Genetic regulation of ductular reaction in liver injury and regeneration

项目总结 肝脏表现出显著的再生能力，但慢性损伤或严重的急性损伤可能 压倒代偿反应，导致肝功能衰竭。定期需要可移植的肝脏 超过捐赠者池，需要开发基于再生医学的新疗法和 更深入地了解肝脏的内源性修复机制。这项建议寻求一种机械化的 理解导管反应(DR)的基因调控网络，这是一种相关的损伤反应 有广泛的肝脏损伤和疾病。DR的定义是胆管上皮的增殖性扩张 细胞(BECs)，并可涉及成熟肝细胞和BECs之间的上下文相关的谱系转换， 有助于组织再生。DR的基因调控仍然知之甚少，包括BECs是如何 平衡增殖和表型可塑性。SOX9是干/祖细胞所必需的转录因子 在许多上皮组织中起作用，并已被证明通过全基因组建立细胞身份 对染色质景观的影响。在肝脏中，Sox9是BEC规范在发育过程中的定时所必需的 在成体BEC中广泛表达。我们实验室最近利用Sox9egfp转基因来研究bec的异质性。 结果表明，Sox9在BECs和胆管周围杂交瘤细胞亚群中的表达水平不同 动态平衡和胆汁淤积期间的肝细胞(HybHeps)。我们实验室的新发现也证明了 成年Sox9基因敲除小鼠骨髓内皮细胞的异常这一提议的中心假设是Sox9起作用 作为DR的主要调节者，通过抑制增殖和促进BEC的特性。以下是具体目标 将检验这一假设：目标1A将确定Sox9在损伤诱导的BEC增殖中的作用，通过 Sox9EGFP等位基因和(1)BEC/肝细胞或(2)BEC特异性Sox9基因敲除小鼠的应用 模特们。目的1B将确定Sox9在双向BEC到肝细胞可塑性中的作用，通过 补充体内谱系追踪和体外有机物分析。Aim 2A将绘制染色质调控图 通过整合转录学和染色质分析，了解肝损伤期间BEC亚群的DR景观。 AIM 2B将通过将单细胞多组学应用于BEC-2来确定Sox9在DR中的基因组调控作用。 特定的Sox9基因敲除小鼠模型。在这个项目中产生的数据将提供基本的机制 深入了解DR的基因调控并确定调控节点，以提高治疗靶向 终末期肝病的再生。

PROJECT SUMMARY The liver exhibits remarkable capacity for regeneration, but chronic injury or severe acute damage can overwhelm compensatory responses and result in liver failure. The need for transplantable livers regularly exceeds the donor pool, necessitating the development of new regenerative medicine-based therapies and a deeper understanding of the liver’s endogenous repair mechanisms. This proposal seeks a mechanistic understanding of gene regulatory networks underlying ductular reaction (DR), a damage response associated with a broad range of liver injury and disease. DR is defined by the proliferative expansion of biliary epithelial cells (BECs) and can involve context-dependent lineage conversion between mature hepatocytes and BECs that contributes to tissue regeneration. The genetic regulation of DR remains poorly understood, including how BECs balance proliferation and phenotypic plasticity. Sox9 is a transcription factor required for stem/progenitor cell function in a number of epithelial tissues and has been shown to establish cellular identity through genome-wide effects on the chromatin landscape. In the liver, Sox9 is required for timing of BEC specification in development and is broadly expressed in adult BECs. Our lab recently used a Sox9EGFP transgene to study BEC heterogeneity and showed that Sox9 is expressed at distinct levels in subpopulations of BECs and peribiliary hybrid hepatocytes (HybHeps) during homeostasis and cholestasis. New findings from our lab also demonstrate abnormalities in BECs of adult Sox9 knockout mice. The central hypothesis of this proposal is that Sox9 functions as a master regulator of DR, by inhibiting proliferation and promoting BEC identity. The following specific aims will test this hypothesis: Aim 1A will determine the role of Sox9 in damage induced BEC proliferation, through the use of the Sox9EGFP allele and (1) combined BEC/hepatocyte or (2) BEC-specific Sox9 knockout mouse models. Aim 1B will determine the role of Sox9 in bi-directional BEC-to-hepatocyte plasticity, through complementary in vivo lineage tracing and in vitro organoid assays. Aim 2A will map the chromatin regulatory landscape of DR, by integrating transcriptomics and chromatin assays in BEC subpopulations during liver injury. Aim 2B will determine the genomic regulatory impact of Sox9 in DR by applying single cell multi-omics to BEC- specific Sox9 knockout mouse models. The data generated in this project will provide fundamental mechanistic insight into genetic regulation of DR and identify regulatory nodes for therapeutic targeting to enhance regeneration in end stage liver disease.