Negative regulation of selected bHLH proteins by eHAND

The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. In this study, we have identified a novel transcriptional activity of eHAND. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, we show that eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, we propose that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2.