9 Truncation of PITX2 differentially affects its activity on physiological targets

Marie-He´le`ne Quentien1,Ve´ronique Vieira2, Maurice Menasche2, Jean-Louis Dufier2, Jean-Paul Herman1, Alain Enjalbert1, Marc Abitbol2,3 and Thierry Brue1,4 1Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M), UMR6231, Faculte´ de Me´decine Secteur Nord, Universite´ de la Me´diterrane´e, CS 80011, 13344 Marseille Cedex 15, France

2Centre de Recherches The´rapeutiques en Ophtalmologie, E´ quipe d’accueil 2502 MENRT, Universite´ Paris Descartes, Faculte´ de Me´decine Paris-Descartes-campus Necker, Paris, France

3De´partement d’Ophtalmologie, Centre Hospitalier Universitaire Necker-Enfants Malades, Paris, France

4Service d’Endocrinologie, Diabe`te et Maladies Me´taboliques, et Centre de Re´fe´rence des Maladies Rares d’origine Hypophysaire DEFHY, Hoˆpital de la Timone, 264 rue St Pierre, Cedex 5, 13385 Marseille, France

(Correspondence should be addressed to T Brue at Service d’Endocrinologie, Diabe`te et Maladies Me´taboliques, et Centre de Re´fe´rence des Maladies Rares d’origine Hypophysaire DEFHY, Hoˆpital de la Timone; Email: [email protected])

Abstract

The bicoid-like transcription factor PITX2 has been previously described to interact with the pituitary-specific POU homeodomain factor POU1F1 (human ortholog of PIT-1) to achieve cell-specific expression of (PRL) and GH in pituitary somatolactotroph cells. In this work, we have investigated the functional properties of three PITX2 mutants reported in Axenfeld–Rieger syndrome patients relative to the regulation of these , using reporter genes under the control of human PRL (hPRL), hGH, or POU1F1 promoters transfected in nonpituitary and pituitary cell lines. Among the three mutations studied, Y167X and E101X introduce a premature stop codon, and F104L leads to an amino acid substitution. While PITX2(E101X) is not expressed in the cells following transfection, and PITX2(F104L) is functionally inactive, the PITX2(Y167X) mutant keeps its DNA-binding capacity and displays a markedly enhanced activation of the hPRL and POU1F1 promoters, but not of the hGH promoter. Y167X is the first mutation of PITX2 described to result in a differential effect on the activation of its different physiological targets, hPRL and POU1F1 on one hand and hGH on the other hand. The differential effect of the Y167X mutation might be linked to an interaction of PITX2 with different transcription factors or cofactors when bound to the hPRL and POU1F1 or the hGH promoters. These results might form the basis for the identification of the PITX2 complex necessary for the differential GH or PRL expression. Journal of Molecular Endocrinology (2011) 46, 9–19

Introduction The bicoid transcription factors, PITX1 and PITX2, are the early markers of development. The pituitary gland is a key regulator of growth, Their expression patterns are similar, but differ in reproduction, and homeostasis. Its development is several ways. They recognize the same bicoid binding the result of a highly regulated pattern of expression of sites and activate the promoters of a number of genes signaling molecules and transcription factors. In mice, expressed in the pituitary gland, including those in which this development has been well investigated, encoding a glycoprotein subunit (aGSU), TSHb, a complex genetic cascade leads to the formation of LHb, FSHb, GnRH receptor (GnRHR), PRL, and GH this organ, which secretes six from five (Tremblay et al. 2000). We have shown that in humans, different cell types: corticotrophs producing ACTH; several pituitary promoters, including the human thyrotrophs producing TSH; gonadotrophs producing PRL (hPRL), hGH, and POU1F1, are the targets for LH and FSH; somatotrophs producing GH, and PITX2 (Quentien et al. 2002b) and that PITX factors are lactotrophs producing prolactin (PRL; Zhu et al. involved not only in the basal activity of the hPRL 2007). Transcription factors essential for this process promoter but also in its activation by forskolin, include PIT-1, the murine ortholog of human POU1F1 epidermal growth factor (EGF), and TRH (Quentien (Li et al. 1990), RPX (Hermesz et al. 1996), LHX3 et al. 2002a). The Pitx2 gene is expressed in the brain, (Netchine et al. 2000, Machinis et al. 2001) and LHX4 , pituitary gland, mandibular and maxillary (Bach et al. 1997), PROP1 (Sornson et al. 1996), SOX2 regions, eye, and umbilicus. It consists of six exons (Fantes et al. 2003, Kelberman et al. 2008) and SOX3 and encodes a homeodomain protein with a lysine (Rizzoti et al. 2004, Kelberman et al. 2006), and PITX1 residue at position 50, typical of bicoid-related homeo- and PITX2 (Drouin et al. 1998). domain , which are major developmental

Journal of Molecular Endocrinology (2011) 46, 9–19 DOI: 10.1677/JME-10-0063 0952–5041/11/046–009 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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transcription factors (Muccielli et al. 1996, Semina et al. (Pernasetti et al. 1997), with ubiquitous transcriptional 1996, Gage & Camper 1997, Gage et al. 1999a). Five factors, such as ETS factors (Bradford et al. 2000), or isoforms have been identified, with different N-terminal with the panpituitary transcriptional regulators LHX3 domains but identical C-termini and homeodomains (Bach et al. 1997), and in particular with PITX1 and (Arakawa et al. 1998, Lamba et al. 2008). The regions PITX2 (Drouin et al. 1998, Tremblay et al. 1998). We flanking the homeodomain, including the C-terminal show in this study that one of the mutations studied, part of PITX2 in particular, may modulate the binding Y167X, markedly potentiates the capacity of PITX2 to affinity and/or specificity through protein–protein activate the hPRL and POU1F1 promoters, whereas it interactions with factors such as POU1F1 (Amendt leaves its activity toward the hGH promoter unchanged. et al. 1999). Footz et al. (2009) identified two domains Thus, the mutation differentially affects the activity of inhibiting transactivation in the C-terminal tail. These this transcription factor on its different physiological domains are separated by a domain responsible for targets. This is the first PITX2 mutation for which such increasing transactivation. a strong differential effect on GH and PRL expression Mutations of PITX2 have been identified as being exists. This differential effect suggests that the molecu- responsible for Axenfeld–Rieger syndrome (ARS; lar partners interacting with PITX2 on these three Tumer & Bach-Holm 2009). This autosomal dominant different promoters are not the same and modulate the condition is characterized by a spectrum of ocular functional activity of the mutation. anterior chamber defects, dental hypoplasia, and redundant periumbilical skin. In some cases, other A symptomatic features have also been described, includ- F104L E101X ing mental retardation, cleft palate, and anomalies Y167X of the cardiovascular system (Semina et al. 1996). GH PITX2b 1 84HD 144 317 deficiency (Feingold et al.1969, Sadeghi-Nejad & B C Senior 1974) and short stature (Brooks et al. 1989) Nuclear extracts Whole-cell extracts have also been reported in several cases, with an 0 – PITX2 Y167X E101X F104L 0 – PITX2 Y167X E101X F104L 55 000 55 000 enlarged sella turcica and combined GH/TSH or 43 000 43 000 GH/ACTH deficiency (Polomeno et al.1980). A 34 000 34 000

broad range of PITX2 mutations have been described, 26 000 26 000 with missense mutations usually affecting the homeo- domain and leading, in some cases, to a dominant- 17 000 negative mutant form (Semina et al. 1996, Alward et al. 17 000 1998, Kulak et al. 1998, Perveen et al. 2000, Priston et al.

10 000 2001, Phillips 2002, Xia et al. 2004, Idrees et al. 2006, 10 000 Kniestedt et al. 2006, Vieira et al. 2006, Weisschuh et al. 2006). Splice-site (Semina et al. 1996, Doward et al. 1999, Perveen et al. 2000, Maciolek et al. 2006), frameshift, and nonsense mutations have been found throughout 123456 123456 the gene and may result in truncated proteins (Semina 0 – PITX2 Y167X E101X F104L

et al. 1996, Perveen et al. 2000, Priston et al. 2001, Wang 42 000 et al. 2003, Brooks et al. 2004, Lines et al. 2004, Saadi et al. 2006, Vieira et al. 2006). 123456 We report in this study a functional analysis of three Figure 1 Production of the wild-type and mutant forms of PITX2. PITX2 mutations responsible for ARS, which we have (A) Schematic representation of the PITX2 protein domains. described previously and are shown in Fig. 1A (Vieira Arrows show the locations of the three point mutations associated et al. 2006). Indeed, even when patients have no defined with Axenfeld–Rieger syndrome, which were analyzed in this study. (B) Western blot analysis of nuclear extracts of CV1 pituitary axis deficiencies, as is the case here, the expressing wild-type or mutant PITX2 after transient transfection. mutations identified can be used as tools to approach SDS-PAGE analysis of proteins showed the presence of bands the mechanisms by which PITX2 influences its targets. of the expected molecular weight: 32 kDa for PITX2 and Thus, in the present work, we have investigated the PITX2(F104L); 17 kDa for PITX2(Y167X). No translation product was observed for the PITX2(E101X) mutant. (C) Western blot effects of these mutations on the regulation, by PITX2, analysis of whole-cell extracts of CV1 cells expressing wild-type of pituitary target genes, using the human pituitary or mutant PITX2 after transient transfection (upper pannel). promoters of the genes encoding PRL, GH, and SDS-PAGE analysis of proteins showed similar results compared POU1F1. The terminal differentiation of lactotroph to (B). The lower panel shows the actin used as a control for the efficacy of the extraction. The prestained molecular weight cells and the regulation of the Prl or GH genes require standards are indicated. No PITX2-specific band was detected the interaction of POU1F1 with cell-type-specific when the experiment was performed with nontransfected cells (0) partners, such as the nuclear receptor or cells transfected with the empty vector (K).

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Materials and methods monkey fibroblast-like CV1 cells were grown in DMEM supplemented with 10% FCS. CV1 cells were Plasmid constructs and mutagenesis transfected in serum-free medium, with the assistance of Lipofectamine reagent (Invitrogen) according to The previously described (Caccavelli et al. 1998) reporter the manufacturer’s instructions. Cells were plated at a plasmid hPRL-164/Luc, containing 164 bp of the hPRL density of 50 000 cells/well, in 12-well plates, 24 h proximal promoter, was a gift from Dr J Martial before transfection. Transfections were carried out (Universite´ de Lie`ge, Lie`ge, Belgium). The 493 bp with 0.6 mg reporter plasmid, 0.1–1 mg effector plasmid, hGH gene promoter (Pa3-GHp-Luc) was provided by and 20 ng phRL. Cells were incubated with the Dr N L Eberhardt (Mayo Clinic, Rochester, MN, USA) DNA/liposome complexes for 3 h and 1 ml of 20% and the 102 bp human Pit-1 (POU1F1) gene promoter FCS in DMEM was then added. In all transfections, fused to the luciferase gene (Luc) was provided by total DNA levels were kept constant and we controlled Dr M Delhase (University of California, San Diego, CA, for the nonspecific effects of the viral promoters using USA). The human PITX2 isoform b cDNA was obtained the appropriate empty vectors. from the RZPD German Resource Center cDNA library. Full-length human POU1F1 cDNA coding regions were cloned by PCR, using normal pituitary tissues and Dual luciferase assay specific oligonucleotide sequences. PITX2b and POU1F1 were inserted into the CMV-driven eukaryotic GH4C1 and CV1 cells were harvested 48 h after expression vector pcDNA3 (Invitrogen). Mutations of transfection and lysed in 250 ml passive lysis buffer the B1 and B2 sites in the hPRL-164/Luc construct and (Promega). The Dual-Luciferase Assay System (Pro- the Y167X, E101X, and F104L mutations in the mega) was subsequently used to assay the activities of pcDNA3PITX2 construct were generated by PCR, with the firefly and Renilla luciferases, measured sequentially the QuickChange Mutagenesis kit (Stratagene, La Jolla, on the same sample, with a luminometer. For each CA, USA) and the following commercially synthesized control, the total luciferase activity normalized against oligonucleotides (Eurogentec, Seraing, Belgium); Renilla luciferase activity was taken as 1 and the results mutations indicated in the sense strand (in bold): were expressed as fold activation over control. Data are B1mut: 50-GAAGATATCAAAGCGGTATAAAGCCAATA- presented as the meanGS.E.M. of three independent TCTGGGAAAGAG-30; B2mut: 50-GAAATTATGGGGGT- experiments using different plasmid preparations of ACGGTCAATGACGGAAATAGATGACC-30; Y167X: each construct. Statistical significance was determined 50-GCTCATGCAGCCCTAAGACGACATGTACCCAGG-30; by the Wilcoxon nonparametric paired test. Signifi- E101X: 50-GCAGCTCCAGGAGCTGTAGGCCACTTTCC- cance was declared at P!0.05. AGAGG-30; F104L: 50-GGAGCTGGAGGCCACTTTGCA- GAGGAACCGCTACC-30. Total and nuclear cell extracts Plasmid DNA was purified with the Qiafilter Plasmid Maxi kit (Qiagen) and all mutations were confirmed by Total cell extracts were prepared from CV1 cells 48 h DNA sequencing with the CEQ Dye Terminator Cycle following transfection in 100 mm dishes. Six hundred Sequencing with Quick Start kit (Beckman Coulter microliters ice-cold lysis buffer (25 mM Tris–HCl (pH France, Paris, France). 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM NaF, 1 mM phenyl- methylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, Cell culture and transfections and 10 mg/ml leupeptin) were added to each plate and Rat GH4C1 somatolactotroph pituitary cells were gently stirred for 30 min at 4 8C. Nonsolubilized grown in HamF10 medium supplemented with 15% material was removed by centrifugation at 5400 g for horse serum and 2.5% FCS. Cells were transfected in 10 min at 4 8C. Nuclear proteins were extracted from serum-free medium, with the liposome-based trans- the CV1 cells 48 h following transfection in 100 mm fection kit Transfast (Promega Corporation), used dishes. Cells were scraped in 1 ml PBS and centrifuged according to the manufacturer’s instructions. Cells at 400 g for 2 min. Four hundred microliters hypotonic were plated at a density of 250 000 cells/well in buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 12-well plates 24 h before transfection and were 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, transfected with 1 mg DNA (0.3 mg reporter plasmid, 10 mg/ml aprotinin, and 10 mg/ml leupeptin) were 0.1–1 mg effector plasmid, and 20 ng phRL, a Renilla added to the pellet and left on ice for 5 min. luciferase vector displaying constitutive expression Following the addition of 25 ml of 10% Nonidet P-40, (Promega Corporation)). Cells were incubated with nonsolubilized material was precipitated by centri- theDNA/liposomecomplexesfor1hand1.5ml fugation at 15 700 g for 30 s. The pellet was resus- complete medium was then added. African green pended in a 20 mM HEPES, 400 mM KCl, 20% glycerol, www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 46, 9–19

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2 mM dithiothreitol, and 0.5 mM PMSF buffer. After 400 mM KCl, 20% glycerol, 2 mM dithiothreitol, and one freeze–thaw cycle, protein solution was centrifu- 0.5mM PMSF) and 1mg poly(dI-dC). We added gated for 30 min at 16 900 g Protein contents in the 20 000 c.p.m. of the radiolabeled probe to the reaction lysates were measured colorimetrically using Lowry and incubated the mixture for a further 25 min. The protein assay (Bio-Rad Laboratories Inc.). bound proteins were separated from the free probe by electrophoresis in an 8% polyacrylamide gel containing 0.5% Tris-borate EDTA, at 180 V for 3 h at 4 8C. The gel Western blot analysis was then placed against X-ray film for autoradiography. Whole-cell protein extracts (40 mg) or nuclear protein extracts (25 mg) were resolved on a 15% SDS-PAGE using the Laemmli buffer system. After transfer to Results PVDF membrane (PerkinElmer, Waltham, MA, USA), immunodetection of PITX2 was performed using Wild-type PITX2, PITX2(Y167X), and PITX2(F104L) rabbit polyclonal antibody (1/1000; Capra Science, mutant proteins, but not the PITX2(E101X) mutant A¨ngelholm, Sweden) and peroxidase-conjugated goat protein, are produced in vivo anti-rabbit IgG (1/2000; Jackson ImmunoResearch We evaluated the production of PITX2 mutant proteins Laboratories, Inc., West Grove, PA, USA). Immunodetec- by western blot using nuclear extracts (Fig. 1B)or tion of actin was performed with mouse monoclonal whole-cell protein extracts (Fig. 1C) from transfected antibody (1/2000; Sigma–Aldrich Inc., St Louis, MO, CV1 cells. In both types of extract, we detected bands USA) and peroxidase-conjugated goat anti-mouse IgG– of the expected sizes: a band of 32 kDa for wild-type HRP (1/10 000; Santa Cruz Biotechnology, Santa Cruz, (wt) PITX2 and PITX2(F104L), and of about 17 kDa CA, USA). Membranes were developed with the Lumi- for PITX2(Y167X), this protein being smaller due to nata Forte, Western HRP substrate, and immunodetec- the presence of a premature stop codon (Fig. 1B and C, tion system (Millipore Corporation, Billerica, MA, USA). lanes 3 and 4). We were unable to detect the PITX2(E101X) protein in either conditions (Fig. 1B In vitro transcription/translation and C, lane 5). Thus, this mutant is not synthesized in vivo and for this reason it was not further studied. The TNT T7-coupled reticulocyte lysate system (Pro- mega) was used for in vitro transcription/translation. Reactions were carried out in a total volume of 50 ml The PITX2(Y167X) mutant retains the capacity to bind with reticulocyte lysate, 1 mg plasmid DNA, 1 mM amino DNA, whereas the PITX2(F104L) mutant does not acid mixture, RNasin (40 U/ml; Life Technologies, Following the production, by in vitro translation, of the Carlsbad, CA, USA), T7 RNA polymerase, in the pre- wtPITX2 and the PITX2(Y167X) and PITX2(F104L) 35 sence or absence of S-Met (10 mCi/ml; PerkinElmer). mutant proteins (Fig. 2A), they were used to compare 35 S-Met-radiolabeled translation products were separa- their DNA-binding activities, using equal volumes of ted by SDS-PAGE. The gel was then placed against the in vitro translation products. Consistent with previous X-ray film for autoradiography. reports (Lamonerie et al. 1996, Quentien et al. 2002b), the wtPITX2 protein formed a specific complex with the known bicoid CE3 sequence of the POMC promoter Electrophoretic mobility shift assay (Fig. 2B, lanes 3 and 4). No binding was detected Electrophoretic mobility shift assays (EMSA) were for PITX2(F104L) (Fig. 2B,lanes7and8),whereas carried out with wild-type and mutant PITX2 and the PITX2(Y167X)/CE3 complexes were detected in these 32P-labeled double-stranded oligonucleotide 50-ACC- conditions (Fig. 2B, lanes 5 and 6). Thus, the Y167X AGGATGCTAAGCCTGTGTC-30, containing the CE3 mutation retains the ability to bind DNA, consistent with PITX-specific binding site (in bold) of the proopiome- the conservation of the homeodomain in this truncated lanocortin (POMC) promoter (Lamonerie et al. 1996). protein while the substitution of a Leu residue for a We 5 0 end-labeled 100 ng of annealed double-stranded Phe residue in the homeodomain of the PITX2(F104L) DNA by incubation with T4 polynucleotide kinase mutant impaired binding to the bicoid-specific CE3 probe. (Invitrogen, Paisley, UK) and 2 ml 32P-gATP (Perkin- Elmer) for 10 min at 37 8C. The reaction was stopped The PITX2(Y167X) mutant activates the hPRL-164 by adding EDTA to a concentration of 5 mM. The promoter, whereas the PITX2(F104L) mutant does not radiolabeled probe was then purified on a Sepharose G25 column. We incubated 2.5or5mlofin vitro We investigated the ability of the PITX2 mutants to translated proteins on ice for 25 min in a 20 ml reaction activate the hPRL promoter, by cotransfecting the mixture containing 1! binding buffer (20 mM HEPES, nonpituitary CV1 cell line with various amounts of

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A TNT proteins – PITX2 Y167X F104L construct and 30 ng of the different PITX2 expression 32 000 constructs alone or together with 30 ng wtPITX2 expression vectors. The PITX2(F104L) mutant did 25 900 not significantly modify the activation of the hPRL promoter by the wtPITX2 when both constructs 19 400 were cotransfected, as the observed activation by a 14 800 factor of 1.7 is about half of that observed with . B double dose of wtPITX2 (2 8 fold induction). By PITX2 Y167X F104L contrast, the PITX2(Y167X) mutant significantly TNT proteins (µl) 0 – 2·5 5 2·5 5 2·5 5 increased the PITX2-induced activation of the hPRL NS promoter (Fig. 4A). PITX2 In all, 100 ng wtPITX2 and PITX2(Y167X) induced . . Y167X hPRL activation by a factor of about 2 6 and 5 5 respectively, when cells were transfected singly with the corresponding constructs (Fig. 4B). Following Free probe cotransfection with half the quantities of each of the 12 3 4 5 678 wtPITX2 and PITX2(Y167X) expression vectors, trans- CE3* activation by a factor of about 3.5 was observed, this Figure 2 DNA-binding properties of wild-type and mutant PITX2. level of activation being intermediate between that (A) In vitro translation reactions were performed in the presence of induced by the double amount of wtPITX2 and 35S-Met. SDS-PAGE analysis of radiolabeled protein reactions showed the presence of bands of the expected molecular weight: PITX2(Y167X) each. 32 kDa for PITX2 and PITX2(F104L); 17 kDa for PITX2(Y167X). (B) EMSA with the CE3 PITX-binding site from the POMC gene promoter used as radiolabeled probe and two different volumes of The Y167X mutation increases the synergy between in vitro translation products for wild-type or mutant PITX2 forms. PITX2 and POU1F1 for the activation of hPRL Note that, as expected, no complex was formed in control promoter conditions, with the probe alone (0) or in presence of empty vector K ( ) (lanes 1 and 2 respectively). NS, nonspecific band. PITX2 PITX transcription factors are known to cooperate designated the wild-type protein, Y167X, the PITX2(Y167X), and F104L, the PITX2(F104L). CE3*, radiolabeled CE3 probe. with several other transcription factors in the pituitary gland. These factors include the pituitary-specific wtPITX2 and mutant PITX2 expression vectors together with this reporter construct (Fig. 3). Both 7 PITX2 wtPITX2 and PITX2(Y167X) activate the hPRL 6 Y167X * promoter in a dose-dependent manner. However, F104L while 25 ng of wtPITX2 vector activates the hPRL 5 E101X * promoter by a factor of about two, much stronger 4 * activation (factor of 3.7) was observed with the same

* * amount of the PITX2(Y167X) mutant. This enhanced 3

activity of the PITX2(Y167X) mutant relative to that induction Fold * 2 of the wtPITX2 could be observed at all levels of DNA input tested: transfection with 50 and 75 ng 1 of expression vector resulted in 2.7- and 3-fold (for . . 0 wtPITX2) or 4 3- and 5 6-fold (for PITX2(Y167X)) 0 255075 induction respectively. Thus, the Y167X mutation DNA amount (ng) induced a mean 75% increase in transactivation K activity over that of wtPITX2. The PITX2(F104L) and Figure 3 Differential transcriptional activation of the 164hPRL promoter by wild-type and mutant PITX2 proteins. Nonpituitary PITX2(E101X) constructs did not activate the hPRL CV1 cells were transfected using the hPRL-164/Luc reporter promoter at any of the doses tested. plasmid and various amounts of pcDNA3 expression vector containing full-length wild-type or mutant PITX2 cDNAs. A phRL plasmid was used as an internal control for transfection efficiency. The activities of wtPITX2 and PITX2(Y167X) on the Cells were harvested after 48 h and assayed for firefly luciferase hPRL promoter are additive activity. Results were normalized with respect to Renilla luciferase activity and are expressed as fold activation over control. We investigated the possible interference of the Transfections were performed in triplicate for each set of conditions in each experiment. The data shown are the PITX2(Y167X) and PITX2(F104L) mutants with the meanGS.E.M. of three independent experiments. Statistical transactivation of the hPRL promoter by the wtPITX2, significance was determined by the Wilcoxon nonparametric test. by cotransfecting CV1 cells with the hPRL-164/Luc *Significantly different from empty vector, P!0.05. www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 46, 9–19

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A ** The PITX2(Y167X)-induced transactivation of the 5 ** hPRL promoter requires the B1 and/or B2 bicoid 4·5 4 binding sites * 3·5 3 * We have previously identified two bicoid binding sites, 2·5 2 B1 and B2 (proximal and distal respectively, relative to

Fold induction Fold 1·5 K 1 the transcription start site (Fig. 5A) in the 164hPRL 0·5 promoter, both of which are required for PITX 0 transcription-factor-mediated transactivation of this PITX2 Y167X F104L PITX2 Y167X F104L pcDNA3 PITX2 promoter (Quentien et al. 2002a)). Disruption of the B B1 or B2 bicoid binding site decreased the activation of ** 6 hPRL-164/Luc by wtPITX2 by 10 and 45% (Fig. 5B). 5 ** 4 3 A hPRL/Luc 2 Fold induction Fold 1 A sequence 0 PITX2 0 100 50 0 Luc Y167X 0050 100 DNA amount (ng) B2 B1 C ** 18 16 14 12 PITX2 binding sites 10 * 8 6 *

Fold induction Fold 4 * POU1F1 binding sites 2 0 2 X CRE PITX2 Y167X PIT Y167X POU1F1 ETS consensus motif

PITX2 + Y167X POU1F1 AP1 binding site Figure 4 The Y167X mutation increases the transactivation of hPRL by PITX2. (A) CV1 cells were cotransfected with the hPRL- B 164/Luc construct and 30 ng of the indicated PITX2 constructs, 120 alone or together with an equal amount of wtPITX2 construct. PITX2 (B) CV1 cells were cotransfected with the hPRL-164/Luc 100 Y167X construct and various amounts of the wild-type PITX2 and * PITX2(Y167X) constructs, as indicated. (C) CV1 cells were 80 * cotransfected with the hPRL-164/Luc construct, PITX2(Y167X), and PITX2 constructs, together with the POU1F1 construct, to 60 * * assess the ability of PITX2(Y167X) to act in synergy with POU1F1 and the effect of this mutant on PITX2/POU1F1 synergy. The data 40

shown are the meanGS.E.M. of three independent experiments. to hPRL-164) 20 * * Statistical significance was determined by the Wilcoxon non- Promoter activity (relative parametric paired test. *Significantly different from wtPITX2, P!0.05. **Significantly different from PITX2, P!0.05. 0 hPRL/Luc B1mut B2mut B1B2mut Constructs transcription factor POU1F1, which is a major Figure 5 The PITX2 and PITX2(Y167X) proteins transactivation regulator of the somatolactotroph lineage and acts in activity of K164hPRL is decreased when either one or both of the synergy with the PITX transcription factors to activate bicoid binding sites (B1 and/or B2) were mutated. (A) Schematic the hPRL promoter (Quentien et al. 2002b). In our representation of the two PITX2 bicoid binding sites (B1 and B2) on the K164hPRL proximal promoter (ovals). POU1F1-binding conditions, cotransfection with 25 ng of each construct sites (rectangles) and consensus motifs for ETS factors, CRE, encoding wtPITX2 and POU1F1 activated the hPRL and AP1 are shown. The A sequence is indicated by a black line. promoter by a factor of 13 (Fig. 4C), whereas activation (B) CV1 cells were transfected with different hPRL-164/Luc by a factor of 17 was observed when cells were reporter constructs: wild-type, B1mut/Luc with mutated B1 site; B2mut/Luc with mutated B2 site, and B1/B2mut/Luc with mutated cotransfected with constructs encoding POU1F1 and B1 and B2 sites. The results are expressed as luciferase activity the PITX2(Y167X) mutant. Furthermore, cooperation with respect to that of the control wild-type hPRL-164/Luc between wild-type and mutant PITX2 was observed construct, arbitrarily set at 100%. Transfections were performed in if cells were cotransfected with both forms together triplicate for each set of conditions in each experiment. The data presented are the meanGS.E.M. of three independent experi- with POU1F1. The PITX2/POU1F1 synergic activation ments. Statistical significance was determined by the Wilcoxon of the hPRL promoter is therefore conserved with the nonparametric paired test. *Significantly different from hPRL- Y167X mutation. 164/Luc construct, P!0.05.

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The PITX2-induced transcriptional activity of the of this promoter as with wtPITX2. Similar results were double B1/B2 mutant was 80% lower than that obtained when pituitary GH4C1 cells were transfected observed with the native construct. The results were in the same conditions (Fig. 6B). the same for the activation by PITX2(Y167X).

The Y167X mutation increases the transactivation of Discussion hPRL and POU1F1 but does not modify the trans- K activation of the hGH promoter in the nonpituitary The 164 bp hPRL proximal promoter region was CV1 cell line and the pituitary GH4C1 cell line previously shown to be sufficient to drive the basal activity in somatolactotroph cells and to mediate the PITX2 transactivates the hPRL, POU1F1, and GH responses to almost all second messengers (Lemaigre promoters (Quentien et al. 2002b). Transfection of et al. 1989, Peers et al. 1991, 1992, Berwaer et al. 1993, 50 ng of wtPITX2 expression vector activated the hPRL Pernasetti et al. 1997, Caccavelli et al. 1998, Manfroid and POU1F1 promoters by factors of 1.5and2 et al. 2001). We have previously demonstrated that respectively, in CV1 cells (Fig. 6A), whereas the PITX2-driven hPRL promoter activation is dependent PITX2(Y167X) mutant induced a markedly potentiated on two functional PITX-regulator elements (B1 and B2) activation of both promoters: 3.5- and 4-fold induction residing in the K164 bp proximal promoter. The B2 respectively. Conversely, transfection with the hGH/ PITX-binding site is critical for synergic interaction Luc reporter construct and the PITX2(Y167X) with POU1F1 and for responsiveness to forskolin, expression vector resulted in the same activation level EGF, and TRH (Quentien et al. 2002a). The 500 bp of the proximal promoter of the human GH gene A pcDNA3 contain binding sites for POU1F1, SP1, and a Zn finger PITX2 protein as well as for PITX2 ( Jones et al. 1995). The Y167X ** 4·5 functional role of these PITX2 sites, however, has not ** * 4 * been investigated yet. 3·5 The aim of our study is to analyze the functional 3 effects of the PITX2 mutations found in ARS patients. 2·5 We have used, for this purpose, human pituitary * 2 promoters as target genes, the CV1 heterologous cell 1·5 Fold induction Fold * * * line, and the pituitary somatolactotroph GH4C1 cell 1 line (expressing PITX2, PITX1, POU1F1, PRL, and GH) 0·5 to characterize the three PITX2 mutations found in ARS 0 hPRL/Luc hGH/Luc POU1F1/Luc patients (Vieira et al. 2006). We have used PITX2 isoform b, which gives the strongest activation of pituitary B pcDNA3 PITX2 targets, including the PRL promoter, of the five isoforms 3 ** Y167X described (Cox et al. 2002, Lamba et al. 2008). The E101X mutation introduces a premature stop 2·5 * ** * codon resulting in a truncated protein that lacks a large 2 part of the DNA-binding domain and the carboxy- 1·5 * * * * terminal region. However, this protein could not be 1 produced in transfected CV1 cells. PITX2 mRNAs have Fold induction Fold 0·5 a short half-life and are unstable (Briata et al. 2003). This mutation may affect RNA stability, resulting in the 0 hPRL/Luc hGH/Luc POU1F1/Luc degradation of mRNAs in vivo by nonsense-mediated mRNA decay, a degradation pathway that targets Figure 6 The PITX2(Y167X) mutant protein enhanced activation of the hPRL/Luc and POU1F1/Luc promoter, but had no mRNAs with premature termination codons (Silva & transactivating effect on the hGH/Luc promoter in CV1 and Romao 2009). GH4C1 cells. (A) CV1 cells were transfected with the reporter The impairment of transactivation observed with the plasmids hPRL/Luc, hGH/Luc, or POU1F1/Luc and with wild-type PITX2(F104L) mutant can be explained by the loss of or mutant PITX2 constructs. (B) Homologous GH4C1 cells were transfected with the Transfast method, with the same reporter and DNA-binding activity. This Phe104 residue has been expression vectors as described above in (A). Transfections were implicated in the intramolecular interactions, ensuring performed in triplicate for each set of conditions in each the correct formation of the PITX2 homeodomain experiment. The data presented are the meanGS.E.M. of three (Fraenkel et al. 1998). The first helix is required to independent experiments. Statistical significance was determined contact the DNA and stabilize the protein/DNA by the Wilcoxon nonparametric paired test. *Significantly different from promoter basal activities, P!0.05. **Significantly different structure. Thus, the replacement of the Phe residue from PITX2, P!0.05. by a Leu residue in position 20 of the first helix might www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 46, 9–19

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affect the stability of the DNA/protein complex, these two transcription factors (Amendt et al. 1999). resulting in defective binding. Surprisingly, the cotransfection experiments resulted Our data show that the PITX2(F104L) mutant is in synergistic activation of the hPRL promoter by transcriptionally inert, while the PITX2(E101X) the PITX2(Y167X) mutant protein. This finding mutant is not produced in vivo. Thus, the observed shows that a direct physical interaction between these pathology in these cases might be due to haploinsuffi- two factors is not required for synergic transactivation ciency. The observations reported by Gage et al. (1999b) of the hPRL promoter and confirms the similar for transgenic mice are consistent with this hypothesis, conclusions by Saadi et al. (2001). On the other hand, as mice heterozygous for a hypomorphic allele of Pitx2 the binding of PITX2 to its specific binding sites is display a disturbed development of some organs and essential for synergic activation of the promoter as mimic most features of the ARS phenotype. shown by the loss of its activity when these sites are The Y167X mutation generates a PITX2 mutant mutated. This result confirms our previous demon- protein that activates the proximal hPRL promoter stration that the integrity of the B2 bicoid element and more strongly than wtPITX2. This mutant protein lacks of the P1 or P2 POU1F1-binding sites located in the most of the C-terminal tail beneath the homeodomain, hPRL proximal promoter is required for synergy retaining only 23 amino acids from this region. The between PITX2 and POU1F1 (Quentien et al. 2002a). C-terminal part of PITX2 has been implicated in the Studies showing that the PITX2 dose is critical modulation of the activity of the protein through during development (Diehl et al. 2006), the ARS conformational changes (Amendt et al. 1999), through ocular phenotype of mice with an overexpression of the inhibition of DNA binding and protein–protein PITX2 in the eye (Holmberg et al. 2004) and the interactions. Analysis of this part of the protein description of gain-of-function PITX2 mutations caus- indicated that the first 61 aa of the C-terminal region ing ARS (Priston et al. 2001, Saadi et al. 2006) support (ID1) inhibit the transactivation brought about by the the hypothesis that an ARS phenotype can be caused N-terminus domain of PITX2, while the AD2 domain, by a gain-of-function mutation. Thus, the ARS pheno- comprising residues 62–134, enhances the transactiva- type observed in the patient carrying the PITX2 tion (Footz et al. 2009). A second inhibitory domain, (Y167X) mutation may be due to the fact that this ID2, includes the last 39 aa of the C-terminal tail. When mutation leads to an increase in the activation of some a protein, such as POU1F1, binds to ID2, DNA binding of its ocular targets. An alternative explanation, and transcriptional activities are stimulated, probably invoking nonsense-mediated RNA degradation or due to masking of this inhibitory domain (Amendt et al. protein degradation, causing ARS by happloinsuffi- 1999). In our conditions, PITX2(Y167X) conserved its ciency, seems less likely, given that we could observe DNA-binding capacity. The PITX2(Y167X) mutant functional effects after transfection in the cells. activated the hPRL promoter very strongly in CV1 We assessed the transactivation properties of the cells and GH4C1 somatolactotrophs. We suggest that, PITX2(Y167X) mutant protein on two other pituitary despite the concurrent loss of the AD2 activatory somatolactotroph promoters, hGH and POU1F1, in domain, the disappearance of the two inhibitory CV1 and GH4C1 cells. On the POU1F1 promoter, domains is sufficient to lead to enhancement of the we found the same result as for the hPRL, i.e. a effect of the N-terminal activation domain and that, as a potentiation of the transactivation by PITX2 by the consequence, PITX2(1–166), comprising the intact mutation. Note that this phenomenon can be observed homeodomain of PITX2 allowing normal DNA binding also in the case of a minimal promoter, consisting and the activatory domain conferring transactivation only of the consensus bicoid binding site (Footz et al. properties, brings about a stronger transactivation of 2009). On the contrary, the effects of PITX2 on the the hPRL promoter than that observed for the full- hGH promoter were left unchanged by the mutation. length, functionally regulated protein. Note that a This mutation of the PITX2 gene is the first mutation similar enhancement of transactivatory activity has been reported to have different activities with the hPRL reported by Saadi et al. (2006) for a similar deletion and GH promoters in somatolactotroph cells. mutant, although slightly longer than the present one Transcription factors exert their functional activity (PITX2(W178X)), and the mechanisms involved are through interactions with different cofactors assembled likely the same as those for the Y167X mutation. Finally, into a complex around them. The composition of this wtPITX2 and the PITX2(Y167X) mutant compete for complex depends on various parameters such as the the same binding sites, as intermediate transactivation structural variations in the transcription-factor-binding levels were observed in cells cotransfected with both site pair. Transcription-factor-binding sites have specific wild-type PITX2 and PITX2(Y167X) constructs. allosteric effects on the transcription factor and The 39 aa domain in the PITX2 C-terminal is also different binding sites and/or DNA sequence context known to be required for synergy between PITX2 and around the binding sites may impose different confor- POU1F1 and mediates the physical interaction between mation on the transcription factor, as it has been

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Downloaded from Bioscientifica.com at 09/24/2021 11:29:15AM via free access PITX2 somatolactotroph target-gene regulation . M-H QUENTIEN and others 17 demonstrated, for example, in the case of POU1F1 Funding (Scully et al. 2000). It is also established that transcrip- tion factors can, in turn, induce structural changes, This work was supported by a grant from Pfizer to the Association pour such as bending of the DNA sequence to which they le De´veloppement de la Recherche Me´dicale au Centre Hospitalier de Marseille, ADEREM, and by a grant from Association Retina, France bind, in a manner specific to a given factor (Kerppola & to M A. V V’s PhD was supported by a fellowship from ‘Ministe`re de Curran 1993).Thereisthusacomplexdynamic l’Enseignement Supe´rieur et de la Recherche’. interplay between a given transcription factor and its binding site, leading to structural modifications of both that will influence the composition of the complex build around the transcription factor by associated References cofactors. Sequence analysis and a comparison with the bicoid-related homeodomain binding site TAATCC led Alward WL, Semina EV, Kalenak JW, Heon E, Sheth BP, Stone EM & to the identification of two putative PITX2-binding sites Murray JC 1998 Autosomal dominant iris hypoplasia is caused by a in the hGH promoter, at positions K119 (TTATCC) mutation in the Rieger syndrome (RIEG/PITX2) gene. American K Journal of Ophthalmology 125 98–100. (doi:10.1016/S0002- and 151 (TGATCC) (Quentien et al. 2002b) and these 9394(99)80242-6) sites seem indeed to correspond to the site of action of Amendt BA, Sutherland LB & Russo AF 1999 Multifunctional role of PITX2 (Quentien et al. preliminary results). These the Pitx2 homeodomain protein C-terminal tail. Molecular and sequences are different from the B1 (TAAACC) and B2 Cellular Biology 19 7001–7010. (TAATCT) PITX2-binding sites in the hPRL promoter. Arakawa H, Nakamura T, Zhadanov AB, Fidanza V, Yano T, Bullrich F, Shimizu M, Blechman J, Mazo A, Canaani E et al. 1998 Identification Thus, different conformation of the PITX2 factor and characterization of the ARP1 gene, a target for the human bound to these specific binding sites in the hPRL or acute leukemia ALL1 gene. PNAS 95 4573–4578. (doi:10.1073/ the hGH promoters may modulate the specific pnas.95.8.4573) interaction of PITX2 with the various factors required Bach I, Carriere C, Ostendorff HP, Andersen B & Rosenfeld MG 1997 to favor the production of PRL or GH and lead to A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes and different composition of the cofactor complexes. Development 11 1370–1380. (doi:10.1101/gad.11.11.1370) To explain the increase in the transactivating activity Berwaer M, Peers B, Nalda AM, Monget P, Davis JR, Belayew A & of PITX2(Y167X) on the hPRL of hPOU1F1 promoter, Martial JA 1993 Thyrotropin-releasing and epidermal we suggest the existence of a repressor regulating growth factor induce human prolactin expression via identical multiple cis elements. Molecular and Cellular Endocrinology 92 1–7. 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(doi:10.1076/opge.25.1.57.29002) Recent data have highlighted the major role of Caccavelli L, Manfroid I, Martial JA & Muller M 1998 Transcription combinatorial interactions of PITX2 with pituitary factor AP1 is involved in basal and okadaic acid-stimulated activity of transcription factors, but this network has been only the human PRL promoter. Molecular Endocrinology 12 1215–1227. partly characterized. Further studies with the truncated (doi:10.1210/me.12.8.1215) Cox CJ, Espinoza HM, McWilliams B, Chappell K, Morton L, Hjalt TA, PITX2(Y167X) mutant protein will help to identify the Semina EV & Amendt BA 2002 Differential regulation of gene PITX2-interacting proteins required to favor the expression by PITX2 isoforms. Journal of Biological Chemistry 277 production of PRL or GH expression in a particular 25001–25010. (doi:10.1074/jbc.M201737200) cell type. Diehl AG, Zareparsi S, Qian M, Khanna R, Angeles R & Gage PJ 2006 Extraocular muscle morphogenesis and gene expression are regulated by Pitx2 gene dose. Investigative Ophthalmology & Visual Science 47 1785–1793. (doi:10.1167/iovs.05-1424) Declaration of interest Doward W, Perveen R, Lloyd IC, Ridgway AE, Wilson L & Black GC 1999 A mutation in the RIEG1 gene associated with Peters’ The authors declare that there is no conflict of interest that could be anomaly. Journal of Medical Genetics 36 152–155. (doi:10.1136/jmg. perceived as prejudicing the impartiality of the research reported. 36.2.152) www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 46, 9–19

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Journal of Molecular Endocrinology (2011) 46, 9–19 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/24/2021 11:29:15AM via free access PITX2 somatolactotroph target-gene regulation . M-H QUENTIEN and others 19

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