Abstract
The muscle creatine kinase gene enhancer contains two regulatory elements (MCK-R and MCK-L) with the consensus E-box sequence (CAnnTG). A myocyte specific protein complex, MEF1, binds the MCK-R site. MEF1 contains several basic H-L-H myogenic determination factors (MDFs), each dimerized with ubiquitous members of the bH-L-H family (e.g. E12/E47). We now demonstrate that the ubiquitous bH-L-H factor E2-2 is a major component of the endogenous MCK-R site specific complex. Previous studies described the MCK-L site as a similar but low affinity MDF/bH-L-H heterodimer binding site. However, we find that the MCK-L site exhibits preferential binding of an unknown ubiquitous factor which contains neither E12/E47 nor E2-2, and that it exhibits differential transcriptional activity with muscle and non-muscle cells. The differential behavior of the MCK-L and MCK-R sites may be a general trait of E-box elements since one among several E-boxes in the MLC 1/3 enhancer also binds preferentially to the MCK-L factor. From our studies we now propose separate consensus sequences for MCK-R and MCK-L E-box types: AACAc/gc/gTGCa/t and GGa/cCANGTGGc/gNa/g. Our results suggest that while many muscle gene E-boxes are capable of binding the previously characterized spectrum of MDF/bH-L-H heterodimers in vitro, MCK-L type E-boxes probably bind qualitatively different factors in vivo.
Highlights
Previous studies described the muscle creatine kinase (MCK)-L site as a similar but low affinity myogenic determination factors (MDFs)/bH-L-H heterodimer binding site
The MCK-R and MCK-L Sites Differ in Their Transcriptional Activities—To determine whether the MCK-L and MCK-R sites exhibit functional differences we examined the activity of artificial genes containing four contiguous MCK-L or MCK-R sites fused to the thymidine kinase promoter and chloramphenicol acetyltranferase reporter gene (CAT)
The transient expression of these constructs was examined in replicating and differentiated skeletal muscle cells (MM14 myoblasts and myocytes) as well as in mitogen-deprived non-muscle (NIH 3T3 cells). The purpose of the latter comparison was to test the expression of the L- and R-MCK E-boxes in a non-myogenic cell that had been subjected to similar growth arrest conditions as those required for converting replicating myoblasts to the differentiated state
Summary
Previous studies described the MCK-L site as a similar but low affinity MDF/bH-L-H heterodimer binding site. We find that the MCK-L site exhibits preferential binding of an unknown ubiquitous factor which contains neither E12/E47 nor E2–2, and that it exhibits differential transcriptional activity with muscle and non-muscle cells. In gel shift assays the MCK-R site binds a myocyte specific complex called MEF1 [2] that contains the myogenic determination factors (MDFs), MyoD, and/or myogenin [2, 8, 9].
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