DNA methylation profiles of cytokine and transcription factor genes in immune cells

Abstract

The past years have seen an enormous scientific interest in the field of DNA methylation. Numerous studies have documented the functional significance of DNA methylation in the regulation of gene expression and the maintenance of local chromatin structure. A large body of evidence underlines the implication of aberrant DNA methylation in nearly all types of human cancers.<br /> The focus of the current work was to investigate the role of DNA methylation in the human immune system at candidate immune-relevant genes by emphasising two key biological aspects. <br /> First, the role of DNA methylation as an epigenetic mark, possessing the function to memorize the expression phenotype, was analyzed within key cytokine genes. Large-scale methylation profiling experiments of these genes were carried out in designated CD4+ memory T-cells subsets. DNA methylation in the IL10 gene, encoding the anti-inflammatory cytokine IL-10 and in the IFNg gene, encoding the pro-inflammatory cytokine IFN-g were of special interest due to the key role of these molecules in the immune response.<br /> Cell type specific differentially methylated regions were found in the genes encoding the cytokines IL-13 and IL-4 in DNA sequences that were previously shown to harbor regulatory elements. This finding suggests a role for DNA methylation in the expression regulation of these genes.<br /> As for the gene encoding the cytokine IFN-g, low levels of DNA-methylation were measured in human ex vivo IFN-g expressing cells at several CpGs located proximal to the transcription start site and within an enhancer element 5.0 kb upstream. At the same time, IFN-g non-expressing cells were shown to be strongly upmethylated at the same CpG sites. The specific role of this methylation pattern as a heritable epigenetic mark for rapid expression induction was demonstrated through experiments carried out in vitro. Further in vitro cultivation experiments were performed to investigate the dynamics between site-specific demethylation and expression induction in the IFNg gene.<br /> In contrast, DNA methylation does not seem to play any role in the regulation of the IL10 gene. With the exception of two CpGs located in the proximal promoter, slightly demethylated in IL-10 producing cells, no differential methylation was measured between IL-10 expressing and non-expressing cells. The two questionable CpGs do not harbour the epigenetic memory mark for expression since in vitro cultivation of IL-10+ cells under expansive conditions rapidly results in the loss of IL-10 expression without measurable methylation changes at these sites. Further epigenetic analysis at the level of histones revealed covalent modifications like hyperacetylation and trimethylation that are associated with an open chromatin status in IL-10 producing cells as compared to their non-expressing counterparts. These findings led to the conclusion, that the IL10 gene is excluded from the functional cytokine memory in human Th cells, preventing the generation of memory cells with an inherited program to secrete IL-10, possibly to ensure a limited effect of IL-10 in downregulation of adaptive pathogen-specific immunity.<br /> As a second biological aspect, variations in the level of DNA methylation during development and maturization of the B- and T-cell lineage were monitored in key transcription factors. A set of cell types, resembling major steps of the human hematopoietic development were employed for this analysis.<br /> It could be shown that variations in the local methylation of several transcription factor genes (SPI1, TCF7, c-maf, Etv5, TBX21 and GATA3) is an accompanying feature during development and lineage maturation. For some genes (SPI1, Eomes and Etv5), an inverse correlation between promoter methylation and the respective gene expression could be demonstrated, suggesting an implication for DNA methylation in the expression of these genes.<br /> Some of the hereby-discovered regions of differential methylation between individual cell types were tested for their use in diagnostic applications such as rheumatoid arthritis, endometriosis and lymphoma.<br /> Specific differentially methylated CpGs in the IFNg gene were successfully used to measure balance shifts of T-helper cell compartments in the peripheral blood of patients with rheumatoid arthritis. By means of the same marker, no differential methylation was found in the endometrium of women with endometriosis as compared to non-endometriosis controls.<br /> Methylation information from specific factors (SPI1, TCF7, c-maf and eomes) was applied to show aberrant methylation patterns in a B-cell malignancy of the lymph node when compared to healthy tissue. Thus, transcription factors may represent a novel class of aberrantly methylated genes in hematopoietic cancer. In addition, the used candidate gene approach is a valid one for identifying novel tumor biomarkers.