The Epigenetic Mechanisms Of RFX1 Regulating CD11a And CD70 Expression In Lupus T Cells

Purpose Previous study has confirmed that aberrant epigenetic modifications play important role in the occurrence and development of systemic lupus erythematosus (SLE). The overexpression of autoimmune-related genes such as CDlla and CD70 is closely related with DNA hypomethylation and histone H3 hyperacetylation in CD4+T cells from SLE patients. Furthermore, we demonstrated that the expression level of RFX1 is down-regulated in SLE CD4+T cells compared with healthy controls. Overexpression of RFX1 in SLE CD4+T cells up-regulates the DNA methylation level, and down-regulates histone H3 acetylation level, and represses mRNA and protein expression of CDlla and CD70, and reverses the self-reactivity of SLE CD4+T cells. Except DNA methylation and histone H3 acethylation,we want to know whether RFX1 is involved in regulating histone methylation status, which have not been reported presently. In this study, we will investigate whether RFX1 regulates the histone H3K9 tri-methylation levels on the promoter of CDlla and CD70 genes in CD4+T cells from SLE patients in order to demonstrate the epigenetic mechanisms in the pathogenesis of SLE.Methods CD4+T cell samples were isolated from 15 SLE patients and 15 healthy controls. H3K9 tri-methylation levels were measured by chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. CD4+T cells were transfected with plasmids using the Human T cell Nucleofector Kit. RFX1 and histone methyltransferase suppressor of variegation 3-9 (Drosophila) homolog 1 (SUV39H1) interaction was determined by co-immunoprecipation (co-IP) and Western blot and immunofluorescence staining. CDlla and CD70 mRNA levels were measured by real-time RT-PCR. The protein level of SUV39H1 was detected by western blot.Results The tri-methylation levels on the promoter regions of CDlla and CD70 were down-regulated in CD4+T cells from SLE patients compared with healthy controls (P<0.01). co-IP experiment and indirect immunolofluorescence staining confirmed that RFX1 interacts with SUV39H1 in CD4+T cells and Jurkat cells. Overexpression of RFX1 in SLE CD4+T cells increased the H3K9 tri-methylation levels of CDlla and CD70 genes promoter(P<0.01), thereby down-regulated the expression of CDlla and CD70(P<0.01). In contrast, down-regulation of RFX1 in normal CD4+T cells decreased the H3K9 tri-methylation levels of CDlla and CD70(P<0.01) and enhanced the expression levels of CDlla and CD70 genes(P<0.01).Conclusion Down-regulation of RFX1 leads to the decreased H3K9 tri-methylation level on the promoter regions of CDl1la and CD70 genes by recruitment of histone methyltransferase SUV39H1. The above contributes to demonstrating the molecluar mechanisms under RFX1 regulating auto-immune related genes CD 11 a and CD70 and provides the important clues for uncovering the epigenetic mechanisms in the pathogenesis of SLE.