FIG. 1.

JMJ represses ANF gene expression. (A) JMJ inhibits ANF gene expression in cardiomyocytes. The ANF reporter gene −3003ANF-Luc or −638ANF-Luc (2 μg) was cotransfected with 0.1 μg of the JMJ expression vector into rat neonatal primary cardiomyocytes. Relative luciferase activity was calculated when luciferase activity with the reporter gene alone was set at 1. (B) JMJ inhibits activation of the ANF gene by Nkx2.5 or GATA4 or both. The ANF reporter gene −638ANF-Luc (2 μg) was cotransfected with 2 μg of various transcription factors in the expression vectors into 10T1/2 cells grown on 60-mm plates by calcium phosphate precipitation methods. Luciferase activity was normalized with β-galactosidase activity to correct transfection efficiency. Relative luciferase activity was expressed as the activation level (n-fold) above that of the reporter gene alone. Filled bars indicate the means and T bars indicate standard errors of the means for four separate transfection assays with duplicate plates. (C) Cotransfection of JMJ does not affect the expression level of Nkx2.5 or GATA4. Western blots show expression levels of exogenous genes in the same 10T1/2 cell extracts transfected for the reporter gene assays described for panel B. After cotransfection of the Myc-tagged Nkx2.5 and/or GATA4 expression vector with JMJ as indicated, equivalent amounts of the cell extract (100 μg/lane) were subjected to immunoblotting for Nkx2.5 and GATA4 or JMJ.