Transcriptional regulatory regions for expression of the rat pyruvate kinase M gene

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Abstract
To study the regulatory mechanism of pyruvate kinase M gene transcription, we analyzed its chromatin structure and cis‐acting DNA regions. Two DNase‐I‐hypersensitive sites were detected in dRLh‐84 hepatoma cells, but not in hepatocytes, which coincides with expression of the M gene in the two types of cells. These sites, designated HS2 and HS1, were located around the major transcription start site and about 2.9 kb downstream from this site, respectively. A transient chloramphenicol acetyltransferase expression assay indicated that the region around HS1 did not show any activity, whereas the upstream region up to ‐457 had promoter activity in hepatoma cells. Most of this activity was lost by a 5′‐deletion from ‐286 to ‐225. Further analysis identified a cluster of three cis‐acting regions from ‐279 to ‐216, which are named boxes A, B and C. These regions did not have any independent effect, but the inclusion of all regions were synergistic. These regions were not active in hepatocytes, suggesting that they have cell‐type specificity. A gel mobility shift assay indicated that unidentified, but distinct, nuclear proteins bound to the three boxes. These results suggest that transcriptional regulation of the M gene involves alteration of chromatin structure and binding of proteins to three cis‐acting elements.