Abstract
Expression of the muscle creatine kinase (MCK) gene in skeletal and heart muscle is controlled in part by a 5' tissue-specific enhancer. In order to identify new regulatory elements, we designed mutations in a previously untested conserved portion of this enhancer. Transfection analysis of these mutations delineated a new control element, named Trex (Transcriptional regulatory element x), which is required for full transcriptional activity of the MCK enhancer in skeletal but not cardiac muscle cells. Gel mobility shift assays demonstrate that myocyte, myoblast, and fibroblast nuclear extracts but not primary cardiomyocyte nuclear extracts contain a trans-acting factor that binds specifically to Trex. The Trex sequence is similar (7/8 bases) to the TEF-1 consensus DNA-binding site involved in regulating other muscle genes. To determine if TEF-1 interacts with Trex, selected TEF-1 binding sites such as GTIIc and M-CAT and two anti-TEF-1 antisera were used in gel shift assays. These experiments strongly suggest that a factor distinct from TEF-1 binds specifically to Trex. Thus it appears that MCK transcription is regulated in skeletal muscles through a Trex-dependent pathway while Trex is not required for MCK expression in heart. This distinction could account partially for the difference in levels of muscle creatine kinase in these tissues.
Highlights
Expression of the muscle creatine kinase (MCK) gene in differentiated muscle cells is controlled by an array of cis-elements and trans-acting factors
The MEF-2 site is the target of MEF2 proteins, which are known as the Related to Serum Response Factor (RSRF) family [27, 28], and BBF-1, a serum-inducible factor identified in cardiac but not in skeletal muscle [29]
Nucleotide alignment of the four mammalian MCK enhancers identifies an additional highly conserved region located between the AP2 and A/T-rich sites (Fig. 1A)
Summary
Expression of the MCK gene in differentiated muscle cells is controlled by an array of cis-elements and trans-acting factors. Analysis, gel mobility shift studies, and footprinting assays have identified six transcriptional regulatory elements in the MCK enhancer (from 5Ј to 3Ј): the CArG, AP2, A/T-rich, left and right E boxes, and MEF-2 sites [6, 10, 11] Each of these MCK gene control elements has been well conserved during mammalian evolution [7, 12,13,14], and similar nucleotide motifs have been implicated in the transcriptional control of numerous other muscle-specific genes [15]. Unlike the other MCK enhancer control elements, the AP2 site seems to repress transcription in cultured skeletal and cardiac muscle cells as its mutation leads to increased expression in both cell types These six enhancer regulatory elements are the target sites for an array of muscle and non-muscle-specific DNA binding factors. The MCK enhancer appears to contain no perfect match to the MCAT sites, key elements in regulating the cardiac troponin T
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