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KMID : 0624219990070000015
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Focus on Genetic Science
1999 Volume.7 No. 0 p.15 ~ p.34
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Cloning of human ACC-β promoter and its regulation by MRFs
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Lee, Jae-Jung
Moon, Young-Ah/Kim, Kyung-Sup
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Abstract
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The 280kDa β-isoform of acetyl-CoA carboxylase (ACC-β) is predominantly expressea in heart and skeltal muscle, whereas the 265kDa α-isoform (ACC-α) is the major ACCin lipogenic tissues. With the expection that the muscle-specific expression of ACC-β might bo explained by the responsiveness of its promoter to muscle-related transcription factors, we have cloned 15.5kb human ACC-β promoter. ACC-β promoter-reporter construct showed strong induction by MyoD in fibroblast cells. Serial deletion of the promoter revealed that MyoD acts on the E-boxes at -503 to -403, and on the proximal region including the transcription initiation site and 5’-UTR. DNase I footprinting with the heterodimer of recombinant MyoD and E47 protected two of the three E-boxes. Destruction of those E-boxes by site-direct mutagenesis resulted in 50% decrease of MyoD responsiveness, and mutation on a GGCGCC motif of the +14 to +19 region and another E-Box resides in the proximal region drastically decreased the Myod responsiveness. MyoD binding to the GGCGCC encompassing region was demonstrated by EMSA, and this finding is unique since DNA binding of MyoD is known as E-box-specific through dimerizstion with E-proteins. We screened human skeletal muscle cDNA library and isolated several transcription factors including Myf4 and Myf6 by yeast one hybrid system. EMSA showed that recombinant Myf4 and Myf6 could bind to this novel cis-element, moreover, transient expression of Myf6 could confer significant activation on the ACC-β promoter reporter construct or an artificial promoter harboring this cis-element. These findings suggest that MRFs, such as MyoD, Myf4 and Myf6, may contribute to the muscle-specific expression of ACC-β via E-boxes and a novel cis-element.
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KEYWORD
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