Genomic Binding Patterns of Forkhead Box Protein O1 Reveal its Unique Role in Cardiac Hypertrophy.

Background: Cardiac hypertrophic growth is mediated by robust changes in gene expression, as well as changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase (pol) II de novo recruitment or loss, while the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site (TSS). The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein (Fox)O1 is an insulin-sensitive transcription factor, which is also regulated by hypertrophic stimuli in the heart, however, the scope of its gene regulation remains unexplored. Methods: To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing (ChIP-Seq) in mouse hearts following 7-day isoproterenol injections (Iso- 3 mg/kg/day), transverse aortic constriction (TAC), or vehicle injection/sham surgery. Results: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 ChIP-Seq results were aligned with those of pol II ChIP-Seq across the chromosomal coordinates of sham- or TAC-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% post-TAC. Interestingly, FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro , endothelin (Et-)1- and, in vivo , pressure overload, -induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 , in vitro ,and Fstl1 and Uck2 , in vivo . Conclusions: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release, as the latter represents the majority (59%) of FoxO1-bound, pol II regulated genes following pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.