Key result
Human MCK enhancer requires cooperative 5' and 3' domains to activate transcription.
Why the study?
The functional organization and interaction of multiple domains within the human M creatine kinase gene enhancer were not fully characterized.
This basic science study demonstrates that the human M creatine kinase gene enhancer relies on multiple interacting functional domains, including MyoD and MEF-2 binding sites, for its activity.
Enhancer mapping in myogenic cells may guide muscle gene studies; leaves open relevance to cardiac CK regulation in vivo.
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.
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Trask et al. (1992) studied this question. Enhancer constructs (5' and 3' fragments of human M creatine kinase gene enhancer) was evaluated on Enhancer activity (activation of SV40 promoter in transient transfection experiments). The human M creatine kinase enhancer comprises multiple functional interacting domains, with 5' and 3' constructs interacting to activate the SV40 promoter.
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