The promoter of the GATA-4 gene was analyzed in P19CL6 cells. A 124 bp segment containing conserved two GC-boxes and E-box was essential for the basal promoter activity, as determined with a transient luciferase reporter gene assay. However, an extended 1312 bp reporter construct but not the 124 bp segment, when ligated to the GFP gene and stably inserted into the chromosome, showed regulated promoter activity since GFP was expressed upon DMSO addition. Mutations of the two GC-boxes and/or E-box significantly impaired the GFP expression. Furthermore, mutation of the distal conserved GATA motif in the 1312 bp sequence decreased the expression of GFP. Chromatin immuno-precipitation assay showed that GATA-6 binds to this conserved GATA motif. These results suggest that the distal GATA motif recognized by GATA-6 together with the GC- and E-boxes may be important for transcriptional activation of the GATA-4 gene in the chromosome. (C) 2011 Elsevier Inc. All rights reserved.