Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.
Abstract: Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates promoter activity. Here, studies of the proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the promoter but also requires the upstream segment of the promoter between -119 bp and -335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.
Publication Date: 2018-04-26 PubMed ID: 29755409PubMed Central: PMC5932191DOI: 10.3389/fendo.2018.00195Google Scholar: Lookup
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Summary
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The research article focuses on how the Glial Cells Missing 1 (GCM1) protein regulates Equine chorionic gonadotrophin beta subunit (eCGβ), a hormone crucial for early horse pregnancy.
Objective of the Study
- The main purpose of the study was to ascertain if GCM1 can govern the activity of the eCGβ promoter, a gene known to give hormones the specificity to bind to the right receptor. The factors that transcriptionally manage this subunit in the trophoblast (the layer of cells that form the outer layer of a blastocyst, providing nutrients to the embryo) aren’t known.
Methodology
- The researchers first studied the eCGβ proximal promoter and identified four binding sites for GCM1, confirming its presence in the equine chorionic girdle and its surrounding tissues through a western blot analysis.
- With the help of luciferase assays, the team showed that there is endogenous activity in the eCGβ promoter within BeWo choriocarcinoma cells, with max activity taking place in a proximal 335 bp promoter fragment.
- Transactivation studies were conducted in COS7 cells using an equine GCM1 expression vector, which displayed that GCM1 could transactivate the proximal 335 bp eCGβ promoter.
- Chromatin immunoprecipitation, using primary ChG trophoblast cells, was performed to reveal that GCM1 had a preference for the most proximal GCM1-binding site over site 2.
Results and Conclusion
- The results demonstrated that when site 1 was mutated, there was a loss of endogenous promoter activity in BeWo cells, and GCM1 failed to transactivate the promoter in COS-7 cells. This showed that GCM1 specifically binds to site 1 in the eCGβ promoter and also requires the upstream segment of the promoter between -119 bp and -335 bp of the translation start codon for activity.
- Four GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, displayed different expressions in the ChG between days 27 and 34, making them good candidate partners for GCM1.
- Overall, the data suggested that GCM1 drives the eCGβ promoter activity, primarily through binding to a predicted GCM1-binding site, but another factor(s) needs to bind the proximal promoter for this function to be executed.
- The researchers thus hypothesize that ETV7 and HOXA13 work with GCM1 to initiate eCGβ transcription between days 30 and 31, whereas ETV1 partners with GCM1 to maintain this transcription.
Cite This Article
APA
Read JE, Cabrera-Sharp V, Kitscha P, Cartwright JE, King PJ, Fowkes RC, de Mestre AM.
(2018).
Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.
Front Endocrinol (Lausanne), 9, 195.
https://doi.org/10.3389/fendo.2018.00195 Publication
Researcher Affiliations
- Department Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom.
- Department Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom.
- Department Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom.
- St. Georges Medical School, Molecular and Clinical Sciences Research Institute, University of London, London, United Kingdom.
- Centre for Endocrinology, William Harvey Research Institute, Queen Mary University of London, London, United Kingdom.
- Department Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom.
- Department Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom.
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Citations
This article has been cited 3 times.- Read JE, Cabrera-Sharp V, Offord V, Mirczuk SM, Allen SP, Fowkes RC, de Mestre AM. Dynamic changes in gene expression and signalling during trophoblast development in the horse. Reproduction 2018 Oct 1;156(4):313–330.
- Read JE, Cabrera-Sharp V, Offord V, Mirczuk SM, Allen SP, Fowkes RC, de Mestre AM. Dynamic changes in gene expression and signalling during trophoblast development in the horse. Reproduction 2018 Oct 1;156(4):313-330.
- Wang X, Liu R, Chen Z, Zhang R, Mei Y, Miao X, Bai X, Dong Y. Combining Transcriptomics and Proteomics to Screen Candidate Genes Related to Bovine Birth Weight. Animals (Basel) 2024 Sep 23;14(18).
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