Transcriptional regulation of acetyl-CoA carboxylase gene expression
Abstract
The gene for acetyl CoA carboxylase (ACC), the rate-limiting enzyme in the biosynthesis of long-chain fatty acids, contains two distinct promoters, PI and PII. Multiple forms of ACC mRNA with 5$\sp\prime$-heterogeneity are generated as a result of differential splicing of two primary transcripts formed under the control of these two promoters. PI is responsible for the generation of the class I mRNA of ACC which is induced in tissue specific manner under lipogenic conditions. PII generates the class II mRNA constitutively. Transcription from PI is normally repressed by the presence of a negative cis-element (a 28 CA repeat sequence located 220 bp upstream from the transcription initiation site) and the repression is overcome when C/EBP-$\alpha$ binds to the GCAAT sequence in the CCAAT box of PI promoter. Repression by the 28 CA repeat sequence requires the GCAAT sequence in the CCAAT box. Insertion of the 28 CA repeat sequence into the thmidine kinase promoter results in repression that can also be relieved by the C/EBP-$\alpha$. The same sequence, however, exerts no effect on the ACC PII which does not contain the CCAAT box. During the differentiation of 30A5 preadipocytes into adipocytes, the expression of the class I ACC mRNA and the C/EBP-$\alpha$ mRNA is coordinately increased. Therefore, it is believed that the presence of the CA repeat sequence in the promoter is involved in the inactivation of PI and that C/EBP-$\alpha$ is one of the factors that is responsible for the activation of PI under lipogenic conditions. C/EBP-$\beta$, a second member of the C/EBP family, involves in the PI activation by cAMP. Transcription of the reporter gene (the CAT gene) under the control of PI promoter increased about 60 fold by treatment of 8-CPT cAMP and IBMX for 48 hrs in confluent 30A5 cells. The level of the C/EBP-$\beta$ mRNA reached at maximum within 3 hr by treatment of cAMP in confluent 30A5 cells. On the other hand, the mRNA increase of C/EBP-$\alpha$ required longer period of incubation (24 to 48 hrs) and it reached at maximum level after being differentiated. The induction of the reporter gene expression by cAMP was inhibited by overexpression of the mutant regulatory subunit of cAMP dependent protein kinase (PKA) and by treatment of H8, the protein kinase inhibitor. The expression of the C/EBP-$\beta$ mRNA was also inhibited by treatment of H8. C/EBP-$\beta$ shares the binding site on PI with C/EBP-$\alpha.$ The bindings of C/EBP-$\alpha$ and C/EBP-$\beta$ to the DNA fragment, containing the CCAAT box element of PI, increased in the nuclear extract, prepared from 30A5 cells treated with 8-CPT cAMP and IBMX for 48 hr. However, the increase of the C/EBP-$\beta$ binding was greater than C/EBP-$\alpha$. The induction of the reporter gene expression and the increase of C/EBP-$\beta$ mRNA by cAMP were not affected by treatment of TNF, unlike C/EBP-$\alpha$ mRNA, indicating that C/EBP -$\beta$ may contribute to the cAMP responsiveness of PI promoter. To get insight into the control mechanism of the ACC gene transcription by PI, the negative cis-element in PI of ACC that leads to the repression of PI was characterized and the trans-activators which involve in PI activation during differentiation of 30A5 preadipocyte.
Degree
Ph.D.
Advisors
Kim, Purdue University.
Subject Area
Biochemistry|Molecular biology
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