A conserved MRF4 promoter drives transgenic expression in Xenopus embryonic somites and adult muscle

Abstract

The muscle regulatory factor MRF4 is expressed in both embryonic and adult vertebrate skeletal muscle cells. In mammals the MRF4 gene has a complex cis-regulatory structure, with many kilobases (kb) of upstream sequence required for embryonic expression in transgenic mice. Here, initial functional comparison between Xenopus and mammalian MRF4 genes revealed that 610 base pairs (bp) of the XMRF4a proximal promoter drove substantial transgenic expression in X. laevis myogenic cells, from somites of neurula embryos through adult myofibers, and as little as 180 bp gave detectable expression. Over 300 bp of XMRF4a proximal promoter sequence is highly conserved among three X. laevis and X. tropicalis MRF4 genes, but only about 150 bp shows significant identity to mammalian MRF4 genes. This most-conserved XMRF4a region contains a putative MEF2 binding site essential for expression both in transgenic embryos and in transfected mouse muscle cells. A rat MRF4 minimal promoter including the conserved region also was active in transgenic X. laevis embryos, demonstrating a striking difference between the mouse and Xenopus transgenic systems. The longest XMRF4a promoter construct tested, with 9.5 kb of 5′-flanking sequence, produced significantly greater expression in transfected mouse cells than did promoters 4.3-kb or shorter, suggesting that the intervening region contains an enhancer, although no increased expression was evident when this region was included in transgenic X. laevis embryos. Further identification and analysis of Xenopus MRF4 transcriptional control elements will offer insights into the evolution of this gene and of the myogenic gene regulatory network.

Making muscle: Morphogenetic movements and molecular mechanisms of myogenesis in Xenopus laevis

“…In mammals, the mrf4 gene has a complex cis-regulatory structure that includes proximal and distal regulatory elements that pattern mrf4 gene expression in early and late myogenic cells [110,111]. Functional comparisons with mammalian mrf4 indicates that X. laevis mrf4 has a 610bp proximal promoter that includes an enhancer; over 300bp of this region is conserved in both X. laevis and X. tropicalis and contains a MEF2 binding site essential for expression of the protein [112]. Mrf4 knockout in mice results in normal myogenesis and upregulation of Myogenin, suggesting that Myogenin can compensate for Mrf4 loss.…”

“…For instance, within the 610bp X. laevis mrf4 promoter region, only about 150bp is conserved in mammals; this difference in transcriptional control elements indicates divergent evolution of myogenic gene regulatory elements [112]. During the formation of neuromuscular connections in X. laevis mrf4 expression may be induced by innervation; in fact, denervation of adult muscle results in reduced mrf4 but not myoD RNA levels [114,115].…”

Structure and dynamics of stress fibers in adult stem cells

“…Although it remains transcriptionally inactive in the non-muscle cell, it leads to formation of a myogenic phenotype eventually [56]. MRF4 is envolved in myofiber differentiation [57], and has the ability to determine whether MyoD and Myf5 are absent [58]. These genetic manipulations via cDNA are highly specialised techniques, but other biochemical methods to induce myogenic differentiation have been proven to be successful as well.…”