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

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.