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Identification and Analysis of Prophet of Pit-1-Binding Sites in Human Pit-1 Gene

Department of Basic Allied Medicine (N.I., M.K., H.S., K.T., T.Y., D.Y., Y.S., Y.O.), Kobe University School of Medicine, Kobe 654-0142, Japan; Division of Endocrinology/Metabolism, Neurology, and Hematology/Oncology (G.I., K.I., Y.T., K.C.), Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; and College of Nursing Art and Science (H.K.), University of Hyogo, Kobe 673-8588, Japan

Address all correspondence and requests for reprints to: Yasuhiko Okimura, Department of Basic Allied Medicine, Kobe University School of Medicine, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0142, Japan. E-mail: okimuray{at}kobe-u.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prophet of Pit-1 (Prop1) is a transcription factor that regulates Pit-1 gene expression. Because Pit-1 regulates the differentiation of pituitary cells and the expressions of GH, prolactin and TSHβ genes, Prop1 mutation results in combined pituitary hormone deficiency in humans. However, Prop1-binding sites in human Pit-1 gene and the mechanism leading to combined pituitary hormone deficiency have remained unclear. In this study, we identified and analyzed Prop1-binding elements of the human Pit-1 gene. Prop1 stimulated the expression of the reporter plasmid containing Pit-1 gene from translation start site to –1340 dose dependently in GH3 cells. The activation by Prop1 was observed in GH3 and TtT/GF cells but not COS7, HeLa, JEG3, and HuH7 cells. Deletion analysis of Pit-1 gene showed that the Prop1-responsive elements were present within the –257-bp region. Within the –257-bp region, there are four elements similar to consensus sequence of paired-like transcription factors. Because Prop1 is a member of paired-like transcription factors, we assessed the elements. EMSA and transient transfection assay using the mutation of the elements revealed that the element from –63 to –53 (the proximal Prop1 binding element) was essential to Prop1-binding and Prop1-induced activation of Pit-1 reporter plasmid. A region at –8kb of human Pit-1 gene is similar to the distal region containing Prop1-binding elements in mouse Pit-1 gene. We showed the region functioned as an enhancer. Furthermore, chromatin immunoprecipitation assay showed that the proximal element could bind Prop1 in vivo cultured cells. Taken together, these findings indicated the novel functioning binding elements of Prop1 in human Pit-1 gene.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE PITUITARY GLAND develops from a middle structure that is contiguous with the primordium of the ventral diencephalons. Contact of the primordial stomadial ectoderm and the neuroepithelium during head folding results in the initial organ determination, forming Rathke’s pouch (1, 2). In Rathke’s pouch, several transcriptional factors including LIM homeodomain transcription factor 3 (3, 4), pituitary homeobox (5, 6), and gene expressed in embryonic stem cells (7, 8) appear subsequently. After the proliferation of the pituitary cells, different hormone-producing cells arise from distinct spatial and temporal fashion (9).

Differentiation of GH-producing cells is regulated by Pit-1, a pituitary-specific transcription factor (10, 11). Pit-1 has been shown to play a pivotal role in the cell type-specific expression of the prolactin (PRL) (10), TSHβ (12), and GHRH receptor gene (13) in addition to the GH gene and appears at embryonic day (e) 13.5 in mice before GH gene expression. The expression of Pit-1 is regulated by a paired-like transcription factor, Prophet of Pit-1 (Prop1) that appears at e10 before the expression of Pit-1 (14). The expression of Prop1 is transient and disappears at e14.5. It is reported that Prop1 is required for the initial expression of Pit-1, and after the initial expression of Pit-1, Pit-1 expression is activated by factors other than Prop1.

The Ames murine dwarf (df) that shows GH, PRL, and TSHβ deficiency has a mutated Prop1 in which Ser of codon 83 is replaced with Pro (14, 15). The mutation is located in DNA binding domain, and the mutant Prop1 is reported not to bind PRDQ9, a consensus element of paired-like transcription factors including Prop1. As for mouse Pit-1 gene, there are two Prop1-binding sites at –7.8 and –7.9 kb from the transcription start site (14). In human Pit-1 gene, however, there are no reports concerning Prop1-binding elements. Prop1 mutation is not rare among the patients with combined pituitary hormone deficiency (CPHD) (16, 17). In humans the prevalence of Prop1 mutation among the patients with CPHD has been reported at 48% (16), and many of these mutations are thought to be located within the putative DNA bindings sites (16, 18). Because studies have been conducted showing defects in DNA binding activity of mutant Prop1 by EMSA using consensus elements of paired-like transcription factors (19, 20, 21, 22), it is thought the CPHD is due to the inability of Prop1 to bind and activate the Pit-1 gene. However, this has not been directly tested because the Prop1 binding sites have not been identified in the human Pit-1 gene. Vieira et al. (19), however, reported that Prop1 stimulated the human Pit-1 reporter plasmid containing proximal promoter site of 1.2 kb, suggesting Prop1-binding elements might be located near the transcription start site in human Pit-1 gene. To clarify the mechanism of Prop1 to activate Pit-1, it is important to identify and analyze Prop1-binding elements. In the present study, we attempted to identify Prop1-binding elements of human Pit-1 gene and analyzed the function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture
GH3 and JEG3 cells were obtained from American Type Culture Collection (Manassas, VA), and TtT/GF, COS7, HeLa, and HuH7 cells were obtained from Riken (Saitama, Japan). These cells were cultured in DMEM with 10% (vol/vol) calf serum. Culture media contained penicillin G (100 µg/ml) and kanamycin (15.5 µg/ml).

Plasmids
The 1.4 kb of the 5'-flanking region (from –1340 to +30, when the translation start site was numbered as +1) of the human Pit-1 gene (National Center for Biotechnology Information accession no. NM_001122757, gene ID 5449) was PCR amplified from human genome library (CLONTECH, Mountain View, CA) using 5'-GGGTACCG-CTGCTTACTGACAAAGTGTTC-3' and 5'-GGGAGCTCATCAGCCG-AAGTAAAAGCTTGG-3' as primers. The amplified 1.4-kb Pit-1 gene was inserted into KpnI and SacI sites of multicloning site of pGL3 basic vector (Promega Japan, Tokyo, Japan), which was named –1340-Pit-1-Luc. For deletion study to identify important sites for Pit-1 gene expression, a series of PCR-amplified Pit-1 genes (from –996, –646, –492, –257, or –142 to +30) were inserted into multicloning site of pGL3 basic vector, and these plasmids were named –996-, –646-, –492-, –257-, and –142-Pit-1-Luc, respectively. All the DNAs amplified by PCR were sequenced with a DNA sequencer [model ABI PRISM 377; PerkinElmer Japan (Tokyo, Japan); Applied Biosystems Japan (Tokyo, Japan)] to confirm DNA sequence. A Prop1-binding site (–63 to –53) of –257-Pit-1-Luc and –142-Pit-1-Luc was mutated with a site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. In the site-directed mutagenesis of both plasmids, mutant sense primer (5'-CGGCCCTTTGAGACAGTAGGATAATAAAACT-CTGATTTGGGG-3') and mutant antisense primer (5'-CCCCAAATCAGAGTTTTATTATCCTACTGTCTCAAAGGGCCG-3') were used. Bold characters indicate the mutated sites. The resulting vectors were named –257M-Pit-1-Luc and –142M-Pit-1-Luc.

To evaluate the function of proximal Prop1-binding element (–63 to –53) in the presence or absence of the distal Prop1-binding elements, two sets of plasmid pairs were made. One was distal A+-142Pit-1-Luc and distal A+-142M-Pit-1-Luc, and the other was distal B+-142Pit-1-Luc and distal B+-142M-Pit-1-Luc. Distal A region (–8111 to –7954) that have two distal Prop1-binding elements was PCR amplified from human genome library (CLONTECH) using 5'-CGGGGTACCGAATCAATGTAGTATAAGGCAGGT-3' (KpnI site is underlined) and 5'-TTGGCGCGCGCTGTCTAGATAATAAAGCTCTGA-3' (BssHII site is underlined) as primers. The DNA region between –257 and –143 of –257-Pit-1-Luc or –257M-Pit-1-Luc was displaced with the amplified 158-bp DNA. The resulting plasmid had a 158-bp DNA region containing two distal Prop1-binding elements just upstream –142-Pit-1-Luc or –142M-Pit-1-Luc because KpnI and BssHII digestion cut out DNA region between –257 and –143. The plasmid finally had three Prop1-binding elements (two distal elements and one proximal element). To make distal B+-142Pit-1-Luc and distal B+-142M-Pit-1-Luc, double-strand oligonucleotides containing a Prop1-binding element (–8002 to –7988) were inserted into –142-Pit-1-Luc or –142M-Pit-1-Luc. Human Prop1 expression vector (pcDNA3.1/Prop1) was provided by Dr. T. Usui (National Hospital Organization, Kyoto Medical Center, Kyoto, Japan). Human Prop1 cDNA that had been cut out from pcDNA3.1/Prop1 was inserted into the HindIII and SmaI sites of multicloning site of pFlag-CMV-2 expression vector (Sigma-Aldrich Japan, Tokyo, Japan).

Transient expression assays
pcDNA3.1/Prop1 (1.2 µg) and a series of Pit-1 reporter plasmids (0.4 µg) were transfected to GH3, TtT/GF, COS7, HeLa, JEG3, or HuH7 cells using Lipofectamine 2000 (Invitrogen Japan, Tokyo, Japan). Five nanograms of pRL-CMV containing the cDNA encoding Renilla luciferase (Promega) were also cotransfected to evaluate transfection efficiency. Cells were harvested 24 or 48 h after transfection, and luciferase activity was measured with Luminescencer-PSN (ATTO, Tokyo, Japan) using the dual-luciferase assay system (Promega). For dose-response experiments, 0.3, 0.6, or 1.2 µg of pcDNA3.1/Prop1 was transfected to GH3 cells with 0.4 µg of –1340-Pit-1-Luc. Luciferase activity was measured 48 h after transfection. The luciferase activity was normalized with the activity of cotransfected pRL-CMV. Values were expressed as multiples of induction relative to the activity of the pGL3 basic vector and represent mean ± SD of at least four determinations, and the whole experiment was repeated three times.

Prop1 protein
The Prop1 cDNA (National Center for Biotechnology Information accession no. NM_006261) was amplified by PCR using primers (forward, 5'-ATGGAAGCAGAAAGGAGGCG-3'; reverse, 5'-AGAGGATCCTCAGTTCCAGGACTTGGATG-3') and pcDNA3.1/Prop1 as a template. The resulting cDNA was inserted into EcoRV and BamHI sites of the pTD1 (Shimadzu, Tokyo, Japan) to create Prop1 mRNA expression. The cDNA was transcribed using Thermo T7 transcription kit (TOYOBO, Osaka, Japan) and was purified with DyeEx 2.0 spin kit (QIAGEN). The resulting mRNA was translated using the Transdirect insect cell (Shimadzu) according to the manufacturer’s instruction.

EMSA
Mobility shift assays were performed to assess the Prop1-binding activity of Pit-1 gene promoter. Four elements, the sequences of which were similar to that of paired-like transcription factors-binding motif, were located within –257 bp of Pit-1 gene. To clarify which element is important for Prop1-binding, four oligonucleotide probes (probe A, from –257 to –228; probe B, from –227 to –198; probe C, from –189 to –145; probe D, from –88 to –42) were made. To compare Prop1-binding to putative distal elements (–8068 to –8058 and –8000 to 7989) and the proximal element (–63 to –53), probe E (–8079 to –8047) and probe F (–8011 to –7979) were also made. These oligonucleotides were labeled with digoxigenin (DIG) using terminal transferase. Binding reactions contained 62 fmol of the labeled probe, varying amounts of Prop1, 1 µg poly(deoxyinosine-cytosine), 0.1 µg poly-L-lysine, 20 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM (NH₄) ₂SO₄, 1 mM dithiothreitol, 2% Tween 20, and 30 mM KCl in a total volume of 20 µl. Reaction mixtures were incubated for 15 min at room temperature. For binding competition assays, 8 pmol unlabeled oligonucleotides were added to the reaction mixture. After incubation, the reaction mixtures were applied to nondenaturing polyacrylamide gel. After electrophoresis, DIG-labeled DNA fragments were transferred onto a Hybond-N-membrane (GE Healthcare, Tokyo, Japan) and detected using the DIG gel shift assay kit (Roche Diagnostics, Tokyo, Japan).

Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using a ChIP assay kit (Upstate, Lake Placid, NY) and described elsewhere (20). Briefly, 24 µg of Flag-CMV-Prop1 was transfected to GH3 cells in a 10-cm dish. After 48 h, GH3 cells were fixed with 1% formaldehyde and incubated for 10 min at 37 C. Cells were washed using ice-cold PBS and pelleted by centrifugation for 4 min at 2000 rpm and resuspended in lysis buffer. The nuclei of the cells were sonicated for 30 min to reduce the DNA length to between 200 and 600 bp. Before immunoprecipitation, the chromatin was precleared with unblocked protein A Sepharose. The precleared chromatin was divided into three equal parts. One part was used immunoprecipitation with anti-Flag antibody (Sigma) and the other for negative control with or without normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Immunocomplex were precipitated with protein A Sepharose and were washed. The immunoprecipitated DNA-protein complex was eluted in 250 µl of elution buffer containing 200 mM NaCl, and the Prop1-DNA cross-links were reversed by 4 h incubation at 65 C. Then proteins were digested by proteinase K at 45 C. The DNA from samples was extracted through phenol/chloroform and ethanol-precipitated with 20 µg of glycogen. The immunoprecipitated DNA was analyzed by PCR. For the analysis, the following primers were used: rat Pit-1 –112- to –1-bp region primers (forward, 5'-GCCGCCCTGATGTATATATG-3'; reverse, 5'-CCCACAAGAGAGTAGAGAAC-3') and –3659 to –3537-bp region primers (forward, 5'-TTTAGCTCAGTGGTAGAGCG-3'; reverse, 5'-CCTAGGTTGATTAGCCATCC-3'). These PCR products were verified with 2% agarose gel electrophoresis.

Statistical analysis
All data are presented as means ± SD. After ANOVA analysis where appropriate, Tukey-Kramer test was used to analyze differences between groups. P < 0.05 was considered as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of Prop1-responsive regions in the human Pit-1 gene
pcDNA3.1/Prop1 stimulated the expression of –1340-Pit-1-Luc time and dose dependently in GH3 cells (Fig. 1, A and B). Interestingly, the activation of –1340-Pit-1-Luc by Prop1 was observed in GH3 and TtT/GF cells but not COS7, HeLa, JEG3, and HuH7 cells (Fig. 1C). To identify Prop1-responsive elements in human Pit-1 gene, we generated a series of reporter plasmids that have different 5'-end (from –1340, –996, –646, –492, or –257 to +30) and examined the effect of these deletions on the reporter activity in GH3 cells. Basal luciferase activity of a series of deletion constructs was not different (Fig. 2A). Prop1 stimulated luciferase activity of all the reporter plasmids examined (Fig. 2B), suggesting that the Prop1-responsive elements may be present within –257-bp region from the Pit-1 translation start site.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Dose-dependent and cell-specific activation of human Pit-1 reporter plasmid. A, GH3 cells were transfected with 0.4 µg of –1340-Pit-1-Luc in the absence or presence of 1.2 µg of pcDNA3.1/Prop1. Luciferase activity was measured 24 and 48 h after transfection. The results are shown as relative fold increase ± SD of luciferase activities compared with the control transfected with pcDNA3.1. B, Dose-response activation of –1340-Pit-1-Luc by pcDNA3.1/Prop1 was assessed in GH3 cells. pcDNA3.1/Prop1 (0.3, 0.6, or 1.2 µg) was transfected to GH3 cells with 0.4 µg of –1340-Pit-1-Luc. Luciferase activity was measured 48 h after transfection. C, –1340-Pit-1-Luc (0.4 µg) or pGL3 basic vector was transfected into GH3, TtT/GF, COS7, HeLa, JEG3, and HuH7 cells with 1.2 µg of pcDNA3.1/Prop1 or empty expression vector. Promoter activity was assayed by measuring luciferase activity 24 h after transfection. The effect of pcDNA3.1/Prop1 on the activities of –1340-Pit-1-Luc (solid column) and pGL3 basic vector (open column) are shown as relative fold increase ± SD of luciferase activities compared with the activity of respective reporter plasmids transfected with pcDNA3.1, and fold induction by Prop1 was compared between –1340-Pit-1-Luc and pGL3 basic vector. *, P < 0.05.

View larger version (12K): [in this window] [in a new window]   FIG. 2. Effect of Prop1 on luciferase activity of a series of human Pit-1 deletion constructs. The schematic diagram on the left represents each deletion construct of the human Pit-1 gene fused to luciferase gene. The indicated numbers represent bases from ATG codon as +1. GH3 cells were cotransfected with 0.4 µg of each luciferase construct in the absence or presence of 1.2 µg of pcDNA3.1/Prop1. A, Basal luciferase activity of a series of deletion constructs is shown as relative fold increase ± SD compared with that of control (pGL3 basic vector). Basal luciferase activity of the constructs was not different. B, Induction of luciferase activity of deletion constructs by Prop1 is shown as relative fold increase ± SD of luciferase activities compared with basal activity of each deletion construct.

View larger version (69K): [in this window] [in a new window]   FIG. 3. Localization of Prop1-responsive elements in the human Pit-1 promoter. A, The nucleotide sequences of the –257-bp region from the translation start site of the human Pit-1 gene. The elements similar to PRDQ9 are denoted by bold characters. B, Diagram of the human Pit-1 gene promoter, showing the location of oligonucleotides (probes A–D) used in the EMSA. The open rectangles show the location of the putative Prop1-responsive element in the Pit-1 promoter. C, EMSA was performed using Prop1 protein and the probes A–D. Prop1 protein was incubated with 62 fmol of DIG-labeled probes A–D or PRDQ9 (positive control) in the presence or absence of 130-fold excess of unlabeled oligonucleotide. Probes C and D showed a band of protein-DNA complex. Addition of excess of unlabeled oligonucleotide decreased Prop1-binding of probes C and D.

View larger version (81K): [in this window] [in a new window]   FIG. 4. Mutation of putative Prop1-binding element within probe D resulted in diminished binding to Prop1 in EMSA. A, DNA sequences of wild-type and mutated oligonucleotides are shown. Putative Prop1-binding elements are underlined. The mutated nucleotides (–171A->G and –170A->G in probe C, –61A->G and –60T->G in probe D) were represented by bold characters. B, Prop1 protein was incubated with 62 fmol of DIG-labeled wild-type probes C and D or mutated probes C and D in the presence or absence of 130-fold excess of unlabeled oligonucleotide. There were not any differences of the intensity of the shifted band between wild-type and mutated probe C. On the other hand, when mutated probe D was used, the intensity of the shifted band was dramatically reduced compared with wild-type probe D.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Mutation of the proximal Prop1-binding element resulted in diminished luciferase activity of Pit-1 reporter plasmid. A, –257-Pit-1-Luc contains four PRDQ9-like elements (–255 to –246, –210 to –201, –173 to –163, and –63 to –53). –142-Pit-1-Luc contains one PRDQ9-like element (–63 to –53). Prop1-induced activations of –257M-Pit-1-Luc and –142M-Pit-1-Luc, in which the same mutation (Mut) as mutated probe D was generated, were significantly reduced to 43 and 26% compared with –257-Pit-1-Luc and –142-Pit-1-Luc, respectively, in GH3 cells. B, Prop1-induced activations of –257M-Pit-1-Luc and –142M-Pit-1-Luc were also significantly reduced in TtT/GF cells. *, P < 0.05.

Comparison of Prop1-binding to distal and proximal Prop1-binding elements
There are two Prop1-binding sites at –7.8 and –7.9 kb in mouse Pit-1 gene, and the regions are thought to be involved in early activation of mouse Pit-1 gene. In human Pit-1 gene, two putative distal Prop1-binding elements are also conserved, suggesting that the distal Prop1-binding elements may function in human Pit-1 gene expression. We examined Prop1 binding to the distal and proximal Prop1-binding elements with EMSA. Probe D containing the proximal Prop1-binding element and probe F containing a distal Prop1-binding element (–8000 to 7989) showed definite shifted bands. The band intensity of these probes was stronger than that of probe E that have another putative Prop1-binding element (–8068 to –8058). PRDQ9 was used as positive control (Fig. 6).

View larger version (86K): [in this window] [in a new window]   FIG. 6. Comparison of Prop1-binding to distal and proximal Prop1-binding elements. Prop1-binding to the distal and proximal Prop1-binding elements was examined with EMSA. Prop1 protein was incubated with 62 fmol of DIG-labeled probe D, probe E, probe F, or PRDQ9 (positive control) in the presence or absence of 130-fold excess of unlabeled oligonucleotide. Probe D containing the proximal Prop1-binding element and probe F containing a distal Prop1-binding element (–8000 to 7989) showed definite shifted bands. The shifted band intensity of those oligonucleotides was stronger than that of probe E, which has another distal Prop1-binding element (–8068 to –8058). *, P < 0.05.

View larger version (22K): [in this window] [in a new window]   FIG. 7. Mutation of the proximal Prop1-binding element decreased luciferase activity of Pit-1 reporter plasmid with the distal Prop1-binding elements. A, GH3 cells were cotransfected with 0.4 µg of each luciferase construct in the absence or presence of 1.2 µg of pcDNA3.1/Prop1. Distal A+-142Pit-1-Luc and distal B+-142Pit-1-Luc, both of which contains distal Prop1-binding element in addition to the proximal Prop1-binding element, showed higher luciferase activity than 142Pit-1-Luc that have no distal Prop1-binding element. On the other hand, two nucleotide mutations (from –61 to –60) of the proximal Prop1-binding element (from –63 to –53) resulted in decreased luciferase activity in response to Prop1. The results are shown as relative fold increase ± SD of luciferase activities compared with control transfected with pcDNA3.1. Prop1-induced activations of distal A+-142M-Pit-1-Luc and distal B+-142M-Pit-1-Luc were significantly reduced to 55 and 35% compared with distal A+-142Pit-1-Luc and distal B+-142Pit-1-Luc, respectively. B, In TtT/GF cells, mutation of the proximal Prop1-binding element led to reduced activity in response to Prop1. Distal A+-142M-Pit-1-Luc and distal B+-142M-Pit-1-Luc were significantly reduced compared with distal A+-142Pit-1-Luc and distal B+-142Pit-1-Luc, respectively. *, P < 0.05.

View larger version (11K): [in this window] [in a new window]   FIG. 8. ChIP assay showed that proximal promoter site of Pit-1 gene could bind Prop1 in vivo cultured cells. Soluble chromatin prepared from GH3 cells transfected with Flag-CMV-Prop1 was immunoprecipitated with Flag antibody (Ab) or normal mouse IgG. Precipitated DNA fragments were amplified in PCRs using primer sets amplifying the –112 to –1-bp region or –3659- to –3537-bp region of the rat Pit-1 gene. The functional Prop1-binding element is located within the –112- to –1-bp region, whereas PRDQ9-like elements are not present within the –3659- to –3537-bp region. Lane 1, Immunoprecipitated sample with Flag antibody; lane 2, immunoprecipitated sample with normal mouse IgG; lane 3, sample not immunoprecipitated; lane 4, input in both upper panel (PCR product of the –112- to –1-bp region) and lower panel (PCR product of the –3659- to –3537-bp region). The PCR band corresponding to the –112- to –1-bp region was evident in the sample immunoprecipitated with Flag antibody, whereas no significant band was observed with normal mouse IgG. The PCR band corresponding to the –3659- to –3537-bp region was not amplified well in the samples immunoprecipitated with Flag antibody or normal mouse IgG.

View larger version (94K): [in this window] [in a new window]   FIG. 9. Comparison of wild and mutant Prop1-binding for proximal Prop1-binding element. Wild-type Prop1or mutant Prop1 (W194X Prop1) was incubated with 62 fmol of DIG-labeled probe D or PRDQ9 (positive control) in the presence or absence of 130-fold excess of unlabeled oligonucleotide. Wild and W194X Prop1 did not show difference in the binding to probe D containing the proximal Prop-1 binding element (–63 and –53) as well as PRDQ9.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Mouse Pit-1 gene has proximal and distal enhancer sites. Sornson et al. (14) already reported that the –10 to –5 kb region of the 5'-flanking region of mouse Pit-1 gene is important for its initial expression, whereas the region until –5 kb is not essential to the initial expression of Pit-1 using Lac-Z transgenic mice that were driven by 5'-flanking region of Pit-1 gene. They also showed that there were two Prop1-binding sites at –7.8 and –7.9 kb within the region and that Prop1 stimulated the expression of reporter plasmid that had three –7.8 Prop1-binding sites in transient expression assay. Prop1 expression occurs before Pit-1 expression. Ames mice that have mutant Prop1 show similar phenotype to Snell mice that have Pit-1 mutation and Pit-1 lineage cells (GH, PRL, and TSH cells) are not observed in Ames mice (14). These findings indicate that Prop1 is the activating factor for Pit-1 gene expression. Thus, Prop1-binding site at –7.8 kb is thought as an essential site for the initial activation of Pit-1 gene.

However, it is of note that mouse Pit-1 promoter region until –5 kb in Lac-Z transgenic mice did not contain the element corresponding to –63 to –53 bp shown in the present study (23, 24). The result in Lac-Z transgenic mice by Sornson (14) does not exclude the possibility that the Prop1-binding element from –63 to –53 bp plays a role in Pit-1 expression. Considering the result of present study, the Prop1-binding site is likely to be an element regulating Pit-1 expression. Indeed, the element is conserved in several animals including humans, monkeys, rats, mice, and pigs, suggesting the importance of the element (Fig. 10).

View larger version (19K): [in this window] [in a new window]   FIG. 10. DNA sequences containing the proximal Prop1-binding element in human, monkey, rat, mouse, and pig. Prop1-binding element TAATNNNATAA is conserved in human, monkey, rat, and mouse. One base is different in pig.

Prop1 expression is occurred at e10 in mice and disappeared at e14.5. Prop1 is not present in adult pituitary, although Prop1 mRNA is reported in human normal pituitary (25) and pituitary tumors (25, 26). Because it is difficult to assess whether endogenous Prop1 binds to Pit-1 genome DNA, we used transfection of Prop1 to GH3 cells and ChIP assay to confirm in vivo binding. Consistent with EMSA, the specific region containing the proximal Prop1-binding element was amplified by PCR, suggesting that the Prop1-binding element from –63 to –53 bp indeed binds to Prop1 in vivo.

The prevalence of Prop1 mutation among the patients with CPHD has been reported 48% (16). However, the molecular mechanism how mutant Prop1 causes the disease has remained unclear, although the mechanism was examined in several cases. To date almost all of Prop1 mutations were identified in DNA binding domain. The loss of DNA binding has been examined in EMSA using PRDQ9 because the Porp1-binding element in human Pit-1 gene was not identified. It was recently reported that some patients with CPHD have mutations in the putative transactivation domain but not DNA-binding domain of Prop1 (27, 28). Among the mutations, a mutation W194X Prop1 showed less DNA binding to PRDQ9 than the wild Prop1(27), suggesting the possibility that a domain other than DNA binding domain affects the binding affinity. Because the present study showed that the intensity of shifted probe D was weaker than that of PRDQ9 when wild Prop1 was used (Fig. 3C), W194X Prop1 might show much lower binding to the region –63 to –53, which is included in probe D. This could lead to low expression of Pit-1. However, we did not find a difference in the binding of wild and W194X Prop1 for probe D as well as PRDQ9 (Fig. 7). Thus, we think the defect of interaction with cofactors but not the defect of DNA binding would be responsible for CPHD induced by W194X Prop1.

In summary, we found a Prop1-binding element in the first exon of the human Pit-1 gene. The mutation of the element resulted in diminished binding to Prop1 and decreased expression of the Pit-1 reporter plasmid, suggesting that the Prop1-binding element plays a role in Pit-1 expression. Furthermore, ChIP assay showed that the element could bind Prop1 in vivo cultured cells.

	Acknowledgments

 
We thank Dr. T. Usui (National Hospital Organization Kyoto Medical Center) for the generous gift of pcDNA3.1/Prop1.

	Footnotes

 
This work was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture and grants from the Japanese Ministry of Health, Welfare, and Labor and the Growth Science Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: CPHD, Combined pituitary hormone deficiency; DIG, digoxigenin; e, embryonic day; PRL, prolactin; Prop1, Prophet of Pit-1.

Received January 8, 2008.

Accepted for publication July 15, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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