Transcriptional Activity of the 5' Upstream Region of the Porcine Glycoprotein Hormone .ALPHA. Subunit Gene

Aikawa, Satoko; Sugiyama, Takao; Sato, Takanobu; Kitahara, Kousuke; Katō, Takako; Kato, Yukio

doi:10.1262/jrd.51.117

Cited by 11 publications

(14 citation statements)

References 17 publications

(15 reference statements)

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“…In the distal region, no information on typical regulatory elements has yet been provided, while we recently demonstrated the importance of the distal region of porcine aGSU promoter for the high level and cell-type-specific expression of aGSU gene (Aikawa et al 2005a). Indeed, the study of cis-acting activity revealed two cis-acting elements of Prop-1 located at K1038/K1026 and K942/K928 bp.…”

Section: Discussionmentioning

confidence: 93%

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

Sato¹,

Kitahara²,

Sugiyama³

et al. 2006

Journal of Molecular Endocrinology

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Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone b subunit (FSHb) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone a subunit (aGSU), and luteinizing hormone b subunit (LHb). A series of deletion mutants of the porcine aGSU (up to K1059 bp) and LHb (up to K1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine aGSU and LHb promoters by Prop-1, which was found to activate the aGSU promoter of K1059/C12 bp up to 11 . 7-fold but not the LHb promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, K1038/K1026, K942/K928, K495/K479, K338/K326, K153/K146, and K131/K124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of aGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for aGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of aGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHb gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of aGSU and FSHb gene expressions.

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Section: Discussionmentioning

confidence: 93%

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

Sato¹,

Kitahara²,

Sugiyama³

et al. 2006

Journal of Molecular Endocrinology

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“…While the regulation of PGBE with Lhx3 has been well investigated, we recently demonstrated that the distal upstream region plays a role in the remarkable expression of porcine Cga [4]. The region contains many TAAT/ATTA core motifs, a binding element for multiple members of the homeodomain family [29][30][31][32].…”

Section: Discussionmentioning

confidence: 99%

“…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 promoter shows only moderate sequence homology with that of other mammals.…”

mentioning

confidence: 98%

“…Recently, we reported a prominent transcription activity of the porcine Cga promoter over the PGBE [4]. The distal region of the porcine Cga promoter shows only moderate sequence homology with that of other mammals.…”

mentioning

confidence: 98%

“…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].…”

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

Sugiyama

Ishikawa

Katō

et al. 2009

Journal of Reproduction and Development

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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...

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Reproducible transfection in the presence of carrier DNA using FuGENE6 and Lipofectamine2000

2007

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We have examined transfection conditions of chinese hamster ovary cells using FuGENE6 and immortalized gonadotrope cell line LbetaT2 cells using Lipofectamin 2000 and to obtain reproducible and reliable transfection. The experiments were performed with fluorescent protein expression vectors, pEYFP-C1 and pECFP-C1, or secreted-type alkaline phosphatase vector, pSEAP2, as reporter genes. The number of cells that received reporter plasmid increased in proportion to the amount of DNA and reached a plateau at a large amount. Co-transfection using two fluorescence vectors with a small amount of DNA demonstrated that every transfected cell received both vectors without discrimination. The results further indicate that there is a hierarchy of DNA receptiveness among competent cells. Simultaneously, we observed that a reliable transfection took place at the high dose of DNA. That is, the addition of carrier DNA makes possible a reliable delivery of a small amount of DNA of interest to the competent cells. Similar results were also obtained by pSEAP2 vector. Co-transfection of pEYFP-C1 and pECFP-C1 with various ratios at adequate amounts demonstrated that the fluorescence intensities by each vector are proportional to each amount of vector used with comparable efficiency. In addition, we observed that the variation of the assay using fluorescent vectors or secreted alkaline phosphatase vectors were small enough within the +/- 25% (SD, n = 4), showing that the internal marker often used to normalize the data is not essential, since the vectors used allow us to exclude cell-harvest and cell-lysis. Thus, the present study demonstrates that the addition of carrier DNA during transfection provides reproducible and reliable results.

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Transcriptional Activity of the 5' Upstream Region of the Porcine Glycoprotein Hormone .ALPHA. Subunit Gene

Cited by 11 publications

References 17 publications

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

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

Reproducible transfection in the presence of carrier DNA using FuGENE6 and Lipofectamine2000

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