Transcriptional activation of zebrafish cyp11a1 promoter is dependent on the nuclear receptor Ff1b.
The cytochrome P450scc (cholesterol side-chain cleavage enzyme) encoded by CYP11A1 catalyzes the first step in steroidogenesis by converting cholesterol to pregnenolone, and thus, controls the synthesis rate of steroid hormones. In mammals, steroidogenic factor 1 (SF1) has been implicated in the cAMP-mediated transcriptional activation of CYP11A1 promoter. In zebrafish, Ff1b has been established as the homolog of SF1. To assess the dependency of cyp11a1 expression on Ff1b, the putative promoter of zebrafish cyp11a1, spanning 1.7 kb, was isolated and bioinformatic analysis revealed two conserved FF1 response elements (FREs) that potentially bind Ff1b. Transfection studies in cell lines of different lineages confirmed that this promoter fragment contained the necessary regulatory elements required for its basal transcription. Truncation and mutagenesis studies performed in Y1 adrenocortical cells revealed that only the proximal FRE was essential for transcriptional activation. Electrophoretic mobility shift assay, however, indicated that Ff1b bound to both FREs, while their in vivo occupancy was confirmed using a chromatin immunoprecipitation assay. Lastly, the cyp11a1 promoter was able to direct EGFP expression specifically to the interrenal gland and genital ridge when transiently expressed in microinjected zebrafish embryos, and the promoter activity is potentiated by ff1b overexpression as measured from luciferase reporter activity in zebrafish embryos.
Quek
Sue Ing
SI
Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore 117543.
Chan
Woon Khiong
WK
D000818:Animals; D020029:Base Pairing / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D017124:Conserved Sequence; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*; D015027:Zebrafish / Q000033:anatomy & histology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000378:metabolism*
D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; C478921:nr5a1a protein, zebrafish; D049452:Green Fluorescent Proteins; D002786:Cholesterol Side-Chain Cleavage Enzyme