3D Circadian Enhancer-Promoter Interactions Mediated by ESRRG in NAFLD-to-HCC Progression

Project Summary Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related death worldwide, with the fastest- growing rate. Nonalcoholic fatty liver disease (NAFLD) affects approximately 25% of the world’s population and is becoming a leading cause of HCC. The progression of NAFLD to HCC, including nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis, is well-recognized, but the pathogenesis of NAFLD- associated HCC is not well understood. Circadian rhythms allow mammals to anticipate daily environmental changes and maintain physiological homeostasis. Epidemiologic studies indicate that circadian misalignment contributes to the development of many cancers. Moreover, the concept of chronotherapy is attracting more attention to improving drug efficacy when drugs are provided at the optimized time of the day. We and others unexpectedly uncovered that although core clock genes still retain robust rhythmic expression, the rhythms of thousands of genes are altered in NAFLD, suggesting critical roles of noncanonical clock regulators. In preliminary studies, we performed the first Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET) experiments in healthy and NAFLD livers and identified multiple noncanonical clock transcription factors (TFs), including Estrogen Related Receptor Gamma (ESRRγ), which are enriched in the enhancer- promoter circadian looping anchors. Indeed, the rhythmic expression of ESRRγ is enhanced in NAFLD, and ESRRγ is associated with nearly half of these NAFLD-induced E-P circadian loops. Moreover, hepatocyte- specific overexpression of ESRRγ slows, but hepatocyte-specific knockout of ESRRγ accelerates NAFLD- related HCC progression. This proposal will test the hypothesis that ESRRγ, as a noncanonical clock gene, is a key regulator mediating circadian E-P interactions in NAFLD and is critical for the progression of NAFLD to HCC. In functional Aim 1, We will use adeno-associated virus expression systems to introduce ESRRγ expression in constitutive, in-phase, and anti-phase rhythmic patterns in Esrrγ-knockout livers from multiple preclinical NAFLD–HCC mouse models to define how ESRRγ rhythmicity affects the progression of NAFLD and HCC. In mechanistic Aim 2, we will determine how noncanonical clock transcription factors regulate circadian enhancer-promoter looping and gene expression. In translational Aim 3, we will determine whether ESRRγ agonists affect NAFLD-to-HCC progression in a time-dependent manner by administering them at the peak and trough of ESRRγ expression in newly generated ESRRγ reporter mouse models and liver slice culture tissues from people with NAFLD. Completion of this work will provide novel insights into how a noncanonical clock TF, ESRRγ, remodels the 3D circadian chromatin architecture and gene expression during the NAFLD-to-HCC progression, laying the intellectual groundwork for future chronotherapy strategies that maximize efficacies by considering the timing of drug administration.