Hormone-controlled cooperative binding of transcription factors drives synergistic induction of fasting-regulated genes

Abstract

During fasting, hepatocytes produce glucose in response to hormonal signals. Glucagon and glucocorticoids are principal fasting hormones that cooperate in regulating glucose production via gluconeogenesis. However, how these hormone signals are integrated and interpreted to a biological output is unknown. Here, we use genome-wide profiling of gene expression, enhancer dynamics and transcription factor (TF) binding in primary mouse hepatocytes to uncover the mode of cooperation between glucagon and glucocorticoids. We found that compared to a single treatment with each hormone, a dual treatment directs hepatocytes to a pro-gluconeogenic gene program by synergistically inducing gluconeogenic genes. The cooperative mechanism driving synergistic gene expression is based on ‘assisted loading’ whereby a glucagon-activated TF (cAMP responsive element binding protein; CREB) leads to enhancer activation which facilitates binding of the glucocorticoid receptor (GR) upon glucocorticoid stimulation. Glucagon does not only activate single enhancers but also activates enhancer clusters, thereby assisting the loading of GR also across enhancer units within the cluster. In summary, we show that cells integrate extracellular signals by an enhancer-specific mechanism: one hormone-activated TF activates enhancers, thereby assisting the loading of a TF stimulated by a second hormone, leading to synergistic gene induction and a tailored transcriptional response to fasting.

Transcriptional cascades during fasting amplify gluconeogenesis and instigate a secondary wave of ketogenic gene transcription

“…This is because not all synergistic gene induction was fully recapitulated by ectopic PPARα expression. It is tempting to speculate that assisted loading between GR and PPARα take place at some enhancers, leading to synergistic gene induction, as we and other showed in other synergistic paradigms [17,18,63,74]. Unfortunately, we were unable to profile PPARα via ChIP-seq in primary hepatocytes and therefore this possible mechanism remains a hypothesis for the moment.…”

“…This suggests a synergistic cooperation between the two treatments. To test for synergistic gene induction, we used previously-formulated cutoffs [17] based on the definition of synergy: the effect of the dual treatment is higher than the summed effect of both single treatments. Thus, in order for a gene to be determined as synergistically-induced, it has to fulfill these four conditions: the gene is induced in the dual treatment compared to the: (a) non-treated control, (b) the corticosterone single treatment and (c) the Wy single treatment.…”

“…7A). Of note, this pattern does not only contrast with primary target gene induction patterns, it is also at odds with mechanisms of synergistic induction that are not dependent on transcriptional cascades as these mechanisms show peak synergistic induction at 2 h [17, 63].…”

“…In all box plots, the 10-90 percentiles are plotted. GO terms enrichment analysis: performed by GeneAnalytics [81] Analyses of published data: In addition to data generated in this study, we used data that was previously published by us [17][18][19] and others [60]. Statistical analyses: All conditions in all of the described experiments were performed in three biological replicates.…”

“…The secretion of both hormones prominently increases in prolonged fasting periods when glycogen is depleted and gluconeogenesis as well as ketogenesis assume the major role in providing fuel [6][7][8][9][10]. Glucagon and glucocorticoids work in a synergistic manner to activate gluconeogenesis and supply glucose during fasting [11][12][13][14][15][16][17][18][19].…”