A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development

The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P.R., Coutelle, O., Cox, D., Yee, S.P. and Rigby, P.W.J. (2000) Development 127, 3745-3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated differently in each of a considerable number of populations of muscle progenitors, and they begin to illuminate the evolutionary origins of this complex regulation. We further show that separate elements are involved in the activation and maintenance of expression in the various precursor populations, reflecting the diversity of the signals that control myogenesis. Mrf4 expression requires at least four elements, one of which may be shared with Myf5, providing a possible explanation for the linkage of these genes throughout vertebrate phylogeny. Further complexity is revealed by the demonstration that elements which control Mrf4 and Myf5 are embedded in an unrelated neighbouring gene.J. J. C. was supported by a Research Training Fellowship from the Medical Research Council (UK), which also paid for this work.Peer reviewe

Analysis of a key regulatory region upstream of the Myf5 gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.This work in M.B.'s laboratory was supported by the Pasteur Institute and the CNRS and by grants from the ACI Integrative Biology Programme of the MJER, the AFM and the European Community (QLK3-CT-99/02). J.H. benefited from fellowships from ARC and the AFM, L.B. from funding from the MJER, and T.C. from fellowships from NIH and the AFM. The work in P.R.'s laboratory was supported by a grant from The Institute of Cancer Research.Peer reviewe

The initial somitic phase of Myf5 expression requires neither Shh signaling nor Gli regulation

Myf5, the skeletal muscle determination gene, is first expressed in the dorso-medial aspect of the somite under the control of an element we have called the early epaxial enhancer. It has subsequently been reported that this enhancer is a direct target of Shh signaling mediated by Gli transcription factors (Gustafsson et al. 2002). We here demonstrate that activation of Myf5 expression depends on neither Shh function nor an intact Gli binding site, although the Gli site is necessary for continuation of expression. We suggest that the discrepancy is due to the existence of specific interactions between the enhancer and the Myf5 promoter