Maladaptive epigenetic control of MUC5B transcription in pulmonary fibrosis

肺纤维化中 MUC5B 转录的适应不良表观遗传控制

• 批准号: 10627598
• 负责人: ANTHONY N GERBER
• 金额: $ 50.99万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627598
• 关键词: ATAC-seq; Address; Aging; Area; Binding; Bronchioles; Cell Culture Techniques; Cell Differentiation process; Cells; ChIP-seq; Chromatin; Collaborations; Cues; DNA; Data; Disease; Distal; Enhancers; Epigenetic Process; Epithelial Cells; Epithelium; Etiology; European; Event; Family; Fibroblasts; Gene Expression Regulation; Genetic Transcription; Genomics; Genotype; Human; Injury; Investigation; MUC5B gene; Malignant Neoplasms; Methods; Modeling; Molecular; Mus; Odds Ratio; Particulate; Pathogenesis; Phenotype; Population; Process; Pulmonary Fibrosis; RNA; Regulation; Response Elements; Risk; Risk Factors; Role; Sampling; Site; Smoking; Terminal Bronchiole; Testing; Tissue Sample; Transcript; Transcriptional Regulation; Work; XBP1 gene; activating transcription factor; airway epithelium; biological adaptation to stress; chromatin remodeling; endoplasmic reticulum stress; epigenetic regulation; genome-wide; human model; human tissue; idiopathic pulmonary fibrosis; knock-down; lung injury; mortality; mouse model; new therapeutic target; overexpression; programs; recruit; response; response to injury; transcription factor

Project Summary In this project, we will define epigenetic and transcriptional mechanisms that result in increased MUC5B expression, distal airway epithelial abnormalities, and associated fibroproliferative phenotypes. Idiopathic pulmonary fibrosis (IPF) has a 5-year mortality rate surpassing many cancers. The G-to-T transversion, rs35705950, is the most potent known risk factor for developing IPF and is located ~ 3 kb upstream of the MUC5B gene. This -3 kb region regulates MUC5B expression, which is increased in IPF, especially in terminal bronchiole-like regions. Bronchioles are a primary site of IPF pathogenesis and feature aberrant epithelial differentiation in disease, promoting local recruitment and activation of fibroblasts. Although rs35705950 and MUC5B are key players in IPF pathogenesis, rs35705950 alone is not sufficient to cause IPF, and we don’t fully understand how MUC5B and rs35705950 promote IPF pathogenesis. Our proposal is based on a two- hit hypothesis that addresses these gaps. We hypothesize that MUC5B overexpression, potentiated by epigenetic changes, primes the bronchiolar epithelium such that a second injurious hit results in aberrant epigenetic regulation, detrimental ER stress and fibroblast activation. Three Aims address this hypothesis. In Aim 1, we will determine molecular control of MUC5B transcription in proximal and distal airway epithelia from unaffected and IPF subjects. This work extends our preliminary data on -3 kb MUC5B enhancer function, and we will specifically test whether the pioneer transcription factor, FOXA2, induces chromatin remodeling, enhancer RNA transcription and MUC5B expression through methods such as ChIP and factor knockdown. In Aim 2 we will identify epigenetic and transcription factor mechanisms that drive abnormal distal airway epithelial cell differentiation in relationship to MUC5B expression and genotypes in IPF. Aim 2 is motivated by our data implicating important roles in IPF for two transcription factors involved in normal airway epithelial differentiation, EHF and GRHL2. Our methods in Aim 2 will include ATAC-seq, nascent transcript sequencing, knockdown and ChIP to define mechanistic roles for these factors in IPF, with extension of our findings into human samples and cells from Muc5b overexpressing mice. In Aim 3, we will investigate the role of airway epithelial epigenetics and MUC5B expression on transcriptional control of ER stress and fibroblast activation in IPF. Aim 3 builds on our data supporting an epigenetic mechanism in detrimental ER stress responses where IPF- derived cells direct the master ER stress regulator, XBP1(S) to interact with open chromatin at motifs for the ATF family of transcription factors. We will extend on these findings through applying ATAC-seq and ChIP-seq to study XBP1(S) genomic occupancy and ATF4 crosstalk in IPF-derived human airway epithelial cells. We will also study fibroblast epigenetics in relationship to airway epithelial abnormalities in IPF. Taken together, these studies will advance our molecular understanding of transcriptional control in IPF pathogenesis and identify new targets for restoring distal airway epithelial function in this devastating disease.

项目概要 在这个项目中，我们将定义导致 MUC5B 增加的表观遗传和转录机制 表达、远端气道上皮异常和相关的纤维增殖表型。特发性 肺纤维化（IPF）的 5 年死亡率超过许多癌症。 G 到 T 的颠换， rs35705950 是已知的最有效的 IPF 风险因素，位于 rs35705950 上游约 3 kb 处。 MUC5B 基因。该 -3 kb 区域调节 MUC5B 表达，该表达在 IPF 中增加，尤其是在终末期 细支气管样区域。细支气管是 IPF 发病机制的主要部位，具有异常的上皮细胞特征 疾病分化，促进成纤维细胞的局部募集和激活。虽然 rs35705950 和 MUC5B 是 IPF 发病机制的关键参与者，单独的 rs35705950 不足以引起 IPF，我们不认为 充分了解MUC5B和rs35705950如何促进IPF发病机制。我们的建议是基于两次打击 解决这些差距的假设。我们假设表观遗传增强了 MUC5B 的过度表达 变化，启动细支气管上皮，从而导致第二次伤害性打击导致异常的表观遗传 调节、有害的 ER 应激和成纤维细胞激活。三个目标解决了这一假设。在目标 1 中，我们 将确定未受影响的近端和远端气道上皮中 MUC5B 转录的分子控制 和 IPF 科目。这项工作扩展了我们关于 -3 kb MUC5B 增强子功能的初步数据，我们将 专门测试先锋转录因子 FOXA2 是否诱导染色质重塑、增强子 通过 ChIP 和因子敲低等方法进行 RNA 转录和 MUC5B 表达。在目标 2 中，我们 将识别驱动异常远端气道上皮细胞的表观遗传和转录因子机制 IPF 中与 MUC5B 表达和基因型相关的分化。目标 2 是由我们的数据推动的 暗示两种参与正常气道上皮分化的转录因子在 IPF 中发挥重要作用， EHF 和 GRHL2。我们的目标 2 方法将包括 ATAC-seq、新生转录本测序、敲除 和 ChIP 来定义这些因素在 IPF 中的机制作用，并将我们的发现扩展到人类 来自 Muc5b 过表达小鼠的样本和细胞。在目标 3 中，我们将研究气道上皮细胞的作用 表观遗传学和 MUC5B 表达对 IPF 中 ER 应激和成纤维细胞活化转录控制的影响。目的 3 基于我们的数据，支持有害 ER 应激反应的表观遗传机制，其中 IPF- 衍生细胞指导主 ER 应激调节因子 XBP1(S) 与开放染色质的基序相互作用 ATF 转录因子家族。我们将通过应用 ATAC-seq 和 ChIP-seq 来扩展这些发现 研究 IPF 衍生的人气道上皮细胞中 XBP1(S) 基因组占据和 ATF4 串扰。我们将 还研究了与 IPF 气道上皮异常相关的成纤维细胞表观遗传学。综合起来，这些 研究将促进我们对 IPF 发病机制中转录控制的分子理解，并确定 在这种毁灭性疾病中恢复远端气道上皮功能的新目标。