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Investigating the Role of Epigenetics in Scar Maintenance Andrew William

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Investigating the Role of Epigenetics in Scar Maintenance Andrew William Investigating the role of epigenetics in scar maintenance Andrew William Stevenson BSc. (Hons.) School of Surgery This thesis is presented for the degree of Doctor of Philosophy at the University of Western Australia 2016 A I II DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR PUBLICATION The examination of the thesis is an examination of the work of the student. The work must have been substantially conducted by the student during enrolment in the degree. Where the thesis includes work to which others have contributed, the thesis must include a statement that makes the student’s contribution clear to the examiners. This may be in the form of a description of the precise contribution of the student to the work presented for examination and/or a statement of the percentage of the work that was done by the student. In addition, in the case of co-authored publications included in the thesis, each author must give their signed permission for the work to be included. If signatures from all the authors cannot be obtained, the statement detailing the student’s contribution to the work must be signed by the coordinating supervisor. Please sign one of the statements below. 1. This thesis does not contain work that I have published, nor work under review for publication. Student Signature .................. 2. This thesis contains only sole-authored work, some of which has been published and/or prepared for publication under sole authorship. The bibliographical details of the work and where it appears in the thesis are outlined below. Student Signature .................... III 3. This thesis contains published work prepared for publication, some of which has been co-authored. The bibliographical details of the work and where it appears in the thesis are outlined below. The student must attach to this declaration a statement for each publication that clarifies the contribution of the student to the work. This may be in the form of a description of the precise contributions of the student to the published work and/or a statement of percent contribution by the student. This statement must be signed by all authors. If signatures from all the authors cannot be obtained, the statement detailing the student’s contribution to the published work must be signed by the coordinating supervisor. Manuscripts submitted for review Student’s contribution Contribution of the co-authors Student Signature: …………………………… Date ………………… Coordinating Supervisor Signature: ……………………. Date: ………… IV Dedication I dedicate this to my family – to my parents who supported, encouraged and pushed me to be the best the I can be, my brother who has always been there, and my beautiful wife Kara who encouraged me to do this PhD and patiently supported me throughout. V VI Abstract The reparative response to skin injury in mammals results in the development of scar, underpinned by changes in the dermal matrix structure. Scarring is a significant clinical problem and leads to aesthetic, functional and psychological impacts in patients. Scars are maintained for life and, in the case of children, increase in size during periods of growth. This suggests that the cells producing the scar matrix retain differences to those cells producing normal skin dermal matrix. Epigenetic modification is a heritable alteration to DNA that regulates gene transcription but does not involve changes to the DNA sequence. DNA methylation, an important and stable regulator of gene transcription, is an example of epigenetic modification in DNA methylation, methyl groups are added to specific DNA bases and alter DNA structure. This affects the transcriptome and cell phenotype. Epigenetic changes are known to be critical in tissue differentiation during development and in cancer. In this study, the central hypothesis is that epigenetic modification in fibroblasts during healing, specifically DNA methylation, is the mechanism responsible for long-term changes in collagen metabolism in normotrophic scar fibroblasts and subsequently the dermal matrix. Therefore the aim of this study was to characterise changes in DNA methylation and in the transcriptome of scar fibroblasts compared to normal skin fibroblasts and identify genes which underpin the maintenance of scar dermal matrix. This could lead to the identification of novel therapeutic targets to ameliorate scarring. To identify changes in the epigenome and transcriptome of scar fibroblasts, matched 3mm skin biopsies were taken from both forearms of 6 male burn patients aged 18-34 years who had sustained a unilateral forearm burn injury at least one year prior to biopsy: 6 normotrophic scar samples, and 6 patient-matched normal skin samples. Fibroblasts were cultured from these biopsies using the explant technique and these cells were then used for whole genome methylation and transcriptome profiling. The first study assessed the methylation profile of scar and normal skin fibroblasts. Whole genome methylation data was obtained using the Illumina Infinium 450K array. This measures 485 000 methylation sites across the genome. The data was analysed to identify site specific changes as well as gene and region changes in DNA methylation. Using a pairwise comparison and defining significance as p<0.05 with a Benjamini- Hochberg correction for multiple testing, 0.7% of Cytosine-phosphate-Guanine (CpG) VII sites tested were differentially methylated in the scar fibroblasts, with 63% hypomethylated and 37% hypermethylated. Gene and region analysis showed 836/19 076 genes (4.38%) were differentially methylated within the gene body region. A smaller number of genes were differentially methylated in intragenic regions. Finally, the promoter regions were analysed. This showed that nearly 2% of the promoter regions were differentially methylated, with 44% hypermethylated and 56% hypomethylated. Many genes within this differentially methylated set were identified to have important roles in extracellular matrix metabolism through gene ontology analysis. The next part of the study involved comparative transcriptome analysis of scar and normal skin fibroblasts using the Affymetrix human gene ST 2.0 array. Using a nominal significance of p<0.05 and a fold change of ±1.5, 0.8% of genes were found to be significantly differentially expressed, with 47% increased and 53% decreased in expression in scar fibroblasts. Gene set enrichment analysis (GSEA) was carried out and identified 507 gene sets that were significantly differentially expressed, using a Mann- Whitney U-test with a p<0.05. The most significant changes were in gene sets related to extracellular matrix production and cell adhesion. Further bioinformatics analysis involved the integration of the methylome and transcriptome data to identify potential gene targets involved in maintaining the scar fibroblast phenotype. Datasets were combined into a single database, and a list of 16 genes of interest was generated. These 16 genes had significantly differentially methylated promoter regions and were differentially expressed. This list of 16 genes was then linked to gene ontologies using the UCSC table browser, and genes with DNA- binding or transcriptional activity were selected. This criteria was met by 4 targets. After extensive review, 2 were selected for further modulation – Forkhead Box F2 (FOXF2), and Mohawk Homeobox (MKX). Both genes have been implicated in collagen production and in fibrosis, although they had not previously been investigated in the skin. The final stage of this work involved using phenotypic assays to validate the pro- fibrotic functions of FOXF2 and MKX genes. Initially a ‘scar-in-a-jar’ model was modified to assess both collagen quantity and alignment (coherency) in collagen matrices produced by scar and normal skin fibroblasts. This modified assay was then used to compare untreated scar and normal skin fibroblasts. In this assay only scar fibroblasts cultured from 1/4 patients secreted an increased quantity and more aligned collagen matrix than the matched normal skin fibroblasts. This is most likely due to the requirement for stimulation of cells with TGFβ for this assay. VIII The assay was then used to determine whether FOXF2 or MKX had an impact on collagen matrix deposition in scar fibroblasts. Using siRNA knockdown of the two target genes and the modified scar-in-a-jar assay it was found that the double knockdown of both MKX/FOXF2 significantly decreased the quantity and coherence of the collagen matrix. This suggests these genes are important in scar matrix maintenance and therefore potential targets to ameliorate scarring. This study provides the first evidence for epigenetic and transcriptome changes in fibroblasts isolated from established normotrophic scars that may underpin long-term maintenance of the aberrant dermal matrix. Two genes identified using an integrative genomic approach affected collagen matrix deposition in vitro. These findings add important new knowledge to the understanding of why scars persist throughout the life of the patient. Further work exploring the role of the target genes in scarring and the mechanisms underlying the changes are required to translate these findings into therapeutic intervention. IX Table of Contents Abstract ................................................................................................................................................... VII Table of Contents ......................................................................................................................................
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