Isolation and characterization of the human stearoyl-CoA desaturase gene promoter: requirement of a conserved CCAAT cis-element

Stearoyl-CoA desaturase is the rate-limiting enzyme in the production of mo no-unsaturated fatty acids. We have recently cloned and characterized the h uman Scd cDNA and SCD (the stearoyl-CoA desaturase structural gene) on chro mosome 10, as well as the non-transcribed pseudogene on chromosome 17, In o rder to further define SCD regulation and function, we have isolated and ch aracterized the promoter of the structural gene. Screening of chromosome-10 -specific libraries resulted in the isolation of 4.1 kb of SCD sequence ups tream of the translation start site. Binding sites for transcription factor s critical for mouse Scd1 and Scd2 promoter activity, such as sterol-regula ted-element-binding protein and nuclear factor Y, were present in the human SCD promoter (Scd is the mouse stearoyl-CoA desaturase gene). Deletion ana lysis in HaCaT keratinocytes identified a critical region for promoter acti vity between nts 496-609 upstream of the translation start site. Site-direc ted mutagenesis of binding sites in this region identified the CCAAT box as the critical cis-element for SCD promoter activity. An electrophoretic mob ility-shift assay confirmed that this element binds nuclear proteins from H aCaT keratinocytes. The polyunsaturated-fatty-acid (PUFA) response element, previously identified in the promoters of mouse Scd1 and Scd2, was found t o be conserved in the human SCD promoter, and contained the critical CCAAT cis-element, A minimal promoter construct including this region was respons ive to fatty acids, with oleate and linoleate decreasing transcription and stearate increasing it. These studies indicate that CCAAT-box-binding prote ins activate SCD transcription in cultured keratinocytes and that Fatty aci ds modulate transcription, most likely through the conserved PUFA response element.