at 94 C and 12 cycles consisting of 30 sec of denaturation at 94 C, 1 min of annealing at 55 C, and 2 min of extension at 72 C. After DNA purification and amplification (QIAGEN maxi kit; QIAGEN, Chatsworth, CA), the correct sequence was confirmed by DNA sequencing. We used a PRL reporter construct from the proximal promoter region of the human PRL gene: –250; +34 bp (PRL 250) containing three Pit-1 binding sites, a reporter construct containing the positive autoregulatory site of the human POU1F1 promoter gene, and a Tshß reporter construct containing the mouse Tshß promoter sequence between –6 kb and +40 bp from the transcription initiation site; and finally, the proximal promoter of the human GH1 gene (Pa3-Ghp-Luc) containing two Pit-1 response elements (25).

Cotransfection experiments were carried out in the {alpha}T3 cell line (a Pit-1-deficient gonadotroph lineage). {alpha}T3 cells were cultured in DMEM (4.5 g; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal calf serum and antibiotics at 37 C in 5% CO2 and were harvested at 50 to approximately 70% confluence. Approximately 2.5 x 105 cells were transfected in 12-well dishes with Lipofectamine (Invitrogen, Carlsbad, CA) using 500 ng reporter plasmid and 10–100 ng expression vector of the WT POU1F1 or S179R mutant POU1F1 (S179R), up to a total of 750 ng per assay. To investigate interactions with some of the known protein partners on the PRL promoter (PRL 250), the lowest tested quantities of POU1F1 and S179R mutant DNA (10 ng) were used in cotransfection experiments with CBP and LHX3 to maintain total transfected DNA amount. Moreover, in preliminary experiments (data not shown), such a DNA amount allowed the testing of cofactors in the linear part of the dose-response curve of luciferase activity. LHX3 (open frame) in pTracer-CMV vector (gift of Serge Amselem, Paris, France) and mouse CBP in pcDNA3.1 vector (gift of Sally Radovick, Baltimore, MD) were used in amounts of 10 and 20 ng, respectively, as shown in Fig. 3Go, alone and in cotransfection experiments.


Figure 3
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  		FIG. 3. Interactions of WT and S179R mutant POU1F1 with CBP and LHX3 on the PRL promoter. The WT and S179R POU1F1 expression vectors were cotransfected with the PRL promoter construct (PRL 250) and either CBP or LHX3 expression vectors. Relative luciferase activity is indicated as the mean ± SEM fold of at least three separate experiments performed in duplicate compared with activity of the reporter without Pit-1, CBP, or LHX3 transfection. After cotransfection with CBP, S179R exhibited statistically decreased levels of transactivation compared with WT POU1F1 (cotransfected with CBP or LHX3, respectively; P < 0.05). Cotransfection of S179R with CBP did not induce a modification in transactivation induced by S179R alone.

 
Nonspecific effects of viral promoters were controlled by using the pcDNA3.1 empty vector (EV). The luciferase values were normalized using the ß-galactosidase data to control for transfection efficiency. The coding sequence for ß-galactosidase was inserted into the pCEP4 eukaryotic expression vector (Invitrogen, Cergy Pontoise, France), where it was expressed under control of the CMV promoter. Luciferase activity was measured by spectrophotometer at 48 h after transfection. Transfections were performed in duplicate for each condition within a single experiment, and experiments were repeated three to five times. The relative luciferase activity for each control was set to 1; results were expressed as fold activation and represented the mean ± SEM of three to five experiments.

Western blot analysis. Western blots were performed as described (25) using nuclear extracts and a rabbit polyclonal anti-Pit-1 antibody 1:1000 (gift of Simon Rhodes, Indianapolis, IN).

DNA binding ability. EMSA was performed with POU1F1 recombinant proteins and 32P-labeled DNA fragments as described previously (26). WT POU1F1 and S179R mutant POU1F1 proteins were synthesized using the TNT-coupled transcription-translation reticulocyte lysate system with a T7 polymerase kit (Promega). We used a high-affinity POU1F1 DNA binding site of proximal promoter region of the human PRL gene (P1 site, 5'-AATGCCTGAATCATTATATTCATGAAGAT-ATC-3'). This oligonucleotide was {gamma}32P-labeled with Klenow polymerase. Binding specificity was studied by addition of a human POU1F1 monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY) or of the unlabeled oligonucleotide in excess (25).

Molecular modeling analysis. A three-dimensional model of the S179R mutant of the POU1F1 POU-specific domain was analyzed on the basis of the crystallographic structure of the rat Pit-1 POU domains dimer bound to a 24-bp cognate DNA element (24) using the program MODELLER (27). The modeling process used the same rat Pit-1 POU-specific domain template for deriving distance constraints to be used to drive the human POU1F1 model building, according to the consensus sequence (28). All computations were achieved on an Indigo R 3000 workstation (Silicon Graphics, Inc., Mountain View, CA) (25).

Nuclear accumulation. Chimera constructs of green fluorescent protein (GFP) with the WT POU1F1 and S179R mutant POU1F1 were synthesized using a CT-GFP Fusion TOPO TA Expression kit (Invitrogen) and transiently transfected into COS-7 cells (Pit-1 deficient). Briefly, 3 µg of each fusion plasmid with GFP was transiently transfected into COS-7 cells in 35-mm dishes with Lipofectamine (Invitrogen). COS-7 cells were maintained in DMEM with 10% fetal calf serum and were grown at 37 C in 5% CO2. Forty-eight hours after transfection, fluorescent images of the expressed fusion proteins were analyzed using a fluorescence microscope. Cells transfected with only the GFP expression vector were also analyzed as a control.


   Results
Top
 Abstract
 Introduction
 Patient and Methods
 Results
Discussion
 References
 
Index case

The propositus was a 20-yr-old Japanese man who was referred for evaluation of hypopituitarism. He was born by normal delivery after a 41-wk pregnancy from a nondiabetic mother. He was the only child of nonconsanguineous parents of normal height. At birth, his body length was 50.5 cm (+0.5 SD), and his body weight was 4550 g (+3.38 SD). He had been investigated because of poor feeding and failure to thrive at the age of 1 month, and congenital hypopituitarism was diagnosed. Serum basal levels of GH, PRL, and TSH were all undetectable (<0.05 ng/ml, 1.0 ng/ml, and 0.05 µIU/ml, respectively). Free T3 and free T4 were 1.02 and 6.57 pmol/liter, respectively. He was then started on L-T4. Blunted GH responses to either insulin or arginine stimulation tests (peak values < 0.05 ng/ml) were observed at the age of 4 yr. Therefore, GH therapy was given from 4.8–18 yr of age. The onset of puberty was at the age of 12 yr, and his pubertal development was normal.

On admission to our university hospital, his height was 157.0 cm (–2.39 SD), and his body weight was 56.8 kg (body mass index, 23.0 kg/m2). His blood pressure was 100/62 mm Hg. All other clinical findings were normal. Off-treatment serum basal levels of GH, PRL, and TSH were all undetectable (<0.05 ng/ml, 1.0 ng/ml, and 0.05 µIU/ml, respectively). Free T3 and free T4 were 1.24 and 8.78 pmol/liter, respectively (normal range of free T3 and free T4, 3.0–9.0 and 10–30 pmol/liter, respectively). Basal plasma ACTH and cortisol were normal. GHRH and TRH tests showed no responses of GH, PRL, and TSH. In contrast, LHRH and CRH tests showed normal responses of LH, FSH, ACTH, and cortisol. Cervical ultrasound examination showed an atrophic thyroid gland. Magnetic resonance imaging of the brain revealed a hypoplastic anterior pituitary gland. Based on these findings, congenital CPHD involving GH, PRL, and TSH was diagnosed.

Identification of a novel POU1F1 mutation

As shown in Fig. 1Go, direct sequencing of the patient’s genomic DNA showed a novel homozygous mutation in exon 4 that predicted a change of amino acid 179 from Ser to Arg (AGC to AGG; S179R). It is located in the POU-specific domain of the POU1F1 gene. His mother was heterozygous for the S179R allele. However, his father was not investigated because he refused to participate in the study.


Figure 1
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  		FIG. 1. Sequencing analysis of amplified exon 4 of the POU1F1 gene of the patient, his mother, and a control. Direct sequencing showed a novel missense mutation in exon 4 of the patient that changed amino acid 179 from Ser to Arg (AGC to AGG; S179R). The patient’s mother was heterozygous for the S179R allele.

 
Sequence alignment of the POU-specific domains in POU transcription factors showed that the serine residue at position 179 (28) represents a highly conserved amino acid among several distinct species (rat Pit-1, human Oct-1, human Brn-3a, mouse Brn-1, Caenorhabditis elegans ceh-18, cow Pit-1, human Oct-3, Drosophila I-Pou).

Functional analysis of the S179R mutant

As shown in Fig. 2Go, when compared with WT POU1F1, the S179R mutant POU1F1 showed decreased transcriptional activity in {alpha}T3 cells with at most 30% residual activity on a PRL promoter construct (Fig. 2AGo), as well as on POU1F1 and Tshß promoters (Fig. 2Go, B and C). Moreover, this mutant had no significant transcriptional activity on a GH1 promoter construct (Fig. 2DGo) despite maintained nuclear expression of both the WT and S179R mutant POU1F1 vectors as confirmed by Western blot analysis (Fig. 2EGo). Thus, transfection studies showed that the transactivation capacity of the S179R mutant POU1F1 was markedly decreased on all four target genes. We also tested the interaction of WT and S179R mutant POU1F1 with CBP and LHX3 on the PRL promoter (Fig. 3Go). CBP alone had a very low intrinsic activity on the PRL reporter gene at the amount used. Nevertheless, cotransfection experiments showed an enhancement of WT POU1F1 transcriptional activity on the PRL promoter by CBP. The lower transcriptional activity of the S179R mutant was not modified by CBP cotransfection. LHX3 activated the PRL promoter to a similar extent as WT POU1F1. LHX3 and POU1F1 displayed a strong synergistic activation of the PRL promoter. The S179R mutant induced a synergistic transactivation of the PRL gene when cotransfected with LHX3, albeit at a lesser level than WT POU1F1. In summary, the S179R mutant had completely lost its ability to synergize with CBP on the PRL promoter, whereas its synergy with LHX3 was decreased compared with the WT.


Figure 2
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  		FIG. 2. Transactivation capacities of the PRL (A), POU1F1 (B), Tshß (C), and GH1 (D) promoters in {alpha}T3 (Pit-1-deficient) cells. {alpha}T3 cells were transiently transfected by Lipofectamine (Invitrogen) with 10, 50, or 100 ng of an effector plasmid [WT POU1F1 or S179R mutant POU1F1 (S179R)] and 500 ng of a reporter plasmid [PRL (A), POU1F1 (B), Tshß (C), or GH1 (D) promoter constructs]. The pcDNA3.1 EV was used as a control. Transfections were performed in duplicate for each condition within a single experiment, and experiments were repeated three to five times. Results are expressed as fold activation over the control and represent the mean ± SEM of duplicates in a representative experiment. White box, Control. E, Western blot analysis. After transient transfection and nuclear protein extraction, equivalent amounts of WT and S179R mutant proteins of the expected 31- and 33-kDa isoforms were detected using a polyclonal Pit-1 antibody (gift of Simon Rhodes).

 
To examine the binding ability of the S179R mutant POU1F1 protein, we next performed EMSA analysis. As shown in Fig. 4Go, a gel shift assay using WT POU1F1 revealed a strong specific POU1F1/DNA complex to the labeled P1 binding site (a high-affinity POU1F1 binding site) of the PRL proximal promoter. In contrast, the S179R mutant POU1F1 had a markedly decreased specific binding. Binding specificity was confirmed by the displacement obtained both using a POU1F1 monoclonal antibody or an excess of cold probe.


Figure 4
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  		FIG. 4. EMSA of the human PRL response element (hP1) using the WT POU1F1 and S179R mutant POU1F1 proteins. Arrow, Specific POU1F1/DNA complexes. hP1, P1 site protein; EV, pcDNA3.1 EV protein; WT, WT POU1F1 protein; S179R, S179R mutant POU1F1 protein; Ab, human POU1F1 monoclonal antibody; Oligo, oligonucleotide of P1 site (5'-AATGCCTGAATCATTATATTCATGAAGATATC-3'). The binding was displaced by addition of either the POU1F1 antibody or the unlabeled oligonucleotide in excess. The antibody did not generate a new band (supershift) presumably by direct interaction and displacement of the POU1F1 binding to the labeled probe.

 
Consistent with this result, the S179R residue is located in the fourth helix of the POU1F1 POU-specific domain, close to the DNA binding cleft in the crystallographic structure of the POU1F1 DNA binding domain (Fig. 5Go). Consequently, it is likely that the replacement of Ser179 by Arg causes an alteration of the three-dimensional structure of the POU1F1 dimer associated with the double-stranded DNA helix. Molecular modeling analysis indeed confirmed that this mutation may directly interfere with DNA binding.


Figure 5
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  		FIG. 5. Crystallographic structure of the POU1F1 DNA binding domain as derived from the data of Jacobson et al. (24 ). The S179R mutation is located in the fourth helix of the POU1F1 POU-specific domain, close to the DNA binding cleft.

 
To ascertain that the mutant transcription factor was able to reach its target genes, we further investigated the nuclear accumulation of the WT POU1F1 and S179R mutant POU1F1. In the control cells transfected with the GFP expression vector only, GFP was distributed homogeneously in both the cytoplasm and the nucleus (Fig. 6Go). In contrast, nuclear accumulation of WT-POU1F1-GFP and S179R mutant POU1F1-GFP was observed in all cells analyzed.


Figure 6
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  		FIG. 6. Expression of the fusion protein of WT POU1F1 or the S179R mutant POU1F1 with GFP in COS-7 cells. Nuclear accumulation of the S179R mutant POU1F1 as well as the WT POU1F1 was observed in all cells analyzed. In the control cells, GFP was distributed homogeneously in both the cytoplasm and the nucleus.

 

   Discussion
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
References
 
POU1F1 mutations are rare. Turton et al. (23) recently reported that causative POU1F1 mutations were identified in 10 of 129 individuals with CPHD and isolated GH deficiency (7.8%). Since the first identification of human mutations of the gene encoding POU1F1 in 1992, at least eight recessive mutations of the POU1F1 gene in exons 3 and 4 (F135C, R143Q, A158P, R172X, R172Q, E174G, W193R, and L194Q) have been identified in patients with CPHD involving GH, PRL, and TSH with either a small or normal anterior pituitary gland (8, 9, 13, 14, 16, 19, 22, 23). These patients were homozygous for each mutation. Exons 3 and 4 encode the POU-specific domain of POU1F1, which is important for its ability to dimerize and bind to DNA with high affinity. However, functional analysis has been performed in only a small number of patients with mutations (8, 9, 25, 29, 30). We have previously studied the functional properties of the F135C mutant. It was unable to transactivate POU1F1 target genes despite preserved DNA binding capacity to a specific POU1F1 response element. Furthermore, a structural study based on the crystallographic analysis of the DNA/POU1F1 POU domain complex suggested that this mutant was deficient in its ability to interact with other proteins (25).

With regard to mutations located in the transactivation domain of POU1F1, on the other hand, P14L and P24L have been reported in patients with CPHD (18, 19). Both of these were heterozygous. The patient with P24L POU1F1 mutation described by Ohta et al. (19) was a 5-yr-old boy whose plasma GH and PRL levels were undetectable, and basal TSH level was also low. Although P14L and P24L may dominantly inhibit the transcriptional activity of the WT POU1F1, Kishimoto et al. (31) have shown that the P24L mutant had reduced transcriptional activity, whereas P14L exerted transcriptional effects similar to that of the WT. Furthermore, they found that codon 24 proline in the transactivation domain as well as the POU domain of POU1F1 was crucial to recruit the coactivator, CBP, and that P24L could not recruit CBP into the complex containing POU1F1 in the cultured cells (31).

In the study presented here, we identified the novel S179R homozygous mutation in exon 4 of the POU1F1 gene. This POU1F1 gene mutation caused complete deficiencies of GH, PRL, and TSH with pituitary hypoplasia. We showed that this mutation induced partial or complete loss of function (depending on the target genes studied) associated with markedly decreased DNA binding and possibly altered interaction with CBP. Our data confirmed that replacement of the highly conserved Ser residue at position 179 by an arginine was responsible for the CPHD phenotype involving GH, PRL, and TSH, although this mutant showed normal nuclear translocation.

Recently, nuclear localization of WT POU1F1 and mutant forms of POU1F1 was investigated by Kishimoto et al. (31). Their data suggest that the POU domain but not the transactivation domain is involved in nuclear localization. Furthermore, Sock et al. (32) reported that a basic cluster (RKRKKR) preceding helix 1 of the POU homeodomain was shown by deletion mutagenesis to be involved in the nuclear localization of Tst-1/Oct 6, a member of the POU protein family. Interestingly, our study demonstrated that the S179R POU1F1 mutation albeit affecting the POU-specific domain did not alter the nuclear localization. In previous studies, it was also found that a naturally occurring splice variant of the POU1F1 protein that did not contain a POU-specific domain entered the nucleus and served as a dominant repressor of POU1F1 function (33, 34). These and our data suggest that the POU-specific domain is not crucial for POU1F1 nuclear entry. Further studies including characterization of the nuclear localization signal of POU1F1 will be necessary to clarify the mechanism underlying POU1F1 nuclear entry, which may open new directions in the functional analysis of POU1F1 abnormalities.

Thus, our study showed that homozygous mutations in the POU-specific domain of the POU1F1 gene such as S179R caused a functional defect of the POU1F1 protein through decreased DNA binding to target genes and altered synergism with the coactivator CBP, despite the retention of nuclear accumulation. Consistent with the location of the Ser179 residue in the DNA binding cleft of the POU1F1 POU domain, the S179R mutant POU1F1 has decreased DNA binding ability. Because CBP cotransfection (Fig. 3Go) did not improve S179R stimulation of the PRL promoter, whereas it enhanced transcriptional activity of WT POU1F1, CBP interaction interface may be modified by this mutation. The fact that another mutant protein (carrying the P24L missense mutation) was similarly unable to recruit CBP is of particular interest, especially because proline 24 and serine 179 do not belong to the same protein domains, suggesting that both the so-called transactivation and POU-specific domains might be involved in such interactions with CBP. Computational and direct protein-protein interaction studies are necessary to confirm this observation, which is similar to what was found for the R143Q mutant POU1F1 (30). As already reported by others (6), LHX3 cotransfection with WT POU1F1 resulted in a synergistic transactivation of the PRL reporter gene. In this first study assessing the transcriptional capability of a POU1F1 mutant in the presence of LHX3, we found that LHX3 cotransfection with the S179R mutant also resulted in a synergistic transactivation of the PRL reporter gene, but to a lesser extent, compatible with the lower transactivation induced by S179R alone. This suggests maintained LHX3-S179R protein-protein interactions similar to the preserved protein-protein interactions shown for LHX3 mutants and WT POU1F1 (6).

Finally, our study indicates that functions and three-dimensional structures of abnormal POU1F1 proteins are variously influenced by the types and locations of the mutations of the POU1F1 gene. Together with the variability in genetic background, this may explain why the phenotypes caused by mutations of the POU1F1 gene are likely to be variable in humans. This and other similar studies will help decipher the mechanisms whereby alterations of the POU1F1 protein can affect the functionality of this key pituitary-specific transcription factor that plays essential roles in the control of growth, homeostasis, and lactation.


   Acknowledgments
 
We thank Serge Amselem and Marie-Laure Sobrier (Paris, France), Sally Radovick (Baltimore, MD), and Simon J. Rhodes (Indianapolis, IN) for kindly providing biological tools for the present study.


   Footnotes
 
The GENHYPOPIT network for the study of genetic determinants of hypopituitarism, coordinated by T.B., was funded by the Groupement d’Intérêt Scientifique Institut des Maladies Rares (Grant GISMR0201) and by the Programe Hospitalier de Recherche Clinique (Grant PHRC 25/2003, French Ministry of Health). The present study was supported by the Association pour le Développement des Recherches Biologiques et Médicales au Centre Hospitalier Régional de Marseille and by Pfizer France. This study was also supported by The Jikei University Research Fund.

Disclosure statement: The authors have nothing to declare.

First Published Online September 12, 2006

1 I.M. and S.V.-K. contributed equally to this work. Back

Abbreviations: CBP, cAMP response element-binding protein-binding protein; CPHD, combined pituitary hormone deficiency; EV, empty vector; GFP, green fluorescent protein; LHX3, LIM homeodomain transcription factor 3; Pit-1, pituitary-specific transcription factor 1; PRL, prolactin; WT, wild type.

Received October 18, 2005.

Accepted September 1, 2006.


   References
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
 References
 

  1. Ingraham HA, Chen R, Mangalam HJ, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519–529[CrossRef][Medline]
  2. Cohen LE, Wondisford FE, Radovick S 1996 Role of Pit-1 in the gene expression of GH, PRL, and TSH. Endocrinol Metab Clin North Am 25:523–540[CrossRef][Medline]
  3. Theil LE, Karin M 1993 Transcriptional control of GH expression and anterior pituitary development. Endocr Rev 14:670–689[Abstract/Free Full Text]
  4. Chen R, Ingraham HA, Treacy MN, Albert VL, Wilson L, Rosenfeld MG 1990 Autoregulation of Pit-1 gene expression mediated by two cis-active promoter elements. Nature 346:583–586[CrossRef][Medline]
  5. Xu L, Lavinsky RM, Dasen JS, Flynn SE, Mclnerney EM, Mullen TM, Heinzel T, Szeto D, Korzus E, Kurokawa R, Aggarwal AK, Rose DW, Glass CK, Rosenfeld MG 1998 Signal-specific co-activator domain requirements for Pit-1 activation. Nature 395:301–306[CrossRef][Medline]
  6. Sloop KW, Parker GE, Hanna KR, Wright HA, Rhodes SJ 2001 LHX3 transcription factor mutations associated with combined pituitary hormone deficiency impair the activation of pituitary target genes. Gene 265:61–69[CrossRef][Medline]
  7. Li S, Crenshaw EB, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG 1990 Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347:528–532[CrossRef][Medline]
  8. Tatsumi K, Miyai K, Notomi T, Kaibe K, Amino N, Misumo Y, Kohno H 1992 Cretinism with combined hormone deficiency caused by a mutation in the Pit1 gene. Nat Genet 1:56–58[CrossRef][Medline]
  9. Radovick S, Nations M, Du Y, Berg LA, Weintraub BD, Wondisford FE 1992 A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 257:1115–1118[Abstract/Free Full Text]
  10. Pfäffle RW, DiMattia GE, Parks JS, Brown MR, Wit JM, Jansen M, Nat HVd, Brande JLVd, Rosenfeld MG, Ingraham HA 1992 Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 257:1118–1121[Abstract/Free Full Text]
  11. Cohen LE, Zanger K, Brue T, Wondisford FE, Radovick S 1999 Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency. Mol Endocrinol 13:476–484[Abstract/Free Full Text]
  12. Pernasetti F, Milner RDG, Al Ashwal AAZ, De Zegher F, Chavez VM, Muller M, Martial JA 1998 Pro239Ser: a novel recessive mutation of the Pit-1 gene in seven middle eastern children with GH, PRL, and TSH deficiency. J Clin Endocrinol Metab 83:2079–2083[Abstract/Free Full Text]
  13. Pellegrini-Bouiller I, Belicar P, Barlier A, Gunz G, Charvet JP, Jaquet P, Brue T, Vialettes B, Enjalbert A 1996 A new mutation of the gene encoding the transcription factor Pit-1 is responsible for combined pituitary hormone deficiency. J Clin Endocrinol Metab 81:2790–2796[Abstract/Free Full Text]
  14. Brown MR, Parks JS, Adess ME, Rich BH, Rosenthal IM, Voss TC, Vander Heyden TC, Hurley DL 1998 Central hypothyroidism reveals compound heterozygous mutations in the Pit-1 gene. Horm Res 49:98–102[CrossRef][Medline]
  15. Irie Y, Tatsumi K, Ogawa M, Kamijo T, Preeyasombat C, Suprasongsin C, Amino N 1995 A novel E250X mutation of the Pit-1 gene in a patient with combined pituitary hormone deficiency. Endocr J 42:351–354[Medline]
  16. Hendriks-Stegeman BI, Augustijin KD, Bakker B, Holthuizen P, van der Viet PC, Jansen M 2001 Combined pituitary hormone deficiency caused by compound heterozygosity for two novel mutations in the POU domain of the Pit1/POU1F1 gene. J Clin Endocrinol Metab 86:1545–1550[Abstract/Free Full Text]
  17. Gat-Yablonski G, Lazar L, Pertzelan A, Phillip M 2002 A novel mutation in PIT-1: phenotypic variability in familial combined pituitary hormone deficiencies. J Pediatr Metab 15:325–330
  18. Fofanova OV, Takamura N, Kinoshita E, Yoshimoto M, Tsuji Y, Peterkova VA, Evgrafov OV, Dedov II, Goncharov NP, Yamashita S 1998 Rarity of PIT1 involvement in children from Russia with combined pituitary hormone deficiency. Am J Med Genet 77:360–365[CrossRef][Medline]
  19. Ohta K, Nobukuni Y, Mitsubuchi H, Fujimoto S, Matsuo N, Inagaki H, Endo F, Matsuda I 1992 Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency. Biochem Biophys Res Commun 189:851–855[CrossRef][Medline]
  20. Parks JS, Abdul-Latif H, Kinoshita E, Meacham LR, Pfäffle RW, Brown MR 1993 Genetics of growth hormone gene expression. Horm Res 40:54–61[Medline]
  21. Blankenstein O, Muhlenberg R, Kim C, Wuller S, Pfäffle RW, Heimann G 2002 A new C-terminal located mutation (V272ter) in the PIT-1 gene manifesting with severe congenital hypothyroidism. Possible functionality of the PIT-1 C-terminus. Horm Res 56:81–86
  22. McLennan K, Jeske Y, Cotterill A, Cowley D, Penfold J, Jones T, Howard N, Thomsett M, Choong C 2003 Combined pituitary hormone deficiency in Australian children: clinical and genetic correlates. Clin Endocrinol (Oxf) 58:785–794[CrossRef][Medline]
  23. Turton JPG, Reynaud R, Mehta A, Torpiano J, Saveanu A, Woods KS, Tiulpakov A, Zdravkovic V, Hamilton J, Attard-Montalto S, Parascandalo R, Vella C, Clayton PE, Shalet S, Barton J, Brue T, Dattani MT 2005 Novel mutations within the POU1F1 gene associated with variable combined pituitary hormone deficiency. J Clin Endocrinol Metab 90:4762–4770[Abstract/Free Full Text]
  24. Jacobson EM, Li P, Leon-Del-Rio A, Rosenfeld MG, Aggarwal AK 1997 Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected arrangement and flexibility. Genes Dev 11:198–212[Abstract/Free Full Text]
  25. Vallette-Kasic S, Pellegrini-Bouiller I, Sampieri F, Gunz G, Diaz A, Radovick S, Enjalbert A, Brue T 2001 Combined pituitary hormone deficiency due to the F135C human Pit-1 (pituitary-specific factor 1) gene mutation: functional and structural correlates. Mol Endocrinol 15:411–420[Abstract/Free Full Text]
  26. Tseng YH, Schuler LA 1998 Transcriptional regulation of interleukin-1ß gene by interleukin-1ß itself is mediated in part by Oct-1 in thymic stromal cells. J Biol Chem 273:12633–12641[Abstract/Free Full Text]
  27. Sali A, Blundell T 1993 Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 234:779–815[CrossRef][Medline]
  28. Marchler-Bauer, Anderson JB, DeWeese-Scott C, Fedorova ND, Geer LY, He S, Hurwitz DI, Jackson JD, Jacobs AR, Lanczycki CJ, Liebert CA, Liu C, Madej T, Marchler GH, Mazumder R, Nikolskaya AN, Panchenko AR, Rao BS, Shoemaker BA, Simonyan V, Song JS, Thiessen PA, Vasudevan S, Wang Y, Yamashita RA, Yin JJ, Bryant SH 2003 CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res 31:383–387[Abstract/Free Full Text]
  29. Liang J, Moye-Rowley S, Murer RA 1995 In vivo mutational analysis of the DNA binding domain of the tissue-specific transcription factor, Pit-1. J Biol Chem 270:25520–25525[Abstract/Free Full Text]
  30. Cohen RN, Brue T, Naik K, Houlihan CA, Wondisford FE, Radovick S 2006 The role of CBP/p300 interactions and Pit-1 dimerization in the pathophysiological mechanism of combined pituitary hormone deficiency. J Clin Endocrinol Metab 91:239–247[Abstract/Free Full Text]
  31. Kishimoto M, Okimura Y, Yagita K, Iguchi G, Fumoto M, Iida K, Kaji H, Okamura H, Chihara K 2002 Novel function of the transactivation domain of a pituitary-specific transcription factor, Pit-1. J Biol Chem 277:45141–45148[Abstract/Free Full Text]
  32. Sock E, Enderich J, Rosenfeld MG, Wegner M 1996 Identification of the nuclear localization signal of the POU domain protein Tst-1/Oct 6. J Biol Chem 271:17512–17518[Abstract/Free Full Text]
  33. Day RN, Day KH 1994 An alternatively spliced form of Pit-1 represses prolactin gene expression. Mol Endocrinol 374–381
  34. Voss JW, Wilson L, Rhodes SJ, Rosenfeld MG 1993 An alternative Pit-1 RNA splicing product reveals modular binding and nonmodular transcriptional activities of the POU-specific domain. Mol Endocrinol 7:1551–1560[Abstract/Free Full Text]




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