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 i s 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 i s 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 i s 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.
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.
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