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Arch Biochem Biophys. 2011 Jul;511(1-2):21-30. doi: 10.1016/j.abb.2011.04.012. Epub 2011 Apr 22.

Transcriptional regulation of the human neutral ceramidase gene.

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  • 1Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA.

Abstract

Ceramidases play a critical role in generating sphingosine-1-phosphate by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. In order to elucidate its transcriptional regulation, we identify here a putative promoter region in the 5'-UTR of the human neutral CDase (nCDase) gene. Using human genomic DNA, we cloned a 3000 bp region upstream of the translational start site of the nCDase gene. Luciferase reporter analyses demonstrated that this 3000 bp region had promoter activity, with the strongest induction occurring within the first 200 bp. Computational analysis revealed the 200 bp essential promoter region contained several well-characterized promoter elements, lacked a conical TATA box, but did contain a reverse oriented CCAAT box, a feature common to housekeeping genes. Electrophoretic mobility shift assays demonstrated that the identified candidate transcriptional response elements (TRE) bind their respective transcription factors, including NF-Y, AP-2, Oct-1, and GATA. Mutagenic analyses of the TRE revealed that these sites regulated promoter activity and mutating an individual site decreased promoter reporter activity by up to 50%. Together, our findings suggest that regulation of nCDase expression involves coordinated TATA-less transcriptional activity.

PMID:
21531200
PMCID:
PMC3762322
DOI:
10.1016/j.abb.2011.04.012
[PubMed - indexed for MEDLINE]
Free PMC Article
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