Increased Set1 binding at the promoter induces aberrant epigenetic alterations and up-regulates cyclic adenosine 5'-monophosphate response element modulator alpha in systemic lupus erythematosus

Abstract

Background: Up-regulated cyclic adenosine 5'-monophosphate response element modulator α (CREMα) which can inhibit IL-2 and induce IL-17A in T cells plays a critical role in the pathogenesis of systemic lupus erythematosus (SLE). This research aimed to investigate the mechanisms regulating CREMα expression in SLE.

Results: From the chromatin immunoprecipitation (ChIP) microarray data, we found a sharply increased H3 lysine 4 trimethylation (H3K4me3) amount at the CREMα promoter in SLE CD4+ T cells compared to controls. Then, by ChIP and real-time PCR, we confirmed this result. Moreover, H3K4me3 amount at the promoter was positively correlated with CREMα mRNA level in SLE CD4+ T cells. In addition, a striking increase was observed in SET domain containing 1 (Set1) enrichment, but no marked change in mixed-lineage leukemia 1 (MLL1) enrichment at the CREMα promoter in SLE CD4+ T cells. We also proved Set1 enrichment was positively correlated with both H3K4me3 amount at the CREMα promoter and CREMα mRNA level in SLE CD4+ T cells. Knocking down Set1 with siRNA in SLE CD4+ T cells decreased Set1 and H3K4me3 enrichments, and elevated the levels of DNMT3a and DNA methylation, while the amounts of H3 acetylation (H3ac) and H4 acetylation (H4ac) didn't alter greatly at the CREMα promoter. All these changes inhibited the expression of CREMα, then augmented IL-2 and down-modulated IL-17A productions. Subsequently, we observed that DNA methyltransferase (DNMT) 3a enrichment at the CREMα promoter was down-regulated significantly in SLE CD4+ T cells, and H3K4me3 amount was negatively correlated with both DNA methylation level and DNMT3a enrichment at the CREMα promoter in SLE CD4+ T cells.

Conclusions: In SLE CD4+ T cells, increased Set1 enrichment up-regulates H3K4me3 amount at the CREMα promoter, which antagonizes DNMT3a and suppresses DNA methylation within this region. All these factors induce CREMα overexpression, consequently result in IL-2 under-expression and IL-17A overproduction, and contribute to SLE at last. Our findings provide a novel approach in SLE treatment.

Keywords: CREMα; DNA methylation; DNMT3a; H3K4me3; Set1; Systemic lupus erythematosus.

Fig. 1

ChIP microarray analysis of H3K4me3 enrichments in SLE and control CD4⁺ T cells. a ChIP microarray panels showing relative H3K4me3 enrichments at various gene promoters in CD4⁺ T cell lysates pooled from five healthy controls (left-hand panel) and five patients with SLE (right-hand panel). Anti-H3K4me3 antibody-precipitated DNA and total DNA (input) were respectively labeled with Cy5 (red) and Cy3 (green), and samples were subsequently cohybridized onto microarray panels. Each individual dot shows the Cy3/Cy5 ratio representing relative H3K4me3 enrichment at a specific gene promoter. The CREMα promoter dot (indicated by a blue line) is located in the sixteenth column, seventh row. b Relative H3K4me3 enrichment at the CREMα promoter in SLE and control CD4⁺ T cells, quantified from the results shown in (a)

Fig. 2

H3K4me3 enrichment at the CREMα promoter in SLE and control CD4⁺ T cells. a Relative H3K4me3 enrichment within the CREMα promoter in SLE and healthy CD4⁺ T cells was assessed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). b Positive correlation between the levels of H3K4me3 and CREMα mRNA in SLE CD4⁺ T cells. All reactions were run in triplicate

Fig. 3

Set1 and MLL1 binding at the CREMα promoter in SLE and control CD4⁺ T cells. a, b Relative levels of Set1 (a) and MLL1 (b) binding within the CREMα promoter region in SLE and healthy CD4⁺ T cells were analyzed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). c Positive correlation between Set1 promoter binding and H3K4me3 level in SLE CD4⁺ T cells. d Positive correlation between Set1 promoter binding and CREMα mRNA level in SLE CD4⁺ T cells. All experiments were repeated three times

Fig. 4

Effects of Set1 down-regulation on CD4⁺ T cells from SLE patients. a, b Relative Set1 and CREMα protein levels were evaluated by western blotting analysis of SLE CD4⁺ T cells 72 h after transfection with Set1-siRNA or control-siRNA. β-actin served as an endogenous control. c, d Relative Set1 (c) and H3K4me3 (d) levels within the CREMα promoter in SLE CD4⁺ T cells transfected with Set1-siRNA or control-siRNA were confirmed by ChIP and real-time PCR 72 h after transfection. Results were normalized to input DNA (total chromatin). e, f Relative IL-2 (e) and IL-17A (f) concentrations in the supernatants of SLE CD4⁺ T cells were measured by ELISA 72 h after transfection with Set1-siRNA or control-siRNA. g Relative DNA methylation level at the CREMα promoter in SLE CD4⁺ T cells transfected with Set1-siRNA or control-siRNA was assayed by MeDIP and real-time PCR 72 h after transfection. h,i, j Relative enrichments of H3ac (h), H4ac (i), and DNMT3a (j) within the CREMα promoter region in SLE CD4⁺ T cells were tested by ChIP and real-time PCR 72 h after transfection with Set1-siRNA or control-siRNA. Results were normalized to input DNA (total chromatin). All experiments were performed in triplicate

Fig. 5

Relationships between H3K4me3, DNA methylation, and DNMT3a. a Negative correlation between H3K4me3 enrichment and DNA methylation level at the CREMα promoter in SLE CD4⁺ T cells. b Relative level of DNMT3a binding within the CREMα promoter region in CD4⁺ T cells from 20 SLE patients and 20 healthy controls were detected by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). All data are representative from three independent experiments. c Negative correlation between H3K4me3 enrichment and DNMT3a binding at the CREMα promoter in SLE CD4⁺ T cells