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Molecular Cloning of LIM Homeodomain Transcription Factor

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Molecular Cloning of LIM Homeodomain Transcription Factor Journal of Reproduction and Development, Vol. 58, No 1, 2012 —Original Article— Molecular Cloning of LIM Homeodomain Transcription Factor Lhx2 as a Transcription Factor of Porcine Follicle-Stimulating Hormone Beta Subunit (FSHβ) Gene Takako KATO1), Akio IshikAWA2), Saishu YOshiDA2), Yoshiya SANO2), Kousuke KitAHARA2), Michie NAKAYAMA 2), Takao SUSA2) and Yukio KATO1,2,3) 1)Institute of Reproduction and Endocrinology, Meiji University, Kanagawa 214-8571, Japan 2)Laboratory of Molecular Biology and Gene Regulation, Division of Life Science, Graduate School of Agriculture, Meiji University, Kanagawa 214-8571, Japan 3)School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan Abstract. We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone β subunit gene (Fshβ) by the Yeast One-Hybrid Cloning System using the upstream region of –852/–746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LβT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions –835/–829, –818/–812 and –806/–800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LβT2 and others. Since LHX2 was previously identified as a transcription factor forCga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshβ, it is possible that LHX2 controls the synthesis of FSH at the transcription level. Key words: FSH, Gene regulation, Glycoprotein hormone, Lhx2, Pituitary (J. Reprod. Dev. 58: 147–155, 2012) ollicle-stimulating hormone (FSH) is a member of the pituitary observed that GnRH significantly stimulates the gene expression Fglycoprotein hormone family that includes both a luteinizing of c-Jun and c-Fos, components of AP-1 factor [6]. Multiple pro- hormone (LH) and a thyroid-stimulating hormone (TSH). Each of gesterone response elements (PREs) have been identified in the these hormones shares a glycoprotein hormone common α subunit proximal region of ovine [7] and rat Fshβ [8]. Similarly, estrogen (αGSU) and contains a unique β subunit that confers biological response elements (EREs) were also identified in –105/–72 b of specificity on its respective hormone function. The synthesis and ovine Fshβ [9]. It was reported that the –270/–248 b region of rat secretion of FSH and LH are restricted to pituitary gonadotropes. Fshβ is responsive to activin-dependent activation mediating the Since the regulatory mechanisms of the three subunit genes that transcription factors Smad and PITX2 [10]. A similar region is form gonadotropins are of special interest, several approaches also responsible for Pitx1, the homologue of both PITX2 [11] and have already achieved and provided a better understanding of the FOXL2 [12]. Nevertheless, the mechanism underlying the cell/ molecular mechanisms and regulatory factors governing the basal tissue-specific control of Fshβ-expression remains unclear. and cell-specific expression of the αGSU gene Cga( ) and Lhβ [1]. We have previously reported that the Fd2 region (−852/−746 b On the other hand, our knowledge of the regulation of Fshβ expres- of porcine Fshβ promoter) is a target for many nuclear proteins sion remains limited to the information from extracellular signals. of the porcine anterior pituitary [13] as well as cloned Prop1 [14] GnRH stimulation of FSH is mediated by the protein kinase C and Prx2 [15], both of which belong to the homeodomain tran- signaling cascade via AP-1 sites in the region of –120/–83 bases (b) scription factor family as an Fd2-binding protein. In addition, we from the transcription start site [2–4] and the distal region between have demonstrated that the −852/+10 b region is sufficient for the –4152/–2878 and –2550/–1089 b of ovine Fshβ [5]. We previously pituitary-specific expression of Fshβ [16]. PROP1 is essential for normal production of gonadotropins as well as for the development of PIT1 lineage cells in both humans and mice [17]. This paper Received: July 12, 2011 describes the molecular cloning of an additional transcription Accepted: October 31, 2011 factor for porcine Fshβ, LHX2, which is a member of the LIM- Published online in J-STAGE: December 2, 2011 ©2012 by the Society for Reproduction and Development homeodomain protein family and has been previously identified Correspondence: Y Kato (e-mail: [email protected]) as a transcription factor for Cga [18]. 148 KATO et al. Materials and Methods been described previously [22]. The sequences were aligned using a TAAT motif as a landmark and were submitted for WebLogo One-hybrid cloning of pituitary transcription factors binding analysis (http://weblogo.berkeley.edu/), a graphical representation to the Fd2 region of a nucleotide sequence alignment able to display the relative The integration of Fd2 (–852/–746 b) of the porcine Fshβ pro- frequency of each nucleotide at any position [23]. moter into the chromosome of yeast YM4271 and the screening of Fd2-binding protein from an adult porcine pituitary cDNA library Cell culture, transfection and reporter gene assay constructed in pAD-GAL4 (Stratagene, La Jolla, CA, USA) has A transient transfection assay was carried out using LβT2 cells been described [14]. and Chinese hamster ovary cells (CHO) as already described [21]. LβT2 is the mouse pituitary gonadotrope lineage cell line [24] Production of recombinant protein endogenously expressing gonadotropin genes of Cga, Lhβ and Complimentary DNAs of a full-length porcine LHX2 and a trun- Fshβ [25] and was kindly provided by Dr PL Mellon (University cate LHX2 (ΔLIM-LHX2), which is deleted the N-terminal region of California, San Diego, CA, USA). After incubation for more (amino acid residues 1–203) containing two LIM domains, were than 48 h, an aliquot (5 µl) of cultured medium was assayed for cloned in-frame into the pET32a vector to generate a TrxA-His-Tag secreted alkaline phosphatase activity using the Phospha-Light fusion protein (Novagen, Madison, WI, USA). The cDNAs were Reporter Gene Assay System (Applied Biosystems) according then transformed into BL21-CodonPlus (DE3)-RIPL (Stratagene). to the manufacturer’s instructions with a MiniLumat LB 9506 The recombinant proteins were expressed and prepared using an luminometer (Berthold, Wildbad, Germany). All values were Overnight Express Autoinduction System 1 (Novagen), followed by expressed as means ± SD of quadruplicate transfections in two purification using MagExtractor-His-Tag- (Toyobo, Osaka, Japan). independent experiments. Statistical significance was calculated by Student’s t-test. The reproducibility and reliability of the reporter Electrophoretic mobility shift assay (EMSA) and DNase I assay without internal control used in this study was described in footprinting assay our previous paper [26]. The production of FAM-labeled DNA fragments was described previously [15]. EMSA and DNase I footprinting were also carried Quantitative real-time RT-PCR out as previously described [14, 19]. Total RNAs, which were extracted from the porcine anterior pituitaries of German Landrace pigs with intact gonads of both Construction of reporter vectors and expression vector sexes during the fetal (f40, f50, f65, f82, f95 and f110) and postnatal The following procedures were described previously [15]. periods (p8, p60, p160 (prepuberty) and p230 (sexually matured)), Serial truncated upstream regions of the porcine Fshβ (Accession were kindly supplied by Dr. F. Elsaesser and pooled for 1–6 individu- no. D00621) [20] were obtained by PCR and ligated to the se- als of the respective age and sex for cDNA synthesis as previously creted alkaline phosphatase (SEAP) plasmid vector, pSEAP2-Basic described [27]. Total RNAs were extracted from pituitary tumor (Clontech Laboratories, Inc., Mountain View, CA, USA), result- cell lines of the mouse (αT1-1, αT3-1, LβT2, LβT4, TαT1, MtT/S) ing in the reporter vectors FSHβ (–2320/+10), FSHβ (–1965/+10), and rat (AtT-20) and a nonpituitary mouse cell line, L929. The FSHβ (–985/+10), FSHβ (–596/+10), FSHβ (–238/+10) and FSHβ characteristics of each cell line and their cDNA syntheses were (–103/+10) for FSHβ promoter. A deletion mutant of −745/−104 described in a previous paper [28]. leaving Fd2 and the endogenous proximal promoter region (ΔFSHβ Real-Time PCR was performed on an ABI Prism 7500 Sequence (–985/+10)) was constructed. Detector (Applied Biosystems) using gene-specific primer sets and The expression vector of porcine Lhx2 cDNA was constructed in a TaqMan MGB probe for Lhx2 and cyclophilin A. Primer and probe the mammalian expression vector pcDNA3.1/Zeo+ (Invitrogen, San sequences were as follows: mouse and rat Lhx2 forward primer, Diego, CA, USA), resulting in Lhx2/pcDNA3.1. Reporter vectors 5′-TGAGAGCTTCCGTATTTTCAAAGA-3′; mouse and rat Lhx2 re- consisting of an LHX2-binding site were constructed by ligation of verse primer, 5′-CAATTCATCCAAAGTATGAAGATAAAACAG-3′; synthetic oligonucleotides with an AgeI cutting sequence on both mouse and rat Lhx2 TaqMan probe 5′-TGCCACGTGC- sides of the minimum promoter of Cga fused in the pSEAP2-Basic CTTAG-3′; porcine Lhx2 forward primer, 5′-GGCTAGAGC- vector [21]. Serial mutations in the upstream region were made by TTCTGTATTTTCAAAGAC-3′; porcine Lhx2 reverse primer, PCR with an oligonucleotide primer set composed of a replace- 5′-AATGAACAAGTCATCCAAAGTATGGA-3′; porcine ment (TAAT with GCCG or ATTT with CGGC, respectively) for Lhx2 TaqMan probe, 5′- TGCCACGTGCCTTAG-3′; mouse FSHβ (–985/+10).
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