Different E-box regulatory sequences are functionally distinct when placed within the context of the troponin I enhancer.

Basic helix-loop-helix (bHLH) regulatory proteins are known to bind to a single DNA consensus sequence referred to as an E-box. The E-box is present in the regulatory elements of many developmentally controlled genes, including most muscle-specific genes such as troponin I (TnI). Although the E-box consensus is minimally defined as CANNTG, the adjacent nucleotides of functional E-boxes are variable for genes regulated by the bHLH proteins. In order to examine how E-box regulatory regions containing different internal and flanking nucleotides function when placed within the context of a single regulatory element, the E-box region (14 bp) present within the TnI enhancer was substituted with the corresponding E-box sequences derived from the muscle-specific M-creatine kinase (MCK) and cardiac alpha-actin regulatory elements as well as from the immunoglobulin kappa (Ig kappa) enhancer. Within the TnI enhancer, the E-box sequence derived from cardiac alpha-actin was inactive whereas the corresponding sequence from the MCK right E-box efficiently restored wild-type enhancer activity in muscle cells. Intermediate levels of gene activity were observed for TnI enhancers containing E-boxes derived from the MCK left E-box site or from the Ig kappa E2 E-box. DNA binding studies of MyoD:E12 protein complexes with each substituted TnI enhancer confirmed that DNA binding activity in vitro mimics the relative strength of the enhancers in vivo. These studies demonstrate that the specific nucleotide composition of individual E-boxes, which are contained within the regulatory elements of most if not all muscle-specific genes, contributes to the complex regulatory mechanisms governing bHLH-mediated gene expression.     

Muscle regulatory factor gene: zebrafish (Danio rerio) myogenin cDNA

Myogenin is one of the basic helix-loop-helix proteins that regulate muscle-specific gene expression. Using reverse transciption-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (RACE), zebrafish myogenin cDNA was cloned from mRNA of embryos at 10-96 h post-fertilization. The cDNA, at 1384 base pairs (bp), contained a 771-bp open reading frame with 113- and 500-bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequences of zebrafish myogenin encoded a 256-amino-acid polypeptide. In a comparison with myogenin of carp, trout, Xenopus, chicken and human, zebrafish myogenin shared 90.9, 77.6, 70.3, 62.9 and 51.5% amino acid identity, respectively. The basic helix-loop-helix domains in myogenin are all conserved. The molecular phylogenic tree demonstrated that myogenin of zebrafish is more closely related to that of fish than to the myogenin of other vertebrates.     

An E box comprises a positional sensor for regional differences in skeletal muscle gene expression and methylation.

To dissect the molecular mechanisms conferring positional information in skeletal muscles, we characterized the control elements responsible for the positionally restricted expression patterns of a muscle-specific transgene reporter, driven by regulatory sequences from the MLC1/3 locus. These sequences have previously been shown to generate graded transgene expression in the segmented axial muscles and their myotomal precursors, fortuitously marking their positional address. An evolutionarily conserved E box in the MLC enhancer core, not recognized by MyoD, is a target for a nuclear protein complex, present in a variety of tissues, which includes Hox proteins and Zbu1, a DNA-binding member of the SW12/SNF2 gene family. Mutation of this E box in the MLC enhancer has only a modest positive effect on linked CAT gene expression in transfected muscle cells, but when introduced into transgenic mice the same mutation elevates CAT transgene expression in skeletal muscles, specifically releasing the rostral restriction on MLC-CAT transgene expression in the segmented axial musculature. Increased transgene activity resulting from the E box mutation in the MLC enhancer correlates with reduced DNA methylation of the distal transgenic MLC1 promoter as well as in the enhancer itself. These results identify an E box and the proteins that bind to it as a positional sensor responsible for regional differences in axial skeletal muscle gene expression and accessibility.