Bronchiolar Dysplasia in COPD is Governed by Loss of Polycomb Repressive Complex 2

项目总结 未来的研究问题 作为肯塔基大学毒理学和癌症生物系的研究生，我寻求 了解推动肺部疾病发展的复杂分子过程。我的目标是 论文研究是为了确定多梳抑制复合体2(PRC2)的作用如何正确地引导 肺干细胞的命运，以及这一过程在肺部疾病中是如何失调的。我的中心假设是PRC2 正常情况下抑制细支气管上皮中的基底细胞和杯状细胞的命运，以及PrC2介导的丢失 这些谱系的抑制导致在慢性阻塞性肺泡上皮细胞中观察到表型 肺部疾病(COPD)，包括基底细胞和杯状细胞化生。 背景和方法 多梳抑制复合体2(PRC2)通过组蛋白修饰来调节基因表达。近期 来自Brainson实验室的未发表的数据表明，在慢性阻塞性疾病中，PRC2的活动是失调的 肺部疾病(COPD)。这些发现与该领域中其他显示PRC2丢失的研究结果相吻合 肺发育过程中Krt5、p63等细胞标记物在肺远端的异常表达 在老鼠身上。总之，这些数据表明，不适当的PRC2活性对细胞发育、命运和 功能。他们还需要进一步调查PRC2的S在疾病发展和传播中的作用， 尤其是涉及肺功能的。 方法论 我的建议的目的是：1)阐明在慢性支气管炎中观察到的细支气管上皮细胞的细胞变化。 体外和体内基因调控后的PRC2复合体和2)证实了适当的PRC2基因的缺失 沉默导致人细支气管上皮细胞中基底细胞和杯状细胞命运的扩张。对于目标1， 我正在敲除成年小鼠肺中编码PRC2酶的Ezh2的一个或两个等位基因 4个月后，用卵清蛋白诱导小鼠杯状细胞化生。我的假设是损失 PRC2是否能阻止细胞分解过敏反应将通过肺组织学进行测试。同时，我将使用 肺器官培养和单细胞RNA测序检测PrC2异常时肺细胞的命运 在小鼠的肺细胞中。对于目标2，我将使用EZH2抑制剂，针对PRC2组件的短发夹状RNA，以及 香烟烟雾提取物对气液界面培养的人支气管上皮细胞的影响 PRC2的扰动改变了肺细胞命运的决定。我还将从COPD患者那里建立新的培养体系， 最后探索这些文化中的转录变化以及PRC2如何通过以下方式影响这些变化 染色质免疫沉淀实验。 职业潜力 我的最终目标是通过有效调查生理学问题来影响国家和全球卫生政策 由于环境变化，目前正在发生和已经发生的变化。我的个人 对干细胞生物学和表观遗传学操作的兴趣与布雷森博士阐明肺的目标不谋而合 干细胞命运和注释疾病与正常的人和小鼠肺上皮的表观基因组。

PROJECT SUMMARY Future Research Questions As a graduate student in the Toxicology and Cancer Biology Department of University of Kentucky, I seek to understand the complex molecular processes that drive development of lung diseases. The goal of my dissertation research is to determine how the role of Polycomb Repressive Complex 2 (PRC2) directs proper lung stem cell fate, and how this process is dysregulated in lung disease. My central hypothesis is that PRC2 normally represses basal and goblet cell fates in bronchiolar epithelium, and that loss of PRC2-mediated repression of these lineages leads to phenotypes observed in bronchiolar epithelium of Chronic Obstructive Pulmonary disease (COPD), including basal cell and goblet cell metaplasia. Background and Methodology Polycomb Repressive Complex 2 (PRC2) modulates gene expression through histone modification. Recent unpublished data from the Brainson lab suggests that PRC2 activity is dysregulated in chronic obstructive pulmonary disease (COPD). These findings fit with others in the field that demonstrated loss of PRC2 in developing lung causes aberrant expression of basal cell markers including KRT5 and p63 in distal lung airways in mice. Together these data suggest that improper PRC2 activity is important for cell development, fate, and function. They also warrant further investigation into PRC2's role in disease development and propagation, particularly those involving lung functions. Methodology The Aims of my proposal are to: 1) Elucidate the cellular changes in bronchiolar epithelial cells observed in in vitro and in vivo after genetic modulation of the PRC2 complex and 2) Confirm that loss of proper PRC2 gene silencing leads to expansion of basal cell and goblet cell fates in human bronchiolar epithelial cells. For Aim 1, I am knocking out one or two alleles of Ezh2, the gene encoding the enzyme for PRC2, in adult mouse lung for four months, and challenging the mice with ovalbumin to induce goblet cell metaplasia. My hypothesis that loss of PRC2 will prevent cells from resolving allergic response will be tested by lung histology. In parallel, I will use lung organoid cultures and single cell RNA-sequencing to examine lung cell fates when PRC2 is dysregulated in mouse lung cells. For Aim 2, I will use an EZH2 inhibitor, short hairpin RNAs against PRC2 components, and cigarette smoke extract on human bronchial epithelial cells in air-liquid interphase cultures to examine how perturbation of PRC2 changes lung cell fate decisions. I will also establish new cultures from COPD patients, and finally explore the transcriptional changes in these cultures and how PRC2 influences these changes by chromatin immunoprecipitation experiments. Career Potential My ultimate goal is to influence national and global health policy by effectively investigating the physiological changes that are currently taking and have taken place as a result of environmental changes. My personal interest in stem cell biology and epigenetic manipulation coincides with Dr. Brainson's objective to elucidate lung stem cell fate and annotate the epigenome of diseased versus normal human and mouse lung epithelium.