Identification of the DNA sequence within the mouse inhibin-α promoter required for Wt1A-dependent transcriptional activation.

A, TM4 cells were transiently transfected with luciferase reporter constructs bearing different lengths of the mouse Inhibin alpha promoter sequence, and Renilla luciferase reporter plasmids together with an empty plasmid (pCMV-tag2b) or Wt1A expression vector. The filled and open ovals indicated putative Wt1 response elements (Wt1RE). B, The Wt1-dependent transactivational activity of inhibin-α promoter was significantly decreased after deletion of the ten base pairs (5′- GGC GGG AGT G -3′) sequence including part of the Wt1 consensus binding motif. C, Western blot showed the endogenous Wt1 protein expression in TM4 cells and primary Sertoli cells. TM4 cells were transiently transfected with the empty plasmid or with the expression plasmid for Flag-Wt1A. 48 h after transfection, cells were cross-linked with formaldehyde and cross-linked chromatin was sonicated followed by immunoprecipitation with anti-Flag antibody. Genomic DNA was purified from the immunoprecipitates and subjected to PCR using the primers as indicated in schematic drawing of Inhibin-α promoter and distal primers as negative control. A specific PCR product (black arrow) was amplified in Flag-Wt1A transfected cells with proximal primers, not with the distal primers. Normal mouse IgG served as a negative control.