Transcription regulation of the acetyl-CoA carboxylase gene by glucose

Samira Daniel, Purdue University

Abstract

Acetyl-CoA Carboxylase (ACC), catalyses the rate-limiting reaction in the synthesis of long-chain fatty acids. In the lipogenic tissues of eukaryotes, glucose serves as the primary precursor of these long-chain fatty acids. In this study, we have attempted to study the transcriptional regulation of ACC gene expression by glucose. The rat ACC gene is under the control of two promoters (PI and PII), which function in a tissue-specific manner. We have shown that Promoter II of the ACC gene is activated by high concentrations of glucose. Using stable clones of 30A5 preadipocytes, containing a series of deletion constructs of PII-CAT and measuring CAT gene expression in response to glucose, we established that the region between $-$340 and $-$249 of PII was essential for this induction. When the same sequence was attached to the Thymidine kinase (TK) promoter, the expression of TK also became glucose responsive. By electrophoretic mobility shift assays, competition assays, supershift assays and DNase I footprinting studies, we have shown that only the transcription factor Sp1 (but not MLTF or LFA1) bound to two GC-rich sequences located between $-$340 and $-$249 of Promoter II. Studies of site-directed mutagenesis between $-$340 and $-$181 of the promoter established that the two GC-rich sequences where Sp1 binds, were required for responsiveness to glucose. Occupancy of both sites by Sp1 is essential for glucose induction of Promoter II expression. Mobility Shift assays also indicated the presence of an additional band which could be super-shifted by an antibody against Sp3. Sp3 is a member of the Sp1 family of transcription factors and can recognize the same GC-rich sequence as Sp1. Transfection studies using Drosophila SL2 cells, which are devoid of endogenous Sp1 indicated that cotransfection of Sp1 was required for the activation of PII-CAT gene expression. We have also established that glucose does not change the amount of Sp1 in the cell but induces a nuclear factor which enhances Sp1 binding to the promoter. This in turn leads to increased transcription from Promoter II. It was determined that a 38-42 kDa protein in the nuclear extract was responsible for this enhanced Sp1 binding activity. This factor was found to cross-react with the antibody against protein phosphatase1A. Hence, it appears that the glucose-mediated stimulation of PII by Sp1 is through glucose induction of PP1A or a PP1A-like activity. This was confirmed when the addition of phosphatase 1A could enhance the binding of purified Sp1 to the GC-rich sequences in PII in a concentration-dependent manner. The addition of okadaic acid, a phosphatase inhibitor, to the reaction mixture effectively reduced the Sp1-DNA complex formation. When functional studies were carried out to study the effect of okadaic acid on glucose-treated 30A5 stable cells, no glucose induction of PII was observed. This data put together leads us to believe that the glucose activation of PII is through the glucose-induced dephosphorylation of Sp1 by PP1A.

Degree

Ph.D.

Advisors

Kim, Purdue University.

Subject Area

Molecular biology

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