Fasting massively reorganizes liver chromatin to facilitate dynamic transcription factor crosstalk and synergistic gene expression

The liver is responsible to produce glucose (by gluconeogenesis) and ketones (by ketogenesis) during fasting to satisfy systemic energetic demands. Although this response to fasting is heavily reliant upon transcriptional regulation with many transcription factors (TFs) regulating it, the key TFs with a genome-wide role in dictating it are undefined. Also, TF crosstalk and the contribution of chromatin to the fasting response are unknown. We characterized the genome-wide effect of fasting on hepatic chromatin in mice and found that chromatin organization is extensively reshaped following fasting. Numerous fasting-induced enhancers were exposed, leading to overt changes in gene expression. Employing motif analysis and our newly-developed DNA footprinting computational tool (“BaGFoot”), we implicated four key TFs regulating the fasting response in a genome-wide manner: GR, CREB, PPARα and CEBPβ. We found that these TFs regulate hepatic fuel production by two functionally-distinct modules, one controlling gluconeogenesis and the other ketogenesis. Using genomic and single-molecule-tracking techniques, we found that the gluconeogenic module operates through “assisted loading” in which GR increases accessibility of enhancers, thereby augmenting CREB binding. Importantly, assisted loading of CREB was enhancer-selective, affecting only a subset of CREB-bound enhancers. These events resulted in rapid synergistic gene expression and augmented hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARα levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARα target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to maintain homeostasis upon fasting