Regulation of Porcine Pituitary Glycoprotein Hormone Alpha Subunit Gene with LIM-Homeobox Transcription Factor Lhx3

Abstract

Abstract. The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitaryderived cell line, LβT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LβT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions. Key words: Gene regulation, Lhx3, LIM homeodomain, Pituitary glycoprotein hormone (J. Reprod. Dev. 55: [425][426][427][428][429][430][431][432] 2009) ituitary glycoprotein hormone common alpha subunit (αGSU) plays an indispensable role as a common counterpart by forming a heterodimer with the specific β subunits of follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH) [1]. It is expressed differently in gonadotrope cells producing FSH and LH and in thyrotrope cells producing TSH under cell-specific mechanisms [2]. Understanding the molecular basis of αGSU gene (Cga) regulation would be of scientific interest.We previously cloned and determined up to the -1059 bp upstream sequence of the porcine Cga [3] and demonstrated that transcription factors, Ptx1, Prop1 and Msx1, are potentially capable of modulating this gene [4][5][6]. Several studies have demonstrated that many cis-elements are important for the response of the extracellular signal and cell type-or tissue-specific expression of Cga [7,8]. These cis-elements include pituitary glycoprotein hormone basal element (PGBE, [9], gonadotrope-specific element (GSE, [10], cAMP-responsive element (CRE) [11][12][13], GnRH responsive element [14], upstream regulatory element (URE) [15], trophoblast-specific element (TSE) [16] and alpha basal elements 1 and 2 (alpha BE1 and BE2) [15]. Transcription factors, Lhx2 [9] and Lhx3 [17] for PGBE, Sf1 for GSE [10], CRE binding protein (Creb) for CRE [13,18] and Gata2 for GATA element [19], participate in cell-type or tissue-specific regulation. Thus, in the proximal region, expression of Cga is controlled by various trans-acting factors through binding to the discrete elements.Recently, we reported a prominent transcription activity of the porcine Cga promoter over the PGBE [4]. The distal region of the porcine Cga prom...

Molecular Cloning of Paired Related Homeobox 2 (Prx2) as a Novel Pituitary Transcription Factor

“…DNase I footprinting analysis using the binding mixture described above was carried out as described previously [26,27].…”

Transcription of follicle-stimulating hormone subunit genes is modulated by porcine LIM homeobox transcription factors, LHX2 and LHX3

“…We previously reported a gradual increase in Lhx3 -expression during fetal development by RT-PCR [ 25 ], which was similar to that of Lhx2 -expression [ 14 ]. Real-time PCR during porcine pituitary development revealed that the expression of both isoform genes gradually increased before birth, followed by an appreciable decrease by P230 ( Fig.…”

“…The full-length expression vector of porcine Lhx2 , Lhx3a , Lhx3b , and Lhx3-del (consisting of a 28–406 amino acid region with a primer 5′-GAGAATTCGCGATGGATCCCACTGTGTGCC-3′ based on a previous paper [ 24 ]) was constructed in the mammalian expression vector, pcDNA3.1/Zeo+ (Invitrogen, Carlsbad, CA, USA). For the DNA-binding assay, recombinant proteins were produced by cloning of the truncate cDNAs, ΔLIM-Lhx2 (ΔLIM-LHX2 consisting of amino acid numbers 170–406) and ΔLIM-Lhx3 (ΔLIM-LHX3 consisting of 148–401), into the pET32a vector, followed by expression in Escherichia coli BL21-CodonPlus (DE3)-RIPL (Stratagene, La Jolla, CA, USA), since the LIM domains aggregate by interacting as described previously [ 25 ]. The recombinant proteins were expressed and prepared using an Overnight Express Autoinduction System 1 (Novagen, Madison, WI, USA), followed by purification using His-Tag Mag beads (Toyobo, Osaka, Japan).…”

Molecular Cloning of LIM Homeodomain Transcription Factor <i>Lhx2</i> as a Transcription Factor of Porcine Follicle-Stimulating Hormone Beta Subunit (FSHβ) Gene

“…Taken together with previous studies showing that LHX2 is a transcription factor for Cga [40], the present study suggested a possibility that LHX2 regulates the synthesis of FSH at the transcriptional level. In addition, LHX3 is known to participate in the modulation of porcine Cga [54] and Fshβ [39,55] with common and unique target sites of LHX2. Since the presence of mRNA and the protein for LHX3 in LβT2 cells [39,55] and for LHX2 in αT3-1 cells [18,52] have been reported, it is likely that LHX2 and LHX3 coexist and participate in a different regulation of the same gene with characteristic binding specificity.…”