The E protein HEB is preferentially expressed in developing muscle.

Myogenesis is regulated by the MyoD class of myogenic regulatory factors (MRFs). These basic helix-loop-helix transcription factors dimerize with E proteins to bind conserved E-box sequences in the promoter regions of muscle-specific genes. Perhaps due to their expression in a wide array of tissues, the specific interactions of E proteins with different MRFs have been largely ignored. Likewise, the expression of E proteins in muscle tissue remains mostly uncharacterized. We investigated the expression of the E proteins HEB, E12, and E47 in rat L6 myoblasts, which express only embryonic and fast (2X) myosin heavy chains (MyHCs) in vitro, C2C12 myosatellite cells, and a number of muscle tissues, to determine whether myosin heavy chain diversity is mirrored by diversity in E protein or MRF expression. Although L6 and C2C12 myotubes demonstrate strong expression of embryonic and 2X (fast) MyHCs, immunofluorescence demonstrated the additional expression of type 1 (slow), 2A, and 2B MyHCs in the C2C12 cell line. Immunofluorescence and western blot analyses show that HEB was expressed in differentiating L6 myoblasts, C2C12 cells, and neonatal rat primary myotubes. In contrast, E12 and E47 expression was not detected in either cell line or in any adult muscle tissue examined. These data strongly implicate HEB in the development of skeletal muscle. However, because HEB is expressed in L6 myoblasts, C2C12 myosatellite cells, and neonatal hindlimb muscles, it is unlikely to be involved in a fiber type-specific manner, and may have a more general role in differentiation of myotubes.