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Proximal GATA-binding sites are essential for human HSD3B1 gene transcription in the placenta.

ABSTRACT: The enzyme 3?-hydroxysteroid dehydrogenase/isomerase (3?-HSD) is involved in the synthesis of active steroid hormones. Two human 3?-HSD isoforms are expressed in a tissue-specific pattern. HSD3B1 (type I) expression is essential to produce progesterone for pregnancy maintenance. To understand the mechanisms of human HSD3B1 activation in the placenta, 2.2?kb of 5'-flanking sequence and 5'-deletions were fused to the luciferase reporter gene and transfected into human JEG-3 cells. The proximal -238/+337 sequence had the highest promoter activity. Two GATA elements were identified at -106/-99 and -52/-45. Mutations of either sites greatly reduced promoter activity in JEG-3 cells, demonstrating the importance of GATA sites. EMSA revealed the specific binding of GATA2 and GATA3 to the GATA sequences at -106/-99 and -52/-45. ChIP assays demonstrated the association of GATA2 but not GATA3 with the GATA-binding regions of the HSD3B1 promoter in JEG-3 cells. GATA2 knockdown significantly reduced HSD3B1 expression in JEG-3 cells; however, GATA3 knockdown increased HSD3B1 expression. Western blot analysis revealed high levels of GATA2 but not GATA3 in human placental tissues. This study identified GATA motifs as essential control elements for HSD3B1 transcription and GATA2 as a novel transcriptional regulator of HSD3B1 expression in the human placenta.

SUBMITTER: Lai TC

PROVIDER: S-EPMC5487343 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Proximal GATA-binding sites are essential for human HSD3B1 gene transcription in the placenta.

Lai Tsai-Chun TC Li Hsiao-Fang HF Li Yu-Shian YS Hung Pei-Yu PY Shyu Ming-Kwang MK Hu Meng-Chun MC

Scientific reports 20170627 1

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is involved in the synthesis of active steroid hormones. Two human 3β-HSD isoforms are expressed in a tissue-specific pattern. HSD3B1 (type I) expression is essential to produce progesterone for pregnancy maintenance. To understand the mechanisms of human HSD3B1 activation in the placenta, 2.2 kb of 5'-flanking sequence and 5'-deletions were fused to the luciferase reporter gene and transfected into human JEG-3 cells. The proximal -23 ...[more]

PMID: 28655875

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Project description:The enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD) is required for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each a product of a distinct gene. Two isoforms, 3betaHSD I and II, are expressed in a tissue-specific manner in humans. 3betaHSD I is the only isoform expressed in the placenta, where it is required for the biosynthesis of progesterone and thus essential for the maintenance of pregnancy. We recently identified two transcription factors, activating protein-2gamma (AP-2gamma) and the homeodomain protein, distaless-3 (Dlx-3), that are expressed in both human and mouse trophoblast cells that were shown to be required for trophoblast-specific expression of the orthologous murine 3betaHSD, 3betaHSD VI. Although we identified specific binding sites for AP-2gamma and Dlx-3 in the distal promoter of the human 3betaHSD I gene, HSD3B1, it was found that these transcription factors were not involved in determining placental-specific expression of human 3betaHSD I. Instead, a 53-bp placental-specific enhancer element located between -2570 and -2518 of the HSD3B1 promoter was identified. Within this 53-bp element, two potential placental transcription factor binding sites were found. EMSAs with a 20-bp oligonucleotide containing these two potential placental-specific binding sites identified one of the binding sites specific for the transcription enhancer factor (TEF)-5, which is highly expressed in human placenta and in placental choriocarcinoma-derived JEG-3 cells and the other overlapping binding site, specific for a GATA-like protein. Site-specific mutations in either the TEF-5 binding site or in the GATA binding site, each resulted in complete loss of enhancer activity. The data indicate that TEF-5 and the GATA-like protein act in a coordinate manner to determine the placental-specific expression of the human 3betaHSD I enzyme and therefore are critical for placental progesterone production required for the maintenance of pregnancy.

Dataset Information

Proximal GATA-binding sites are essential for human HSD3B1 gene transcription in the placenta.

Publications

Proximal GATA-binding sites are essential for human HSD3B1 gene transcription in the placenta.

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