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.
Wang
Z
Z
Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Japan.
Takenaka
M
M
Imai
E
E
Yamada
K
K
Tanaka
T
T
Noguchi
T
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D011770:Pyruvate Kinase / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007527:Isoenzymes; D011770:Pyruvate Kinase