Author

Baowei Zhang

Date of Award

7-1993

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Dr. R.A. Rachubinski

Abstract

Structurally diverse peroxisome proliferators induce in rodents a remarkable increase in the activities of peroxisomal fatty acid β-oxidation enzymes. The transcriptional induction of these peroxisomal enzymes in liver has been mechanistically associated with the subsequent pleiotropic responses such as peroxisome proliferation, hypolipidemia, hepatomegaly, and hepatocellular carcinomas. This project was designed to investigate the mechanisms of transcriptional regulation of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) by hypolipidemic peroxisome proliferators.

In search of a peroxisome proliferator responsive element in the promoter region of the HD gene, the 5. 8 kbp upstream region of the HD gene and its deletion mutated fragments were subcloned into a reporter plasmid that contained a luciferase gene. The recombinant plasmids were introduced into H4IIEC3 cells in the presence of ciprofibrate, a hypolipidemic drug. Luciferase activity was measured to assay the inducibility of the promoter mutants. The sequence between nucleotides -3040 and -2845 behaved as a minimal peroxisome proliferator responsive element (PPRE). DNase I footprint analysis of this PPRE fragment with H4IIEC3 nuclear proteins revealed two protected regions. Region I contains consensus hormone responsive motifs: two inverted TGACCT-like sequences followed by a third. Region II contains two protein binding sites: a site containing several CACCC boxes and the other consisting of three directly repeated TGACCT core motifs. In transient transfection assays, the oligonucleotide containing these TGACCT direct repeats, nucleotides -2956 to -2919, conferred a ciprofibrate response, which was independent of its position and orientation relative to the transcription start site. In mobility shift assays, this sequence was recognized by liver specific nuclear proteins.

The three TGACCT-like motifs were further characterized by mutational analysis. Mutations that reduced the intensity of the liver factor binding in mobility shift assays resulted in a decrease in the responsiveness to ciprofibrate in transfection assays. Mutation of a single G residue in the middle core motif completely abolished both the factor binding in vitro and the responsiveness to ciprofibrate in vivo. Modulation of this core HD PPRE involved several known transcription factors. PPAR, RXRα, COUP-TF1, and, weakly, HNF4, were detected to be present in the complex of liver factors with HD PPRE by mobility shift assays with the addition of the respective antibodies. This result was confirmed by similar experiments with the in vitro translated products of mRNAs of these proteins. PPAR and RXRα required the second and third repeats of the HD PPRE whereas COUP-TF1 and, probably also HNF4, recognized the first and second repeats. PPAR and RXRa cooperatively transactivated a reporter gene via this HD PPRE in response to ciprofibrate in several cell lines. COUP-TF1 antagonized the up regulation by PPAR and RXRα.

In conclusion, the major cis element responsible for the ciprofibrate-induction of the ED gene consists of three directly-repeated TGACCT-like motifs. Its function is independent of its position and orientation relative to the transcription start site. Binding of liver specific factors to the element is a prerequisite for its ciprofibrate responsiveness. The overall integrity of the three motifs and the correct spacing between the motifs are critical determinants for the nuclear factor recognition and the transcriptional activation. The transcriptional regulation of the ED gene is achieved by a cooperation of several liver specific transcription factors.

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