Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4

Abstract

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) proteincoding sequence, 8.5 kb of 5−flanking sequence, and 1.0 kb of 3′-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5′ deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5′-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5′-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5′-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.