Genome-wide DNA Hydroxymethylation In CD4~+T Cells From Patients With Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a female-predominant heterogeneous systemic autoimmune disease characterized by the production of a variety of antinuclear autoantibodies and multiorgan involvement. Although the exact etiology of SLE remains unclear at present, an increasing number of studies have shown that epigenetic factors, especially abnormal DNA methylation patterns in CD4+T cells, play essential roles in the development of this disease. Epigenetics refers to the study of heritable changes in gene function that occur without a change in the DNA sequence, and DNA methylation is the most prevalent and best-described epigenetic modification in the field. It is generally considered that DNA methylation represses the expression of relevant genes, whereas DNA demethylation actually results in transcriptional activation. Our previous work has revealed that certain genes in SLE CD4+T cells, such as CD11a (ITGAL) and CD70(TNFSF7), display abnormally low levels of DNA methylation in their regulatory sequences, which lead to the overexpressions of these genes, inducing excessive production of autoreactive T cells as well as autoantibodies, and therefore causing the autoimmune disease. It is thus clear that aberrant DNA hypomethylation in some specific genes of CD4+T cells serves as a key player in the pathogenesis of SLE. However, mechanisms underlying the abnorml DNA methylation status in SLE CD4+T cells have not been fully elucidated so far.DNA hydroxymethylation is a newly-discovered epigenetic phenomenon where5-methylcytosine (5mC) is further oxidated by Ten-eleven translocation (TET) family proteins into5-hydroxymethylcytosine (5hmC), which has been hailed as the "sixth base" in the genome. Mounting evidence has indicated that DNA hydroxymethylation may function as a regulator of DNA methylation pattern and is also closely related to transcriptional regulation. On the one hand,5hmC could be involved in the process of active or passive demethylation as an intermediate; and on the other hand,5hmC may either remove the biological role of5mC by replacing it, or regulate gene expression on its own through recruiting certain functinal proteins. Being a hot area at the epigenetic frontier, DNA hydroxymethylation has been found to play vital roles in dynamically regulating DNA methylation and transcriptional control, and apparently more important biological messages that5hmC carries are to be discovered. Given our previous findings that abnormal DNA methylation status and transcriptional regulation in CD4+T cells are crucial for the development of SLE, we assume that DNA hydroxymethylation is very likely to contribute to the pathogenesis of SLE. Since currently there has not been any information on the role of DNA hydroxymethylation in SLE CD4+T cells, we intend to first investigate the genomic DNA hydroxymethylation status in CD4+T cells from SLE patients and normal individuals by means of hMeDIP-chip to acquire the differential DNA hydroxymethylation profile. Subsequently, genes with discrepant5hmC levels are validated in CD4+T cells from SLE patients as well as healthy controls, and then the correlation between DNA hydroxymethylation and gene expression is analyzed to further explore whether and how DNA hydroxymethylation may exert an influence on the autoimmune responses in SLE. These studies would hopefully deepen our understanding of the pathogenesis of SLE and probably provide a new theoretical basis for more effective diagnosis and treatment of this complicated disease. Part I Genome-wide mapping of DNA hydroxymethylation in CD4+T cells from patients with systemic lupus erythematosusⅠ. DNA hydroxymethylation pattern in CD4+T cells from patients with systemic lupus erythematosusObjective:To investigate the genomic DNA hydroxymethylation pattern in CD4+T cells from patients with systemic lupus erythematosus and normal individuals, uncovering the role of DNA hydroxymethylation in the pathogenesis of systemic lupus erythematosus.Methods:CD4+T cells were isolated from peripheral blood samples of5SLE patients and5healthy controls by Ficoll-Hypaque density gradient centrifugation followed by magnetic bead sorting. DNA hydroxymethylation pattern was determined by hMeDIP-chip.Results:Through hMeDIP-chip, the genomic DNA hydroxymethylation profile was obtained. Altogether3083genes exhibited aberrant DNA hydroxymethylation modifications in their promoters, mostly high-CpG-density promoters, and3030of these genes showed higher levels of5hmC in SLE patients than in controls. In total,4646genes presented discrepant DNA hydroxymethylation status in CpG islands, mainly promoter islands, and4643genes were hyperhydroxymethylated in SLE patients. Most of these genes with differential hydroxymethylation pattern, according to GO analysis, were associated with transcription regulation, metabolic process, development etc, and were linked to signaling pathways in cancer and cell proliferation on the basis of KEGG analysis.Conclusion:DNA hyperhydroxymethylation status was obversed in CD4+T cells from patients with systemic lupus erythematosus, which may be involved in the pathogenesis and development of systemic lupus erythematosus.Ⅱ. DNA hydroxymethylation status in the promoters of PPARG, MALT1and IRF2BP2in SLE CD4+T cellsObjective:To explore the DNA hydroxymethylation status in the promoters of PPARG, MALT1and IRF2BP2in SLE CD4+T cells and thus validate the results of hMeDIP-chip in CD4+T cells from SLE patients and healthy individuals.Methods:Peripheral blood mononuclear cells from10SLE patients and10normal controls were obtained by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. DNA was extracted using cell genomic DNA isolation kit. Hydroxymethylated DNA immunoprecipitation was applied to measure the levels of5hmC in the promoters of gene PPARG, MALT1and IRF2BP2.Results:DNA hydroxymethylation were significantly increased in the promoter regions of PPARG, MALT1and IRF2BP2relative to controls (11.695±2.292vs5.682±1.645, p=0.000;1.838±0.562vs0.989±0.326, p=0.001;0.194±0.049vs0.124±0.031, p=0.001).Conclusion:According to hydroxymethylated DNA immunoprecipitation, levels of5hmC were significantly higher in promoters of gene PPARG, MALT1and IRF2BP2in SLE CD4+T cells than in normal controls, which are in agreement with the results of hMeDIP-chip. Part Ⅱ Gene expression of PPARG MALT1and IRF2BP2in SLE CD4+T cells and its relationship with DNA hydroxymethylationObjective:To investigate the expression of PPARG, MALT1and IRF2BP2in CD4+T cells from patients with SLE and to analyze the correlation between DNA hydroxymethylation pattern and gene expression.Methods:RNA was isolated from CD4+T cells of13SLE patietns and13healthy controls using TRIzol reagent. PPARG, MALT1and IRF2BP2mRNA levels were detected by reverse transcription real-time PCR and correlation between DNA hydroxymethylation status and gene expression was then analyzed.Results:Compared to normal controls, mRNA expression levels of PPARG and MALT1were both significantly higher in CD4+T cells from SLE patients(p=0.001; p=0.001). A rising trend for IRF2BP2mRNA expression was also observed in SLE patients but with no significant difference(p=0.332). Correlation analysis showed that5hmC levels in PPARG and MALT1gene promoters were positively correlated with their mRNA expression levels (PPARG:R=0.713, p=0.021; MALT1:R=0.684, p=0.029).Conclusion:PPARG and MALT1mRNA levels were significantly increased in SLE CD4+T cells, and there was a positive correlation between the gene expression and DNA hydroxymethylation in the promoters, which suggests that enhanced DNA hydroxymethylation in PPARG and MALT1promoters may contribute to the overexpression of the genes in SLE CD4+T cells.