AgRP neuron epigenomes across hunger states reveal that IRF3 mediates leptin's effects

AgRP neurons in the arcuate nucleus of the hypothalamus (ARC) coordinate homeostatic changes in appetite associated with fluctuations in food availability and leptin signaling. Identifying the relevant transcriptional regulatory pathways in these neurons has been a priority, yet such attempts have been stymied due to their low abundance and the rich cellular diversity of the ARC. Here we generated male mouse AgRP neuron-specific transcriptomic and chromatin accessibility profiles during three distinct hunger states of satiety, fasting-induced hunger, and leptin-induced hunger suppression. Cis-regulatory analysis of these integrated datasets enabled the identification of 28 putative hunger-promoting and 29 putative hunger-suppressing transcriptional regulators in AgRP neurons, 16 of which were predicted to be transcriptional effectors of leptin. Within our dataset, Interferon regulatory factor 3 (IRF3) emerged as a leading candidate mediator of leptin-induced hunger-suppression. Gain- and loss-of-function experiments in vivo confirm the role of IRF3 in mediating the acute satiety-evoking effects of leptin in AgRP neurons, while live-cell imaging in vitro indicate that leptin can activate neuronal IRF3 in a cell autonomous manner. Finally, we employ CUT&RUN to uncover direct transcriptional targets of IRF3 in AgRP neurons in vivo. Thus, our findings identify AgRP neuron-expressed IRF3 as a key transcriptional effector of the hunger-suppressing effects of leptin. Overall design: For RNA-seq and ATAC-seq data we generated a NuTRAP^AgRP mouse line, from which we could isolate AgRP neuron-specific mRNA and nuclei We profiled RNA expression and chromatin accessibility in AgRP neurons harvested from the above animals under fed (ad-libitum), fasting (overnight), and “leptin” (fasting plus leptin administration) conditions. RNA-seq samples had an group size of n = 4 (3 in fed), while ATAC was performed in groups of size n = 2. Comparative gene expression and chromatin-accessibility analyses were performed separately for these the above groups. Integrated analyses demonstrating relationships between concordant changes in expressions and chromatin profiles were performed by linking ATAC peaks with gene TSSes at a range of +/- 200kb. Profiling of IRF3 binding sites in AgRP neurons was performed via CUT&RUN in mouse models expressing a constitutively active variant of IRF3 (IRF3-2D).