FIG. 3.

Chromatin immunoprecipitation demonstrates direct binding of FoxO1a to the cGKIα promoter region. (A) Direct binding of FoxO1a to cGKIα enhancer regions was assayed using ChIP. Since the FoxO1a-specific antibody performs poorly in immunoprecipitation assays, FLAG-tagged wild-type FoxO1a was used to perform the ChIP on transduced myoblasts. Upon differentiation, FoxO1a shows rapid binding to cGKIα enhancer regions (−7.0/−6.7 kb and + 6.5/+6.8 kb), which contain FoxO1a binding sites. Cells transduced with an empty MSCV-IRES-GFP vector were used as a negative control. (B) The cGKI promoter area harbors FoxO1a-responsive elements. C2C12 cells transfected with reporter luciferase constructs containing the FoxO1a binding regions (at −7.0/−6.7 kb or +6.5/+6.8 kb from the cGKI start codon) were induced to differentiate by (i) confluence in 10% DMEM (Confl.), (ii) transfer to a medium with 2% horse serum (2% HS), and (iii) transfer to a serum-free medium (Ser. Free). Constructs deleted for (w/o) the FoxO binding sites were used as a negative control. (C) Serum withdrawal did not provoke induction of the reporter due to a massive induction of apoptosis.