M E Ritchie | PubMed Health

PubMed1992

The human M creatine kinase gene enhancer contains multiple functional interacting domains.

Cis-elements (-933 to -641) upstream of the human M creatine kinase gene cap site contain an enhancer that confers developmental and tissue-specific expression to the chloramphenicol acetyltransferase gene in C2C12 myogenic cells transfected in culture. Division of the enhancer at -770 into a 5' fragment that includes the MyoD binding sites (-933 to -770) and a 3' fragment that includes the MEF-2 binding site (-770 to -641) resulted in two subfragments that showed minimal activity but in combination interacted in a position- and orientation-independent fashion to enhance activity of the SV40 promoter in transient transfection experiments. A 5' enhancer construct (-877 to -832) including only one (the low affinity) MyoD binding site was active when present in multiple copies. In contrast, a 3' enhancer construct (-749 to -732) including the MEF-2 binding site was inactive even when present in multiple copies. However, if the 5' construct was extended to include the high-affinity MyoD binding site (-877 to -803) the 5' and 3' constructs interacted in a position- and orientation-independent fashion to activate the SV40 promoter. Thus, the human M creatine kinase enhancer comprises multiple functional interacting domains.

3T3 Cells↗

PubMed1991

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.

3T3 Cells↗