Functional characterization of the mouse melanocortin 3 receptor gene promoter

Keisuke Okutsu, Fumiya Ojima, Naoto Shinohara, Shusuke Taniuchi, Yasusyo Mizote, Kenji Aoki, Toshiyuki Kudo, Maho Ogoshi, Sakae Takeuchi, Sumio Takahashi

Research output: Contribution to journalArticle

Abstract

Melanocortin receptor 3 (MC3R) is expressed in the hypothalamus and pituitary in humans and rodents, and is involved in the control of feeding, energy metabolism, and pituitary function. In the mouse pituitary, MC3R is detected in mammotrophs. This study aimed to clarify the regulatory mechanism for Mc3r expression in the mouse pituitary. The promoter activities of reporter constructs for the MC3R gene 5'-flanking region up to - 4000. bp (transcription initiation site designated as +. 1) were analyzed. The promoter activity significantly increased in the - 86/+. 109 construct, but decreased in the - 38/+. 109 construct, indicating that the minimal promoter required for basal expression of Mc3r is located in the - 86/+. 109 region. Putative binding sites for transcription factors AP-1 and ATF4 were found in the 5'-flanking region of Mc3r. Site-directed mutation or deletion of these sites affected the promoter activities. In gel-shift assays with a nuclear extract of mouse anterior pituitary cells, band-shifts were detected for both sites after the addition of the nuclear extract, and were decreased in the presence of excess unlabeled probe competitors. These results indicated that both sites were involved in the regulation of Mc3r expression in anterior pituitary cells. Estradiol-17β treatment increased the Mc3r promoter activity, indicating that the gene is regulated by estradiol-17β. In conclusion, we have demonstrated the minimum promoter region required for Mc3r expression, and identified two binding sites for AP-1 and ATF4 and in the 5' upstream-flanking region of Mc3r that are essential for Mc3r expression.

Original languageEnglish
Pages (from-to)62-69
Number of pages8
JournalGene
Volume562
Issue number1
DOIs
Publication statusPublished - May 10 2015

Fingerprint

5' Flanking Region
Transcription Factor AP-1
Estradiol
Receptor, Melanocortin, Type 3
Binding Sites
Genes
Sequence Deletion
Transcription Initiation Site
Genetic Promoter Regions
Energy Metabolism
Hypothalamus
Rodentia
Gels
Mouse Mc3r protein

Keywords

  • Gene expression
  • Pituitary
  • Promoter
  • Proopiomelanocortin
  • α-MSH

ASJC Scopus subject areas

  • Genetics
  • Medicine(all)

Cite this

Okutsu, K., Ojima, F., Shinohara, N., Taniuchi, S., Mizote, Y., Aoki, K., ... Takahashi, S. (2015). Functional characterization of the mouse melanocortin 3 receptor gene promoter. Gene, 562(1), 62-69. https://doi.org/10.1016/j.gene.2015.02.043

Functional characterization of the mouse melanocortin 3 receptor gene promoter. / Okutsu, Keisuke; Ojima, Fumiya; Shinohara, Naoto; Taniuchi, Shusuke; Mizote, Yasusyo; Aoki, Kenji; Kudo, Toshiyuki; Ogoshi, Maho; Takeuchi, Sakae; Takahashi, Sumio.

In: Gene, Vol. 562, No. 1, 10.05.2015, p. 62-69.

Research output: Contribution to journalArticle

Okutsu, K, Ojima, F, Shinohara, N, Taniuchi, S, Mizote, Y, Aoki, K, Kudo, T, Ogoshi, M, Takeuchi, S & Takahashi, S 2015, 'Functional characterization of the mouse melanocortin 3 receptor gene promoter', Gene, vol. 562, no. 1, pp. 62-69. https://doi.org/10.1016/j.gene.2015.02.043
Okutsu K, Ojima F, Shinohara N, Taniuchi S, Mizote Y, Aoki K et al. Functional characterization of the mouse melanocortin 3 receptor gene promoter. Gene. 2015 May 10;562(1):62-69. https://doi.org/10.1016/j.gene.2015.02.043
Okutsu, Keisuke ; Ojima, Fumiya ; Shinohara, Naoto ; Taniuchi, Shusuke ; Mizote, Yasusyo ; Aoki, Kenji ; Kudo, Toshiyuki ; Ogoshi, Maho ; Takeuchi, Sakae ; Takahashi, Sumio. / Functional characterization of the mouse melanocortin 3 receptor gene promoter. In: Gene. 2015 ; Vol. 562, No. 1. pp. 62-69.
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AU - Mizote, Yasusyo

AU - Aoki, Kenji

AU - Kudo, Toshiyuki

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N2 - Melanocortin receptor 3 (MC3R) is expressed in the hypothalamus and pituitary in humans and rodents, and is involved in the control of feeding, energy metabolism, and pituitary function. In the mouse pituitary, MC3R is detected in mammotrophs. This study aimed to clarify the regulatory mechanism for Mc3r expression in the mouse pituitary. The promoter activities of reporter constructs for the MC3R gene 5'-flanking region up to - 4000. bp (transcription initiation site designated as +. 1) were analyzed. The promoter activity significantly increased in the - 86/+. 109 construct, but decreased in the - 38/+. 109 construct, indicating that the minimal promoter required for basal expression of Mc3r is located in the - 86/+. 109 region. Putative binding sites for transcription factors AP-1 and ATF4 were found in the 5'-flanking region of Mc3r. Site-directed mutation or deletion of these sites affected the promoter activities. In gel-shift assays with a nuclear extract of mouse anterior pituitary cells, band-shifts were detected for both sites after the addition of the nuclear extract, and were decreased in the presence of excess unlabeled probe competitors. These results indicated that both sites were involved in the regulation of Mc3r expression in anterior pituitary cells. Estradiol-17β treatment increased the Mc3r promoter activity, indicating that the gene is regulated by estradiol-17β. In conclusion, we have demonstrated the minimum promoter region required for Mc3r expression, and identified two binding sites for AP-1 and ATF4 and in the 5' upstream-flanking region of Mc3r that are essential for Mc3r expression.

AB - Melanocortin receptor 3 (MC3R) is expressed in the hypothalamus and pituitary in humans and rodents, and is involved in the control of feeding, energy metabolism, and pituitary function. In the mouse pituitary, MC3R is detected in mammotrophs. This study aimed to clarify the regulatory mechanism for Mc3r expression in the mouse pituitary. The promoter activities of reporter constructs for the MC3R gene 5'-flanking region up to - 4000. bp (transcription initiation site designated as +. 1) were analyzed. The promoter activity significantly increased in the - 86/+. 109 construct, but decreased in the - 38/+. 109 construct, indicating that the minimal promoter required for basal expression of Mc3r is located in the - 86/+. 109 region. Putative binding sites for transcription factors AP-1 and ATF4 were found in the 5'-flanking region of Mc3r. Site-directed mutation or deletion of these sites affected the promoter activities. In gel-shift assays with a nuclear extract of mouse anterior pituitary cells, band-shifts were detected for both sites after the addition of the nuclear extract, and were decreased in the presence of excess unlabeled probe competitors. These results indicated that both sites were involved in the regulation of Mc3r expression in anterior pituitary cells. Estradiol-17β treatment increased the Mc3r promoter activity, indicating that the gene is regulated by estradiol-17β. In conclusion, we have demonstrated the minimum promoter region required for Mc3r expression, and identified two binding sites for AP-1 and ATF4 and in the 5' upstream-flanking region of Mc3r that are essential for Mc3r expression.

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