Neutral cholesterol ester hydrolase (CEH) is a key enzyme in regulating hepatic free cholesterol. Using the CEH specific cDNA sequence in the 5'-untranslated region as a primer, 1.3 kb of sequence upstream of the ATG initiation codon was amplified and cloned. Primer extension analysis with total RNA from rat primary hepatocytes identified a transcription initiation site, 60 bases upstream from the initiation codon. No typical TATA-box sequences were found upstream from the transcription start site. However, a consensus GC-box, which can bind the positive transcription factor SP1, was found 35 bases upstream from the transcription start site. In addition the promoter also contained several hormone responsive half elements, sterol response elements, ubiquitous transcription factor binding sites and liver specific elements.
To determine the promoter activity of the rat CEH gene, the 1.3 kb of 5’-flanking region was fused to a luciferase reporter gene. Smaller 5’-deletion constructs were obtained by generation of unidirectional nested deletion breakpoints in the full-length construct with Exonuclease III. Basal promoter activity, as well as transcriptional regulation by hormones, signal transduction pathways and agents perturbing cholesterol metabolism were studied in human hepatoblastoma HepG2 cells and cultured primary rat hepatocytes by transient transfection assays of the promoter activity of the deletion constructs. Functional glucocorticoid response elements, phorbol ester responsive sequences and sterol responsive sequences were mapped with both the culture systems. Results indicate that the first 599 base pairs upstream of the initiation codon and the region between nucleotides -1317 and -1190 regulate the effects of various physiological stimuli. The effects of various stimuli used in this study were similar in the two cell lines. The rat CEH gene appears to be finely regulated by distinct signals converging to consensus promoter regulatory sequences.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-6302 |
| Date | 01 January 1997 |
| Creators | Natarajan, Ramesh |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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