Histone demethylase JMJD3 regulates CD11a expression through changes in histone H3K27 tri-methylation levels in CD4+ T cells of patients with systemic lupus erythematosus

Abstract

Aberrant CD11a overexpression in CD4+ T cells induces T cell auto-reactivity, which is an important factor for systemic lupus erythematosus (SLE) pathogenesis. Although many studies have focused on CD11a epigenetic regulation, little is known about histone methylation. JMJD3, as a histone demethylase, is capable of specifically removing the trimethyl group from the H3K27 lysine residue, triggering target gene activation. Here, we examined the expression and function of JMJD3 in CD4+ T cells from SLE patients. Significantly decreased H3K27me3 levels and increased JMJD3 binding were detected within the ITGAL (CD11a) promoter locus in SLE CD4+ T cells compared with those in healthy CD4+ T cells. Moreover, overexpressing JMJD3 through the transfection of pcDNA3.1-JMJD3 into healthy donor CD4+ T cells increased JMJD3 enrichment and decreased H3K27me3 enrichment within the ITGAL (CD11a) promoter and up-regulated CD11a expression, leading to T and B cell hyperactivity. Inhibition of JMJD3 via JMJD3-siRNA in SLE CD4+ T cells showed the opposite effects. These results demonstrated that histone demethylase JMJD3 regulates CD11a expression in lupus T cells by affecting the H3K27me3 levels in the ITGAL (CD11a) promoter region, and JMJD3 might thereby serve as a potential therapeutic target for SLE.

Keywords: CD11a; CD4+ T cells; H3K27me3; JMJD3; SLE.

Figure 1. Enhanced JMJD3 mRNA and protein expression in SLE CD4⁺ T cells

CD4⁺ T cells were isolated from SLE patients and normal controls. The JMJD3 mRNA and protein expression levels in CD4⁺ T cells were respectively detected using real-time RT-PCR and western blot analysis. (A) Relative JMJD3 mRNA expression levels normalized to β-actin in SLE (n = 15) and healthy controls (n = 15). (B) Relative JMJD3 protein expression levels in SLE (n = 15) compared with that in healthy controls (n = 15). (C, D) The correlation analysis of SLEDAI score and JMJD3 expression status in SLE patients. The data are shown by dotplot. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2. The relationship among H3K27me3 enrichment, JMJD3 enrichment, and CD11a mRNA expression in SLE CD4⁺ T cells

CD4⁺ T cells were isolated from SLE patients and normal controls. H3K27me3 enrichment and JMJD3 enrichment within the CD11a promoter in CD4⁺ T cells was assessed using ChIP and real-time PCR. CD11a mRNA expression levels were measured using real-time PCR. (A, B) Relative H3K27me3 and JMJD3 enrichment within the CD11a promoter in SLE (n = 15) and healthy controls (n = 15). The results were normalized to input DNA (total chromatin) and represent the means of three independent experiments. (C) Relative CD11a mRNA expression levels in SLE (n = 15) and healthy controls (n = 15). (D, E, F) The correlation analysis of H3K27me3 enrichment, JMJD3 enrichment and CD11a mRNA expression levels in SLE CD4⁺ T cells. The data are shown by dotplot. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3. JMJD3 overexpression up-regulates CD11a expression by reducing H3K27me3 enrichment in healthy CD4⁺ T cells

CD4⁺ T cells were isolated from healthy controls (n = 3) and divided into two groups, which were subsequently transfected with pcDNA3.1-JMJD3 and pcDNA3.1-control, respectively. At 48 h after transfection, relative JMJD3 and CD11a protein levels were assessed using western blot analysis and flow cytometry, respectively. Relative JMJD3 and CD11a mRNA expression levels were measured using real-time PCR. Relative JMJD3 binding and H3K27me3 enrichment at the CD11a promoter in CD4⁺ T cells were assessed using ChIP and real-time PCR. All experiments were performed in triplicate. (A, B) Relative JMJD3 mRNA and protein expression levels at 48 h after transfection with pcDNA3.1-JMJD3 and pcDNA3.1-control. β-actin served as an endogenous control. (C, D) Relative JMJD3 binding and H3K27me3 enrichment at the CD11a promoter at 48 h after transfection with pcDNA3.1-JMJD3 and pcDNA3.1-control. (E, F) Relative CD11a mRNA and protein expression levels at 48 h after transfection with pcDNA3.1-JMJD3 and pcDNA3.1-control. The results of CD11a protein expression are expressed as a percentages (%) of CD4⁺CD11a⁺ T cells. (G) Relative IgG production in B cells stimulated by SLE CD4⁺ T cells transfected with pcDNA3.1-JMJD3 or pcDNA3.1-control. The data are shown as the means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4. Down-regulating JMJD3 restores aberrant CD11a expression by increasing promoter H3K27me3 levels in SLE CD4⁺ T cells

CD4⁺ T cells were isolated from SLE patients (n = 3) and divided into two groups, which were subsequently transfected with JMJD3-siRNA and control-siRNA, respectively. At 48 h after transfection, the relative JMJD3 and CD11a protein expression levels were assessed using western blot analysis and flow cytometry, respectively. Relative JMJD3 and CD11a mRNA expression levels were measured using real-time PCR. Relative JMJD3 binding and H3K27me3 enrichment at the CD11a promoter in CD4⁺ T cells were assessed using ChIP and real-time PCR. All experiments were performed in triplicate. (A, B) Relative JMJD3 mRNA and protein expression levels at 48 h after transfection with JMJD3-siRNA and control-siRNA. β-actin served as an endogenous control. (C, D) Relative JMJD3 binding and H3K27me3 enrichment at the CD11a promoter at 48 h after transfection with JMJD3-siRNA and control-siRNA. (E, F) Relative CD11a mRNA and protein expression levels at 48 h after transfection with JMJD3-siRNA and control-siRNA. The results of CD11a protein expression are expressed as percentages (%) of CD4⁺CD11a⁺ T cells. (G) Relative IgG production in B cells stimulated by SLE CD4⁺ T cells transfected with JMJD3-siRNA or control-siRNA. The data are shown as the means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.