Regulatory mechanisms of gene expression of pyruvate kinase isozymes

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Cis-acting elements responsible for expression of pyruvate kinase L and M genes were studied by transient DNA transfer experiments with chloramphenicol acetyltransferase (CAT) fusion genes. We found two positive regulatory regions required for expression of the L-type isozyme in adult rat hepatocytes. These regions, designated as PKL-I and PKL-II, were located between nucleotides -94 and -76, and -170 and -126, respectively. PKL-II enhanced expression from heterologous as well as homologous promoters in a manner that was independent of its orientation and position relative to the cap site. PKL-I also enhanced the promoter activity. PKL-I and PKL-II showed synergistic enhancer activity. PKL-II contains two notable sequences; one is the sequences that is reported to be the LF-AL binding site and the other is palindrome sequences. PKL-I consists of palindrome sequences which contain a sequence homologous to the LF-Bl binding site. The activities of PKL-I and PKL-II were not detected in K562 erythroleukemia cells, suggesting that PKL-I and PKL-II are very important enhancer elements for hepatocytespecific expression of the L-type isozyme.It was also suggested that the 5'-flanking region of the Retype cap site of the L gene contained cis-acting element responsible for expression of the R-type. The 5'-flanking region of the M gene was suggested to contain two cis-acting elements.Studies on transgenic mice containing CAT fusion genes indicated that the 5'-flanking region of the L-type cap site up to -3000 contained element responsive to insulin.