TRANSCRIPTIONAL REGULATORY REGIONS FOR EXPRESSION OF THE RAT PYRUVATE-KINASE M-GENE

To study the regulatory mechanism of pyruvate kinase M gene transcript ion, we analyzed its chromatin structure and cis-acting DNA regions. T wo DNase-I-hypersensitive sites were detected in dRLh-84 hepatoma cell s, but not in hepatocytes, which coincides with expression of the M ge ne in the two types of cells. These sites, designated HS2 and HS1, wer e located around the major transcription start site and about 2.9 kb d ownstream from this site, respectively. A transient chloramphenicol ac etyltransferase 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 cluste r 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 th e inclusion of all regions were synergistic. These regions were not ac tive 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 chroma tin structure and binding of proteins to three cis-acting elements.