Rules in Regulation: Defining principles of gene expression control by FOXO

Forkhead box O (FOXO) transcription factors are involved in the regulation of the cell cycle, apoptosis and metabolism. In model organisms, FOXO activity affects stem cell maintenance and lifespan as well as age-related diseases, such as cancer and diabetes. Multiple upstream pathways regulate FOXO activity through post-translational modifications and nuclear-cytoplasmic shuttling of both FOXO and its regulators. The diversity of this upstream regulation and the downstream effects of FOXOs, suggest that they function as homeostasis regulators to maintain tissue homeostasis over time and coordinate a response to enable adaptation to environmental changes, including growth factor deprivation, metabolic stress (starvation) and oxidative stress. To gain insight into the mechanisms of FOXO-regulated target gene expression, we studied genome-wide effects of FOXO3 activation. The profiling of RNA polymerase II changes suggests that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator, of which a significant part of gene regulation proceeds through enhancer regions. We investigate features that define the subset of enhancer binding events that actually contribute to gene regulation. From this, we conclude that FOXO3 transcriptional output is determined by the amount of bound FOXO3, with the most prominent gene activation mainly associated with binding to multiple sites within the vicinity of the gene, rather than isolated binding events. FOXO3 binding events are in turn determined by a combination of Forkhead motif presence and pre-existing enhancer activity/accessibility. In this manner, FOXO3 amplifies pre-existing levels of enhancer activity marks (e.g. H3K27ac, p300, RNAPII) and potentiates enhancer-RNA transcription. Since the FOXO3-mediated increases in enhancer activity marks correlate with the regulation of adjacent genes, we conclude that not only enhancer presence and sequence content, but also pre-existing activity dictates the amplitude of FOXO3 gene induction. In addition, we show pre-existing chromatin loops between several FOXO3 bound enhancers and regulated genes. Considering the flexible and cell type specific nature of regulatory regions, their activity and their spatial organization, our observations provide a novel explanation for the context-dependent nature of FOXO transcriptional output and introduce chromatin context as a new player in the regulation of FOXO function. Our results also provide an elegant rationale on how homeostasis can be achieved. The usage of a pre-existing gene program directed by chromatin context of regulatory regions, could allow for a rapid and temporary adjustment in gene transcription, sufficient to re-establish homeostasis, while preventing elaborate time- and energy consuming permanent alterations in the cell’s specific gene expression program.