FIG 2.

MyoR inhibits Gata4-dependent activation of the Cx30.2 enhancer. (A) Schematic depiction of the Cx30.2-Luc reporter construct used in panel B that contains the Cx30.2 enhancer fused to a minimal TATA box and luciferase coding sequence. The 4.1-kb enhancer fragment encompasses the −2.9/−2.3 region that contains the minimal AVC-specific Cx30.2 enhancer (23). (B) Transient-transfection analysis in COS cells. A total of 30 ng of Cx30.2-Luc reporter was transfected, along with 100 ng of Gata4 and/or 100 ng of bHLH transcription factor (MyoR or Hand1). A 10-ng portion of CMV-lacZ was also cotransfected as an internal control. MyoR repressed Gata4-dependent transcriptional activity driven by Cx30.2-Luc, while Hand1, another bHLH transcription factor expressed in the heart, did not. (C) Coimmunoprecipitation analysis between MyoR and Gata4. Myc-Gata4 and either Flag vector alone or Flag-MyoR were cotransfected into COS7 cells. Whole-cell lysates were precipitated with a Flag antibody and probed with a Myc antibody (top panel). A 10% input was also run separately and probed with either a Myc antibody (middle panel) or a Flag antibody (lower panel). Myc-Gata4 was only detected after Flag immunoprecipitation in the presence of Flag-MyoR but not Flag alone. (D) A ChIP experiment was performed in COS7 cells transfected with a Cx30.2 enhancer template and the indicated proteins. MyoR associated with the Cx30.2 enhancer in a Gata4-dependent manner, but the association with the enhancer was strongly diminished in the presence of a MyoR mutant (MyoRΔ138-157) that does not bind to Gata4. (E) MyoR ChIP from dissected E16.5 AVC and non-AVC (atrial and ventricular) tissue showed that MyoR preferentially associates with the Cx30.2 enhancer in AVC tissue compared to non-AVC tissue. (F) Sequential MyoR and Gata4 ChIP experiment from E16.5 heart tissue demonstrated that both MyoR and Gata4 simultaneously associate with the Cx30.2 enhancer in vivo. *, P < 0.05; ns, not significant.