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

项目摘要 未来研究问题 作为肯塔基州大学毒理学和癌症生物学系的研究生，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 论文研究的目的是确定Polycomb Repressive Complex 2（PRC2）的作用是如何正确引导 肺干细胞的命运，以及这个过程是如何在肺部疾病失调。我的核心假设是PRC2 通常抑制支气管上皮中基底细胞和杯状细胞的命运， 这些谱系的抑制导致在慢性阻塞性肺疾病的细支气管上皮中观察到的表型。 肺部疾病（COPD），包括基底细胞和杯状细胞化生。 背景及方法 多梳抑制复合物2（PRC2）通过组蛋白修饰调节基因表达。最近 来自Brainson实验室的未发表数据表明，PRC2活性在慢性阻塞性肺疾病中失调， 肺疾病（COPD）。这些发现与该领域的其他研究结果相吻合，这些研究结果表明， 发育中的肺导致远端肺气道中包括KRT 5和p63在内的基底细胞标志物的异常表达 对小鼠总之，这些数据表明，不适当的PRC2活性对细胞发育、命运和发育是重要的。 功能它们还保证了对PRC2在疾病发展和传播中的作用的进一步研究， 特别是涉及肺功能的那些。 方法 本研究的目的是：1）阐明在肺动脉高压中观察到的细支气管上皮细胞的变化， 在PRC2复合物的遗传调节后的体外和体内，和2）证实适当PRC2基因的丢失 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增。对于目标1， 我正在敲除Ezh2的一个或两个等位基因，Ezh2是编码PRC2酶的基因，在成年小鼠肺中， 4个月，并用卵清蛋白攻击小鼠以诱导杯状细胞化生。我的假设是 将通过肺组织学测试PRC2的浓度将阻止细胞解决过敏反应。同时，我将使用 肺类器官培养和单细胞RNA测序，以检查PRC2失调时的肺细胞命运 在小鼠肺细胞中。对于目标2，我将使用EZH2抑制剂，针对PRC2组分的短发夹RNA， 香烟烟雾提取物对人支气管上皮细胞在气液相间培养，以研究如何 PRC2的扰动改变肺细胞命运决定。我也会从慢性阻塞性肺疾病患者中建立新的培养物， 最后探索这些培养物中的转录变化以及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地调查生理因素来影响国家和全球的卫生政策。 由于环境变化而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操作感兴趣与Brainson博士阐明肺的目标相一致 干细胞的命运和注释的表观基因组的患病与正常的人类和小鼠肺上皮。

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 Recent unpublished data from the Brainson lab suggests that PRC2 activity is dysregulated in chronic obstructive pulmonary disease (COPD).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.