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Biochemical, Molecular, and Genetic Mechanisms

Identification of the Regulatory Region of the L-Type Pyruvate Kinase Gene in Mouse Liver by Hydrodynamics-Based Gene Transfection¹

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

² To whom correspondence should be addressed. E-mail: atsushi{at}agr.nagoya-u.ac.jp

ABSTRACT

Expression of L-type pyruvate kinase (L-PK) is upregulated in the liver by dietary carbohydrate. Previously, 3 carbohydrate/insulin response elements were identified in the 5'-flanking region of the L-PK gene up to bp –170. Studies of the 5'-flanking region beyond bp –183 in transgenic mice suggested that other regulatory elements may be present upstream of bp –183, but the positions of these elements were uncertain. In the present study, the existence of regulatory regions of the L-PK gene responding to stimulation by feeding was examined using in vivo hydrodynamics-based gene transfection (HT) in mouse liver. The firefly-luciferase (FL) gene, fused with various lengths of the 5'-flanking region of the L-PK gene, was introduced into mouse liver by HT. The mice had free access to a high-carbohydrate diet. In liver homogenate, luciferase activity of pL-PK(–1467)-FL (which included the 5'-flanking region from bp –1467 to +17), was markedly stimulated by feeding. 5'-Deletion up to bp –1065 caused only minor changes in luciferase activity, but further deletion up to bp –690 and bp –203 caused significant, gradual decreases in activity. Further analyses utilizing 5'-deletion mutants indicated the existence of positive regulatory regions that respond to stimulation by feeding between bp –1065 and –945, and between –300 and –203 on the L-PK gene. These results suggest that unidentified cis-acting DNA elements exist in the upstream region of the L-PK gene, and that HT is a useful approach for detecting regulatory regions of genes expressed in the liver.

KEY WORDS: • L-type pyruvate kinase • mouse • hydrodynamics-based gene transfection • luciferase