Elucidating the epigenetic regulation of extracellular matrix and virus-induced fibroblast activation

项目总结 虽然伤口愈合过程对成功的组织修复至关重要，但如果不能关闭这些过程 机制导致细胞外基质(ECM)过度积聚，并发展为 纤维化症。尽管最近的研究开始阐明了允许纤维化消退的环境， 大多数纤维性疾病仍未解决；导致器官衰竭或易患癌症。而当 纤维性疾病的诱因分为几类，包括病毒/细菌感染、组织损伤、 和化学损伤，都诱导间充质细胞持续激活为肌成纤维细胞。此外 转化生长因子β(转化生长因子-β)是成纤维细胞的主要激活剂，细胞外基质也有能力 改变成纤维细胞的激活状态。我们和其他人已经证明了这种激活的表型可以持续 尽管细胞从纤维化组织中移除，但这表明成纤维细胞具有后天获得的“表观遗传记忆” 在激活期间并在此后保留。然而，表观遗传调控的分子细节在 肌成纤维细胞的激活以及这些细节是否普遍存在，尽管激活的触发机制尚不清楚。 肌成纤维细胞调节的一个潜在机制是通过细胞外基质依赖的PRO-2的表达。 纤维化基因。基因表达是一个严格调控的过程，需要染色质重塑，结合 转录激活剂和RNA聚合酶的招募以启动转录。因此，结构性的 染色质的重组在基因的时间调控和组织特异性中起着重要作用 表情。Brahma相关基因1(BRG1)是BAF的一个中心催化ATP亚基(BRG1/BRM1-)。 相关因子)复合体，通过核小体驱逐来驱动染色质的可及性。BRG1还拥有 已被证明在健康和病毒诱导的纤维化环境中调节ECM基因的表达。此外， 初步数据发现BRG1缺陷的胰腺成纤维细胞失去了一个关键功能的表达 调节器，Netrin G1。综上所述，这表明BRG1在调控肌成纤维细胞促纤维化基因中发挥了作用。 这项提议的首要目标是测试ECM和诱导纤维化的病毒改变 成纤维细胞的染色质景观以BRG1依赖的方式，并有助于表观遗传记忆 是肌成纤维细胞功能的基础。 为了验证这一假设，Aim 1将首先研究BRG1‘S在胰腺成纤维细胞激活中的作用。 体外，以及这是否由ECM介导的信号调节。在目标2中，实验将重点放在 BRG1在通过体内胰腺炎小鼠模型调节疾病形成中的作用。最后，目标3将在 这些教训和研究纤维化诱导病毒引起肌成纤维细胞激活的机制， 首先是人类常见的病原体--甲型流感病毒。通过使用新的视角来理解 纤维化，这项研究提供了深入的见解，将促进细胞生物学，表观遗传学和病毒学，以及揭示 ECM/病毒如何创造一种促纤维化状态。

PROJECT SUMMARY While wound healing processes are vital for successful organismal tissue repair, failure to turn off these mechanisms lead to the excessive accumulation of the extracellular matrix (ECM) and the development of fibrosis. Although recent research is beginning to illuminate the circumstances that allow for fibrosis resolution, most fibrotic conditions remain unresolved; resulting in organ failure or a predisposition to cancer. While the triggers for fibrotic diseases fall within a handful of categories, including viral/bacterial infection, tissue damage, and chemical insults, all induce the sustained activation of mesenchymal cells into myofibroblasts. Besides transforming growth factor β (TGF-β), which is a major activator of fibroblasts, the ECM also has the ability to alter a fibroblast’s activation state. We and others have demonstrated that this activated phenotype can persist despite the cell’s removal from fibrotic tissues, suggesting that fibroblasts have an “epigenetic memory” acquired during activation and retained thereafter. However, the molecular details underlying epigenetic regulation during myofibroblastic activation and whether these details are universal despite the activation trigger is unknown. One potential mechanism of myofibroblast regulation is through the ECM-dependent expression of pro- fibrotic genes. Gene expression is a tightly regulated process that requires chromatin remodeling, binding of transcriptional activators, and recruitment of RNA polymerase to initiate transcription. As such, the structural reorganization of the chromatin plays a large role in the temporal regulation and tissue specificity of gene expression. Brahma-related gene 1 (BRG1) is a central catalytic ATP-subunit of the BAF (BRG1/BRM1- associated factor) complex which works to drive chromatin accessibility via nucleosome eviction. BRG1 has also been shown to regulate ECM gene expression in both healthy and virally-induced fibrotic contexts. Moreover, preliminary data found that BRG1-deficient pancreatic fibroblasts lost the expression of a key functional regulator, Netrin G1. Taken together, this suggests a role of BRG1 in regulating myofibroblast pro-fibrotic genes. The overarching goal of this proposal is to test the hypothesis that the ECM and fibrosis-inducing viruses alter fibroblasts’ chromatin landscape in a BRG1-dependent manner and contribute to the epigenetic memory that underlies myofibroblastic function. To test this hypothesis, Aim 1 will first investigate BRG1’s involvement in pancreatic fibroblast activation in vitro and whether this is regulated by ECM-mediated signaling. In Aim 2, experiments will focus on the role of BRG1 in regulating disease formation by using an in vivo pancreatitis mouse model. Finally, Aim 3 will build on these lessons and investigate the mechanisms by which fibrosis-inducing viruses cause myofibroblast activation, beginning with the frequent human pathogen, Influenza A virus. By using a novel perspective to understanding fibrosis, this research provides insights that will advance cell biology, epigenetics, and virology, as well as reveal how the ECM/viruses create a pro-fibrotic state.