A Skeletal Muscle-Specific Enhancer Regulated by Factors Binding to E and CArG Boxes Is Present in the Promoter of the Mouse Myosin Light-Chain 1A Gene

Abstract

The mouse myosin light-chain 1A (MLC1A) gene, expressed in the atria of the adult heart, is one of the first muscle genes to be activated when skeletal as well as cardiac muscles form in the embryo. It is also transcribed in skeletal muscle cell lines at the onset of differentiation. Transient transfection assays of mouse skeletal muscle cell lines with DNA constructs containing MLC1A promoter fragments fused to the chloramphenicol acetyltransferase (CAT) gene show that the first 630 bp of the promoter is sufficient to direct expression of the reporter gene during myotube formation. Two E boxes located at bp -76 and -519 are necessary for this regulation. MyoD and myogenin proteins bind to them as heterodimers with E12 protein and, moreover, transactivate them in cotransfection experiments with the MLC1A promoter in nonmuscle cells. Interestingly, the effect of mutating each E box is less striking in primary cultures than in the C2 or Sol8 muscle cell line. A DNA fragment from bp -36 to -597 confers tissue- and stage-specific activity to the herpes simplex virus thymidine kinase promoter in both orientations, showing that the skeletal muscle-specific regulation of the MLC1A gene is under the control of a muscle-specific enhancer which extends into the proximal promoter region. At bp -89 is a diverged CArG box, CC(A/T)6AG, which binds the serum response factor (SRF) in myotube nuclear extracts, as does the wild-type sequence, CC(A/T)6GG. Both types of CArG box also bind a novel myotube-enriched complex which has contact points with the AT-rich part of the CArG box and adjacent 3' nucleotides. Mutations within the CArG box distinguish between the binding of this complex and binding of SRF; only SRF binding is directly involved in the specific regulation of the MLC1A gene in skeletal muscle cell lines.

Comparative transcriptome analysis of muscular dystrophy models Largemyd, Dmdmdx/Largemyd and Dmdmdx: what makes them different?

“…32 Myl4, another regeneration marker overexpressed in the mouse models here studied, is related to myotube formation, as it is transcribed in skeletal muscle during the start of muscle differentiation. 33 Tnnt2, coding for troponin type 2, is also expressed at the beginning of regeneration and was found overexpressed in DMD patients, which was associated with the active regeneration in dystrophic muscle. Figure 5 Enriched functional categories.…”

Expression of the Smooth Muscle Myosin Heavy Chain Gene Is Regulated by a Negative-acting GC-rich Element Located between Two Positive-acting Serum Response Factor-binding Elements

“…Li et al (41) further demonstrated that deletion of a region containing two CArG boxes in the mouse SM22␣ gene (another highly specific SMC marker) resulted in a significant drop in transcriptional activity in cultured SMCs. Thus, similar to several cardiac-and skeletal muscle-specific genes (42)(43)(44), CArG-SRF interaction appears to serve as a common transcriptional activation mechanism for multiple SM-specific genes.…”

Identification of a New Hybrid Serum Response Factor and Myocyte Enhancer Factor 2-binding Element in MyoD Enhancer Required for MyoD Expression during Myogenesis

“…Because the G-to-A 3Ј-end divergence in MyoD-CArG is similar to that reported previously in MLC1A-CArG-box, shown to be functional in binding SRF (Catala et al, 1995), we investigated whether MLC1A-CArG also binds MEF2 proteins during differentiation. Anti-MEF2 supershift experiments with MLC1A probe show that MLC1A-CArG only bound SRF but not MEF2 proteins ( Figure 2B, right lanes).…”

“…Interestingly, a divergent CArG sequence in MLC1A gene, described as functional (Catala et al, 1995), contains the same 3Ј G-to-A divergence as the MyoD-SMB element. In addition, the decanucleotide core sequences are identical in both cases, with the exception of one nucleotide divergence (A to T) in the A/T tract of MLC1A CArG.…”