In this ERC project, we revealed a mechanism for integrating hormonal signals in liver cells, leading to an augmented metabolic response to fasting. We examined the crosstalk between the two major fasting hormones – glucagon and glucocorticoids. Each hormone was previously shown to regulate gene expression by activating transcription factors (glucagon activates CREB and glucocorticoids activate GR), but how these transcription factors crosstalk was undefined. In a paper we published, we found that following hormonal activation, these factors synergistically induce a set of genes important to glucose production. This synergistic gene induction is mediated by cooperative transcription factor binding at enhancers termed ‘assisted loading’. CREB assists the loading of GR on a subset of enhancers through increasing enhancer activity. We showed that CREB activation leads to an increase in the number, intensity and accessibility of GR binding sites, thus facilitating synergistic gene expression and increased glucose production. Thus, this work unraveled critical regulatory mechanisms at play in the liver during fasting.
In addition to this published project, we published an additional paper focusing on the characterization of chromatin accessibility. Chromatin is a DNA-protein structure that enables DNA packaging. Regions in DNA that are transcribed as well as DNA regulatory elements are packaged less densely and are termed ‘accessible’. Because our ERC project heavily relies on genome-wide profiling of chromatin accessibility, we have put significant efforts into improving and optimizing existing protocols to accommodate the special characteristics of liver tissue. Our method was recently published in a peer-reviewed journal.
The third paper from this project explored another key regulatory paradigm: transcriptional cascades. We discovered that fasting induces a network of TFs that, in turn, activate a secondary wave of gene expression. These cascades amplify the gluconeogenic response and also initiate a program that enhances ketogenesis. Our work highlights the importance of these cascades in mediating the body’s acute response to fasting and opens new avenues for studying similar cascades in other physiological processes.
In another paper from the project, we examined the response to refeeding following a period of fasting. When food is consumed again after a period of fasting (i.e. refeeding), a metabolic switch occurs and tissues transition from frugal energy usage and the internal production of fuel to using energy available from food constituents and storage of excess energy in specialized molecules. We found distinct, temporally-organized transcriptional programs occurring upon refeeding with an early wave of transcription followed by a later wave. These programs were driven by enhancer activation that also showed kinetic behavior. We showed that a lipogenic gene program is part of the second wave of transcription and is directly regulated by a TF termed liver X receptor α (LXRα). Interestingly, lipogenesis genes and their associated enhancers markedly overshoot above pre-fasting levels and this is dictated by LXRα. These findings unravel the mechanism behind the long-known phenomenon of refeeding ‘fat overshoot’.
In a recently-published paper, we found that mice undergoing alternate-day fasting (ADF) respond profoundly differently to a following fasting bout compared to mice first experiencing fasting. Hundreds of genes enabling ketogenesis are ‘sensitized’. Liver enhancers regulating these genes are also sensitized and harbor increased binding of PPARα, the main ketogenic transcription factor. ADF leads to augmented ketogenesis compared to a single fasting bout in wild-type, but not hepatocyte-specific PPARα-deficient mice. Thus, we found that past fasting events are ‘remembered’ in hepatocytes, sensitizing their enhancers to the next fasting bout and augment ketogenesis.
