Alternatively spliced MEFV transcript lacking exon 2 and its protein isoform pyrin-2d implies an epigenetic regulation of the gene in inflammatory cell culture models

Abstract

The function of gene body DNA methylation in alternative splicing, and its relation to disease pathogenesis is not fully elucidated. The gene for familial Mediterranean fever (MEFV) encodes the pyrin protein and contains a 998 bp CpG island, covering the second exon, which is differentially methylated in FMF patients compared to healthy controls. Our further observation of increased exon 2-spliced MEFV transcript in leukocytes of FMF patients provoked us to test the role of exon methylation in alternative splicing using inflammatory cell culture models. First, in vitro exon methylation triggered an increased level of exon 2 exclusion using a splicing cassette in a promyelocytic leukemia cell line (HL-60). HL-60 cells subjected to methylating and demethylating agents, as well as cells differentiated to neutrophil-like cells, exhibited different levels of spliced/unspliced transcripts. We observed increased levels of spliced transcripts in neutrophil-like (p = 0.0005), activated (p = 0.0034) and methylated cells (p < 0.0001), whereas decreased levels in demethylated cells (p = 0.0126) compared to control untreated HL-60 cells. We also showed that the protein isoform of pyrin lacking the exon 2 has an adverse subcellular localization in neutrophil-like cells. Therefore, it remains in the cytoplasm rather than the nucleus. This may point to an epigenetic involvement in an important inflammatory gene.

Genetic and Epigenetic Regulation of MEFV Gene and Their Impact on Clinical Outcome in Auto-Inflammatory Familial Mediterranean Fever Patients

“…In agreement with previous studies in the Egyptian population [38,40], the heterozygous genotype was more predominant in our study, followed by homozygous and compound heterozygous. Additionally, no significant difference was shown concerning the association of the MEFV exon 2 methylation% and the pyrin level with genotyping status among FMF patients, which agrees with previous studies [10,26] and may be due to the MEFV exon 2 splicing [15,16].…”

“…In addition, it is involved in NK cell differentiation, where hypo-methylation of promoter regions of killer Ig-like receptor and CD94/NK group 2 member A results in NK cells' maturation and transcription up-regulation [9]. DNA methylation could reduce gene expression by either blocking initiation sites of transcription or interacting with nucleosomes that lead to hetero-chromatinization [10,15]. On the other hand, DNA methylation could enhance gene expression through alternative promoters or by blocking insulators [10].…”

“…In addition, the DNA binding capacity of many transcription factors is affected by the methylation of their binding sequences [16]. Although hyper-methylation of CpG islands, particularly in the promoter region, is highly correlated with transcriptional silencing [10,15,17], it was also reported that methylation in the body of genes acts as a positive regulator of transcription [10].…”

“…MEFV gene methylation and pyrin levels are considered as potential factors in the FMF pathogenesis [10], with previous studies showing controversial results [10,31,32]. Methylation is involved in the FMF pathogenesis through MEFV exon 2 splicing and protein aberrant localization, irrespective of the presence of the MEFV pathogenic variants [15,16]. The fully spliced MEFV second exon shows the highest expression, in comparison to the full-length exon [15,16,33], as the majority of the CpG-islands of 568 bp (41/66) are spanning MEFV gene complete exon 2 [33].…”

Understanding the Relevance of DNA Methylation Changes in Immune Differentiation and Disease

“…The MEFV exon 2 was found to be marginally more methylated in peripheral leukocytes from FMF patients than in healthy controls, its expression level being negatively correlated with methylation [143]. These results have been validated in an in vitro model, which showed aberrant methylation in exon 2, which produces an increase in an aberrant spliced form of pyrin that also occurs at higher levels in FMF patients than in healthy donors [116,117]. Since this analysis of peripheral blood mononuclear cells (PBMC) shows differences in methylation, the results should be interpreted with caution.…”