Regulation of Airway Mucin Gene Expression by Epigenetic Mechanism

描述（由申请人提供）：粘蛋白是粘液分泌粘弹性的主要贡献者，在传导气道的粘纤毛清除中起重要作用。粘蛋白分泌过多导致的异常粘液积聚是各种肺部疾病的主要临床症状之一，如哮喘、囊性纤维化、支气管炎、慢性阻塞性肺疾病等。近年来，气道粘蛋白合成与分泌的相关介质和调控途径的克隆、表达表征、鉴定等方面均取得了重大进展。然而，各种肺部患者的细胞类型特异性和异常黏蛋白分泌的持久性，即使在康复后，仍不清楚。我们假设这种持续现象与黏液蛋白基因的表观遗传修饰以及其他修饰（如气道重塑和气道中炎症细胞因子的升高）有关。初步研究表明，人类MUC5AC上游启动子区（- 4396 bp - 4541 bp）存在CpG岛，而小鼠同源基因中不存在CpG岛。MUC5AC在各种人气道细胞系和原代正常支气管上皮细胞（NHBE）中的表达水平受CpG岛甲基化状态的影响。此外，我们发现全反式维甲酸和烟雾都能够改变MUC5AC CpG岛的甲基化状态，并且这种状态与MUC5AC基因表达呈负相关。基于这些结果，我们假设表观遗传变化，特别是DNA甲基化状态的变化，是粘蛋白基因（尤其是MUC5AC）持续表达的主要机制之一。为了进一步验证这一假设，提出了三个具体目标。第一个目的是确定不同细胞系和原代NHBE细胞中MUC5AC的差异表达是否受到表观遗传机制的调节，包括CpG岛DNA甲基化状态和染色质结构的变化。第二个目的是研究维甲酸诱导细胞系和原代细胞MUC5AC表达是否与表观遗传机制有关，包括CpG岛DNA甲基化状态的改变和染色质结构的改变。第三个目的是确定烟雾诱导MUC5AC表达的持久性是否与人类受试者CpG岛DNA甲基化状态和/或染色质结构的改变有关。为了确定CpG岛DNA甲基化状态，亚硫酸盐测序和定量PCR将使用甲基化和非甲基化序列特异性引物。染色质免疫沉淀（ChIP）方法与抗甲基化脱氧胞嘧啶和各种抗修饰组蛋白抗体将用于评估MUC5AC的DNA甲基化谱和染色质结构。利用siRNA和过表达方法，将建立DNA甲基转移酶（dnmt）对CpG岛甲基化状态和MUC5AC表达的影响。

DESCRIPTION (provided by applicant): Mucins are major contributors to the visco-elastic properties of mucus secretion, which plays an important role in the mucociliary clearance in conducting airways. Aberrant mucus accumulation due to mucin overproduction is one of major clinical symptoms associated with various lung diseases, such as asthma, cystic fibrosis, bronchitis, chronic obstructive pulmonary diseases, etc. Significant progress has been made in the cloning, expression characterization, and the identification of mediators and regulatory pathways involved in airway mucin synthesis and secretion. However, the cell type-specificity and the persistent nature of aberrant mucin secretion in various lung patients, even following recovery, are still unclear. We hypothesize that the persistent phenomenon is related to epigenetic modification on mucin gene in addition to other modifications, such as airway remodeling and elevated presence of inflammatory cytokines in airways. Preliminary studies have shown the presence of CpG islands in the upstream promoter region of human MUC5AC (at -4,396 bp - 4,541bp), which is not present in the mouse orthologue. The level of MUC5AC expression in various human airway cell lines and primary normal bronchial epithelial (NHBE) cells are affected by the methylation status of the CpG islands. Furthermore, we showed both all-trans-retinoic acid and smoke are able to alter the methylation status of MUC5AC CpG islands and that this status is inversely related MUC5AC gene expression. Based on these results, we hypothesize that epigenetic changes, especially changes in DNA methylation status, is one of the major mechanisms involved in the persistence of mucin gene expression, especially MUC5AC. To further test this hypothesis, three specific aims are proposed. The first aim is to determine if differential MUC5AC expression in various cell lines and primary NHBE cells is regulated by epigenetic mechanisms, including changes in CpG island DNA methylation status and chromatin structure. The second aim is to address whether retinoic acid induced MUC5AC expression in cell lines, as well as in primary cells, is associated with epigenetic mechanisms that include alterations in DNA methylation status of CpG islands, and alterations in chromatin structure. The third aim is to determine if smoke-induced persistence of MUC5AC expression is associated with alterations in the DNA methylation status of CpG islands and/or chromatin structure in human subjects. To determine CpG island DNA methylation status, both bisulfite sequencing and quantitative PCR with methylated and non- methylated sequences-specific primers will be used. Chromatin immunoprecipitation (ChIP) approaches with anti-methylated deoxy-cytosine and various anti-modified histone antibodies will be used to assess DNA methylation profiles and chromatin structure of MUC5AC. Using siRNA as well as overexpression approaches, the effects of DNA methyltransferases (DNMTs) on CpG island methylation status and MUC5AC expression will be established. PUBLIC HEALTH RELEVANCE: Aberrant airway mucin expression is a major clinical problem associated with various lung diseases; specifically, diseases airways are associated with a persistent elevation of mucin production. The nature of this persistence is unresolved. We hypothesize that epigenetic changes, especially changes in DNA methylation status and chromatin structure, are involved in the persistent elevation of MUC5AC expression in human airway cells from diseased states. We plan to identify unique molecular signatures that contribute to the regulation of MUC5AC gene expression at the epigenetic level. Understanding the unique aspects of epigenetic mechanisms involved in airway mucin gene expression will lead to a better understanding of aberrant mucin production in the airway, as well as to the development of novel therapeutic approaches to treating airway diseases.