Multiple positive and negative elements regulate human brain creatine kinase gene expression.
We characterized the developmental expression of the brain creatine kinase (BCK) gene in the C2C12 myogenic cell line with the use of isoenzyme, Western blot, and Northern blot analyses. The results show that both BCK subunit protein and mRNA are upregulated early in myogenesis, and then downregulated in fully differentiated myotubes. To characterize the transcriptional regulatory mechanisms, a chimeric construct containing 1.2 kilobase pairs of 5'-flanking DNA from the human BCK gene placed upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVOCAT was transiently transfected into C2C12 cells. In myoblasts and differentiating myotubes, the time course of expression of the constructs paralleled that of endogenous BCK mRNA. Additional constructs prepared by deleting 5'-flanking DNA were also transfected into C2C12 cells. All constructs were preferentially expressed in myoblasts relative to myotubes with absolute levels of expression increasing with deletion of 5'-flanking DNA. In nonmyogenic cells expression of the plasmids also increased with deletion of 5'-flanking DNA. An element from -1150 to -388 was isolated and found to be capable of suppressing expression of the BCK promoter and of heterologous promoters independent of orientation and position and hence to function as a silencer. Thus, BCK expression is mediated by sequences contained in the 5'-flanking DNA, including negative elements active in both C2C12 cells and nonmyogenic cells and elements that mediate the developmental expression of the BCK gene in C2C12 myogenic cells.
Ritchie
M E
ME
Cardiovascular Division, Washington University School of Medicine, St Louis, MO 63110.
Trask
R V
RV
Fontanet
H L
HL
Billadello
J J
JJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003402:Creatine Kinase / Q000235:genetics*; D004247:DNA / Q000235:genetics; D015536:Down-Regulation; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003402:Creatine Kinase