Elsevier

Journal of Autoimmunity

Volume 54, November 2014, Pages 127-136
Journal of Autoimmunity

DNA methylation and mRNA and microRNA expression of SLE CD4+ T cells correlate with disease phenotype

https://doi.org/10.1016/j.jaut.2014.07.002Get rights and content

Highlights

  • We characterized the DNA methylome and the transcriptome in CD4+ T cells of SLE.

  • Integrated analysis of our data highlights the epigenetic mechanism in SLE.

  • The specific DNA methylation changes are correlated with clinical phenotype of SLE.

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease well known for its clinical heterogeneity, and its etiology secondary to a cross-talk involving genetic predisposition and environmental stimuli. Although genome-wide analysis has contributed greatly to our understanding of the genetic basis of SLE, there is increasing evidence for a role of epigenetics. Indeed, recent data have demonstrated that in patients with SLE, there are striking alterations of DNA methylation, histone modifications, and deregulated microRNA expression, the sum of which contribute to over-expression of select autoimmune-related genes and loss of tolerance. To address this issue at the level of clinical phenotype, we performed DNA methylation, mRNA and microRNA expression screening using high-throughput sequencing of purified CD4+ T cells from patients with SLE, compared to age and sex matched controls. In particular, we studied 42 patients with SLE and divided this group into three clinical phenotypes: a) the presence of skin lesions without signs of systemic pathology; b) skin lesions but also chronic renal pathology; and c) skin lesions, chronic renal pathology and polyarticular disease. Interestingly, and as expected, sequencing data revealed changes in DNA methylation in SLE compared to controls. However, and more importantly, although there were common methylation changes found in all groups of SLE compared to controls, there was specific DNA methylation changes that correlated with clinical phenotype. These included changes in the novel key target genes NLRP2, CD300LB and S1PR3, as well as changes in the critical pathways, including the adherens junction and leukocyte transendothelial migration. We also noted that a significant proportion of genes undergoing DNA methylation changes were inversely correlated with gene expression and that miRNA screening revealed the existence of subsets with changes in expression. Integrated analysis of this data highlights specific sets of miRNAs controlled by DNA methylation, and genes that are altered by methylation and targeted by miRNAs. In conclusion, our findings suggest select epigenetic mechanisms that contribute to clinical phenotypes and further shed light on a new venue for basic SLE research.

Introduction

There is increasing evidence that epigenetic modifications play a critical role in the pathogenesis of SLE [1], [2], [3]. Indeed, whereas a decade ago, genomic analysis was proposed as the key to understanding the relationships between susceptibility and environmental factors, it now appears that environmentally-induced epigenetics may also play a critical role in autoimmune immunopathology. This is exemplified not only in SLE, but in several other systemic as well as organ-specific autoimmune diseases [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Such epigenetic modifications include DNA methylation, histone modifications and altered microRNAs (miRNA) expression. Of these, DNA methylation has been the best studied epigenetic feature in patients with SLE. A repressive role for methylation of dC bases in CpG sites located at or near the transcription start sites of genes is well established, particularly in CpGs that are clustered as CpG islands [3]. Methylation of CpGs located in other regions such as gene bodies may also be involved in gene regulation [20]. In contrast to gene regulation, miRNAs, a class of endogenous, small, non-coding regulatory RNA molecules, modulate the expression of target genes at the posttranscriptional level and therefore implicated in diverse cellular processes [21].

Previous work has demonstrated that there are exists hypomethylation of global genomic DNA in several autoimmune related gene promoters in isolated T cells from SLE [1], [2], [3]. Genes with aberrant DNA demethylation identified in lupus T cells include ITGAL [22], CD70 [23], PRF1 [24] and the X chromosome gene CD40LG [25]. Recently, DNA methylation profiling in SLE has been based on Illumina methylation arrays, which identifies genes with differential methylation [26], [27]. Deregulated miRNAs may also influence gene expression and subsequent critical pathways involved in the effector mechanisms of immune-mediated pathology [28], [29], [30]. Interestingly, the aberrant expression of some miRNAs has been associated with SLE disease activity. For example, overexpression of miR-126 and miR-21 induces autoreactivity of CD4+ T cells; down-regulation of miR-142-3P/5P leads to activation of CD4+ T cells and B cell hyperstimulation [31], [32], [33]. These aberrantly expressed miRNAs also reflect methylation-dependent regulation in CD4+ T cells of SLE [31], [33].

In this report we analyzed DNA methylation distribution and variation in genome-wide and mRNA and microRNA transcription profiling by deep-sequencing of CD4+ T cells from SLE patients with different clinical manifestions compared to healthy controls. We also have performed the first integrated comparison of DNA methylation and miRNA expression data, together with mRNA expression data (Fig. 1). Our data demonstrate significant differences of methylation patterns according to clinical phenotype. Further, our data provide a unique resource for identifying epigenetic markers that may be useful in the diagnosis of SLE.

Section snippets

Sample collection

This study was approved by the Human Ethics Committee of Second Xiangya Hospital of Central South University, and written informed consent was obtained from all subjects. SLE patients (mean age 30 ± 8 yrs) were recruited from outpatient clinics in the Second Xiangya Hospital of Central South University. All patients fulfilled at least 4 of the SLE classification criteria of the American College of Rheumatology [34]. SLE patients in this study were divided into three groups according to

DNA methylation profiling of SLE samples with diverse clinical manifestations and normal controls

We isolated genomic DNA from the CD4+ T cells of 4 SLE patients with only skin involvement, 4 SLE patients with skin and renal involvement, 4 SLE patients with skin, renal and joint involvement and 4 normal controls, then put equal amounts of genomic DNA of 4 individuals into one pool for each group. We generated DNA methylomes for 4 seperate pools using MeDIP-seq (Fig. 1). We generated an average of 98 million reads per cohort pool, of which 61.12%–68.52% could be uniquely aligned to the human

Discussion

In this study we identified novel dysregulated genes in SLE CD4+ T cells at the DNA methylation, mRNA and miRNA expression levels. Using high-throughput seqencing and integrated analysis of the DNA methylation, miRNA expression and mRNA expression data we have established the profiling of DNA methylation, mRNA and miRNA in the same samples to investigate the complexity of gene dysregulation in the context of this disease. In this study, CD4+ T cells were isolated from the peripheral blood using

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81220108017, No. 81373205 and No. 81270024), the Hunan Provincial Natural Science Foundation of China (14JJ1009), the programs of Science-Technology Commission of Hunan province (2013FJ4202, 2011FJ2007 and 2012WK3046) and the National Key Clinical Speciality Construction Project of National health and Family Planning Commission of the People's Republic of China.

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    These authors contributed equally to this work.

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