Title: Airway Smooth Muscle (ASM) DNA methylation: A novel target for asthma therapy
Funder: Asthma UK

Abstract: Background Asthma occurs as a result of environmental insults to the lung in genetically susceptible individuals. Environmental factors can leave an epigenetic signature on airway cells, resulting in an altered biological response to future insults. DNA methylation (5-mC), a key epigenetic modification, is a reversible modification of DNA in which a methyl group is added to cytosine residues. 5-mC regulates gene expression. Understanding how 5-mC changes in airway structural cells alter gene expression and function could lead to a major shift in our understanding of the role of environmental factors in driving chronic airway inflammation and remodelling in asthma. Preliminary data During my ERS/CTS Peter Macklem Fellowship (Kobor lab, Vancouver) I trained in genome-wide 5-mC analysis and next-generation sequencing techniques and generated preliminary data showing a reduction in global 5-mC levels in airway smooth muscle (ASM) cells from asthmatic patients. A pilot genome-wide array study identified 67 5-mC sites with differential methylation between asthmatic and non-asthmatic cells. I selected 9 sites for associated steady state gene expression analysis and identified differential 5-mC associated with transcripts that are: not expressed, expressed but show no differential expression, expressed and show positive or negative correlation with expression. I selected 4 genes (RUNX3, KLF6, TBX1, SPINT2) for further investigation based on the differential 5-mC magnitude, the relationship to RNA expression and their potential role in asthma. Hypothesis 5-mC regulates aberrant gene expression in ASM cells isolated from asthmatic individuals, contributing to an asthmatic phenotype and providing a target for novel therapies. Aims 1) To integrate genome-wide 5-mC data with genome-wide expression data from asthmatic and non-asthmatic ASM cells to obtain a genome-wide understanding of the interaction between 5-mC and RNA expression in asthma. I will expand the genome-wide pilot study to include a) a larger 5-mC data set, confirming my preliminary data and facilitating a fuller 5-mC analysis and b) genome-wide gene expression analysis, allowing an integrated understanding of genome-wide 5-mC and gene expression. 2) To investigate the molecular mechanisms of dysregulated DNA methylation in ASM cells in asthma and its role in regulating gene and protein expression. I will interrogate the molecular mechanisms regulating the relationship between 5-mC and gene/protein expression in ASM cells from asthmatic versus non-asthmatic donors. I will also assess the contribution of 5-hydroxymethylcytosine (an intermediate in the removal of 5-mC) to the 5-mC signal/function. 3) To investigate the role of 5-mC dependent differential gene expression in asthmatic and non-asthmatic ASM function. I will combine gene knockdown techniques, genome-wide analysis and functional assays to determine the role of the genes of interest in ASM cell function and contribution to an asthmatic phenotype. Impact This study will combine the track record of the Knox lab in studying ASM cell biology in asthma with the Kobor lab expertise in genome-wide 5-mC analysis allowing me to apply my fellowship training to answer important questions on asthma epigenetics. The studies will identify novel therapeutic targets and may lead to more effective asthma treatments, improving patient quality of life and reducing costs to the NHS.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RESPIRATORY