The gonadotropin-regulated long-chain acyl CoA synthetase gene: A novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1-14. All exon-intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at − 266 / − 216 bp 5′ to the ATG codon. The minimal promoter domain resides within − 254 / − 217 bp 5′ to ATG codon, and upstream sequences to − 404 bp (− 1035 / − 405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of − 217 / − 1 bp, containing a 23 nt GC rich sequence (− 112 / − 90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.