Maladaptive epigenetic control of MUC5B transcription in pulmonary fibrosis

项目摘要 在这个项目中，我们将定义导致MUC 5 B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生表型。特发 肺纤维化（IPF）的5年死亡率超过许多癌症。G到T的颠换 rs35705950是已知的发展IPF的最强风险因子，位于 MUC 5 B基因。该~ 3 kb区域调节MUC 5 B表达，其在IPF中增加，特别是在终末细胞中。 细支气管样区域。细支气管是IPF发病的主要部位， 在疾病中的分化，促进局部募集和活化成纤维细胞。虽然rs35705950和 MUC 5 B是IPF发病机制中的关键参与者，单独的rs35705950不足以引起IPF，我们不认为 充分了解MUC 5 B和rs35705950如何促进IPF发病。我们的提议是基于两次打击 假设解决这些差距。我们假设MUC 5 B的过度表达，通过表观遗传学增强， 改变，引发细支气管上皮，使得第二次损伤性打击导致异常的表观遗传学 调节、有害的ER应激和成纤维细胞活化。三个目标解决了这个假设。目标1： 将确定MUC 5 B在近端和远端气道上皮中转录的分子控制， 和IPF受试者。这项工作扩展了我们关于~ 3 kb MUC 5 B增强子功能的初步数据，我们将 特异性测试先驱转录因子FOXA 2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MUC 5 B表达。在目标2中， 将确定驱动异常远端气道上皮细胞的表观遗传和转录因子机制 在IPF中，与MUC 5 B表达和基因型相关的分化。目标2是由我们的数据驱动的 这暗示了两种参与正常气道上皮分化的转录因子在IPF中的重要作用， EHF和GRHL 2。我们在目标2中的方法将包括ATAC-seq，新生转录测序，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Muc 5 b过表达小鼠的样品和细胞。在目标3中，我们将研究气道上皮细胞在 表观遗传学和MUC 5 B表达对IPF中ER应激和成纤维细胞活化的转录控制。目的 3建立在我们的数据支持有害的ER应激反应的表观遗传机制，其中IPF- 衍生的细胞指导主要的ER应激调节因子，XBP 1（S）与开放的染色质在基序处相互作用， 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的XBP 1（S）基因组占有率和ATF 4串扰。我们将 还研究了成纤维细胞表观遗传学与IPF气道上皮异常的关系。综上所述各项 研究将推进我们对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靶点。

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.