title: Involvement of transcription factor GATA-4 in regulation of CYP19 gene during folliculogenesis and luteinization in buffalo ovary.
abstract: CYP19 gene encode aromatase, the key enzyme of estrogen biosynthesis, is regulated in species- and tissue-specific manner by alternate use of different promoters. Previously, we have reported the cloning and characterization of tissue-specific promoter and transcripts in buffalo ovary and placenta. In human and rat ovary, FSH induces the phosphorylation of transcription factor CREB (cAMP response element binding protein) through PKA pathway which binds to cAMP response element like sequence (CLS) in CYP19 gene ovarian promoter. However, in buffalo as well as in bovine, in silico analysis of ovary specific promoter sequence identified a single base pair deletion in CLS site and is designated as CLS-like sequence. To understand if CLS with a point mutation is still a functional cis-element and is involved in FSH stimulated transactivation of CYP19 gene in buffalo ovary, the present study was thus aimed to functionally characterize the role of buffalo CLS in CYP19 gene transactivation. We also studied the involvement of GATA-4, having consensus binding sites in CYP19 gene ovarian promoter in the vicinity of CLS during different stages of the buffalo estrus cycle. Reporter construct analyses and EMSA results showed that CLS is playing no significant role in CYP19 gene regulation in buffalo ovary. Real time absolute quantification of GATA-4 showed the differential expression of GATA-4 mRNA during folliculogenesis and luteinization with significantly higher transcript abundance in large follicle in comparison to other tissues. Western blot analysis of granulosa cells nuclear protein isolated from different stage of follicular development (small and large follicles) and differentiation (corpus luteum) showed that abundance of phosphorylated GATA-4 (Ser261) was significantly higher in granulosa cell nuclear protein of large follicles as compared to small follicles and corpora lutea. Interestingly, binding studies using ChIP showed significantly enhanced binding to the CYP19 gene promoter in large follicle which was seen to be declined in the luteal tissue. Similar results were obtained in the in vitro experiments as well. Finally, RNAi experiments were performed to validate the involvement of GATA-4 in CYP19 gene regulation. Results of RNAi showed that knockdown of GATA-4 mRNA significantly declined CYP19 gene mRNA as well as 17β-estradiol contents. In conclusion, result of the present study indicated that that in the absence of consensus CRE (cAMP response element); GATA-4 could be a downstream effector of cAMP/PKA pathway in regulation of CYP19 gene during folliculogenesis and luteinization.
authors.affiliation: Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute (NDRI), Karnal, Haryana, India.
authors.forename: Rachna
authors.initials: R
authors.lastname: Monga
authors.forename: Sandeep
authors.initials: S
authors.lastname: Ghai
authors.forename: Tirtha Kumar
authors.initials: TK
authors.lastname: Datta
authors.forename: Dheer
authors.initials: D
authors.lastname: Singh
mesh_terms: D000818:Animals; D001141:Aromatase / Q000235:genetics* / Q000378:metabolism; D002020:Buffaloes / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D034461:Luteinization / Q000235:genetics*; D006080:Ovarian Follicle / Q000378:metabolism*; D010053:Ovary / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D050991:GATA4 Transcription Factor; D012333:RNA, Messenger; D000242:Cyclic AMP; D001141:Aromatase; D017868:Cyclic AMP-Dependent Protein Kinases