Transcription factor interplay as a driving force in the hepatic response to fasting and its dysregulation in metabolic disease

 

The accessibility of different chromatin regions to transcription factors and other DNA-binding proteins is a critical determinant of cell function. Here, we detail a modified assay for transposase-accessible chromatin sequencing (ATAC-seq) protocol which measures chromatin accessibility genome wide. We describe nuclei isolation, tagmentation, PCR amplification, and pre- and post sequencing quality control. Our protocol is optimized for the liver, a tissue where nuclei isolation requires distinct steps. We provide two detailed vignettes: one for bulk ATAC-seq and another for single-nuclei ATAC-seq. Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.