Lugar: Canchas deportivas

Results from microarray analysis of intact colon in this experiment are consistent with other data in this and similar models indicating the suitability of the samples collected in this experiment for studying epithelial cell function in colitis. The findings from this study indicate that when analysed at the pathway level, intact colon is an appropriate tissue in which to examine gene expression changes in the mucosa in fully-developed colitis, as it produces similar gene expression profiles to the epithelium alone. They do indicate, however, that studying epithelial cell function, as opposed to intact colon function, may be more relevant when studying the earlier stages of inflammation because isolation of specific cells may improve sensitivity or the ability to detect early changes in cell function that may lead to the development of inflammation.

The results presented here indicate that the study of gene expression profiles in specific cell types could be of particular benefit in the early stages of colon inflammation to help elucidate the early changes in mucosal function, but perhaps less useful in fully developed inflammation. The expression of genes can be controlled by epigenetic mechanisms, and epigenetic mechanisms such as DNA methylation have been implicated in the development of IBD. To further investigate mucosal changes in the early stages of inflammation, in particular those that could influence gene expression, intact colon samples from the time-course experiment were used to measure DNA methylation in the early and late stages of inflammation development (Chapter 4).

Chapter 4: Global and gene-specific DNA methylation in the Il10-/- mouse colon

Chapter 4: Global and gene-specific DNA methylation in the Il10-/- mouse colon

141

4

Global and gene-specific DNA methylation is

altered in the Il10

mouse colon in early and

late inflammation

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142

4.1

Abstract

The involvement of epigenetic mechanisms in the pathogenesis of IBD is ill-defined, but may be an important factor in understanding the pathogenesis of colitis. The first objective of this study was to measure global DNA methylation using HPLC to test the hypothesis that global methylation levels would be decreased in Il10-/- mice at 12 weeks

of age compared to C57BL/6J mice. The second objective was to measure the methylation levels of selected regions of the Tap2, Stat1 and Ppara genes using

MALDI-TOF mass spectrometry to test the hypothesis that changes observed in Stat1, Tap2 and Ppara gene expression in the Il10-/- mouse model are associated with

differential methylation of CpG sites within key regulatory regions of these genes. Global methylation was increased in Il10-/- mice at 6 weeks of age compared to Il10-/-

mice at 12 weeks of age and C57BL/6J mice, and methylation levels of specific CpG sites within an intronic region, but not the proximal promoter region, of the Stat1 gene

were reduced in Il10-/- mice at 12 weeks of age compared with C57BL/6J mice at 12

weeks of age. This study showed that DNA methylation levels were altered in the Il10-/-

mouse model of IBD and these alterations correlated with changes in the expression of genes that occur in inflammation.

4.2

Introduction

Epigenetic mechanisms are implicated in inflammatory conditions such as IBD. Genetic variation alone accounts for only approximately 25% of IBD heritability [355], indicating that epigenetic mechanisms might contribute to heritability to a large extent. The different incidence of IBD in monozygotic twins is supporting evidence for the role of epigenetic mechanisms in the development of colitis, as are the alterations in global and gene-specific DNA methylation that have been detected in mucosal biopsies of IBD patients [142, 143] and in a mouse model of colitis [145].

Global hypomethylation accompanied by gene-specific hypermethylation is known to occur in both cancer and aging [356]. In a DSS model of colitis, three CpG islands are involved in induction of aberrant DNA methylation in colon epithelial cells, and their methylation levels were increased from 8 weeks after DSS treatment until the development of colon cancers 7 weeks later [357]. Inflammation triggered by the DSS treatment appeared to be responsible for induction of methylation [357], suggesting that

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inflammation-associated changes in DNA methylation are also likely to occur in other models of colitis.

Peroxisome proliferator-activated receptor alpha (Ppara), signal transducer and

activator of transcription 1 (Stat1) and the transporter 2, ATP-binding cassette, sub-

family B (Tap2) genes were shown to have altered expression levels during

inflammation in the Il10-/- mouse model of IBD [110, 118] (Chapter 3) and contain CpG

islands, suggesting their potential for epigenetic regulation by DNA methylation. The

Ppara gene has been identified as a central (hub) gene in the inoculated Il10-/- mouse

model [118]. Ppara expression is known to be modified by DNA methylation.

Methylation at an intergenic CpG island 50 kb upstream of Ppara, a region considered

likely to be an enhancer for Ppara (chr15: 85,514,715-85,514,920), is associated with decreased Ppara gene expression [358]. Increased Ppara mRNA expression is

associated with decreased CpG methylation in the Ppara promoter in the liver [359].

Alterations in methylation state may thus influence the mRNA levels of Ppara, and

these methylation changes may be modulated by colitis.

The Stat1 gene encodes a protein from the STAT family [360]. STAT proteins are

phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. STAT1 can be activated by various ligands including the inflammation- associated cytokines IFNγ and IL6 and mediates the expression of a variety of genes

important for cell viability in response to different cell stimuli and pathogens. STAT1 protein activation and expression is increased in the intestinal mucosa in IBD, although more so in CD than UC [348]. Glucocorticoids, a common therapy during active phases of IBD, inhibit phosphorylation and activation of STAT1, suggesting that inhibition of STAT1 may be an important part of the anti-inflammatory effect of glucocorticoids [348]. Changes in methylation patterns of the Stat1 gene associated with altered STAT1

protein expression have been observed in a cancer study [360].

The Tap2 gene encodes a membrane-associated protein member of the MDR/TAP

subfamily, part of the superfamily of ATP-binding cassette (ABC) transporters. The Tap2 protein is involved in antigen presentation, forming a heterodimer with Tap1 in order to transport peptides from the cytoplasm to the endoplasmic reticulum. The Tap2

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the response of IBD patients to steroid therapy varies with some Tap2 polymorphisms

[361]. Transcription of the Tap2 gene can be increased by demethylation of the

promoter region induced by treatment with 5-azacytidine, an inhibitor of DNA methyltransferase [362].