Regulation of Airway Mucin Gene Expression by Epigenetic Mechanism

描述（由申请方提供）：粘蛋白是粘液分泌的粘弹性的主要贡献者，其在传导气道的粘液纤毛清除中起重要作用。由于粘蛋白过度产生而导致的异常粘液积聚是与各种肺部疾病（如哮喘、囊性纤维化、支气管炎、慢性阻塞性肺病等）相关的主要临床症状之一。气道粘蛋白合成和分泌相关介质和调控通路的克隆、表达特征和鉴定已取得重要进展。然而，细胞类型特异性和异常粘蛋白分泌的持续性质，在各种肺部患者，甚至恢复后，仍然不清楚。我们推测这种持续的现象与粘蛋白基因的表观遗传修饰以及其他修饰有关，如气道重塑和气道中炎性细胞因子的升高。初步研究表明，在人MUC 5AC的上游启动子区（约4，396 bp - 4，541 bp）中存在CpG岛，这在小鼠直向同源物中不存在。MUC 5AC在各种人气道细胞系和原代正常支气管上皮（NHBE）细胞中的表达水平受到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MUC 5AC CpG岛的甲基化状态，并且这种状态与MUC 5AC基因表达呈负相关。基于这些结果，我们假设表观遗传学变化，特别是DNA甲基化状态的变化，是粘蛋白基因表达持续性的主要机制之一，特别是MUC 5AC。为了进一步检验这一假设，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MUC 5AC的差异表达是否受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。第二个目的是解决视黄酸诱导的MUC 5AC在细胞系以及原代细胞中的表达是否与表观遗传机制相关，包括CpG岛的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的MUC 5AC表达的持续性是否与人类受试者中CpG岛和/或染色质结构的DNA甲基化状态的改变相关。为了确定CpG岛DNA甲基化状态，将使用亚硫酸氢盐测序和使用甲基化和非甲基化序列特异性引物的定量PCR。使用抗甲基化脱氧胞嘧啶和各种抗修饰组蛋白抗体的染色质免疫沉淀（ChIP）方法将用于评估MUC 5AC的DNA甲基化谱和染色质结构。使用siRNA以及过表达方法，将确定DNA甲基转移酶（DNMT）对CpG岛甲基化状态和MUC 5AC表达的影响。 公共卫生相关性：异常气道粘蛋白表达是与各种肺部疾病相关的主要临床问题;具体地，疾病气道与粘蛋白产生的持续升高相关。这种持续性的性质尚未得到解决。我们推测，表观遗传学的变化，特别是DNA甲基化状态和染色质结构的变化，参与了人类气道细胞MUC 5AC表达的持续升高。我们计划在表观遗传水平上鉴定有助于MUC 5AC基因表达调控的独特分子特征。了解气道粘蛋白基因表达的表观遗传机制的独特方面，将导致更好地了解气道中异常粘蛋白的产生，以及开发新的治疗方法来治疗气道疾病。

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.