Down-regulation Of HPK1by Aberrant Histone Methylation In CD4~+T Cells And Its Role In The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease which involves multiple organs and systems. It is characterized by T cell overactivation and the overproduction of autoantibodies against multiple self antigens. Although the molecular mechanisms that initiate these autoimmune responses in SLE remain unclear, in recent years, accumulating evidence has demonstrated that epigenetic alterations in the promoters of certain immune-related genes play critical roles in the onset and progression of SLE.The term "epigenetics" refers to stable and heritable changes in gene expression that are not related to changes in the DNA sequence. The major mechanisms of epigenetic regulation include DNA methylation, histone modifications, chromatin modifications, noncoding RNA regulation, and so forth. Now considerable interest in epigenetics of SLE has been focused on DNA methylation. However, studies on histone modifications, especially those about histone modifications in regulatory sequences of special genes in SLE are very limited. Among histone modifications, histone H3lysine27trimethylation (H3K27me3) is a hallmark of gene silencing. It is well known that jumonji domain containing3(JMJD3, a histone demethylase) and enhancer of zeste homolog2(EZH2, a histone methyltransferase) can both regulate H3K27me3level.Hematopoietic progenitor kinase1(HPK1) is a mammalian Ste20-related serine/threonine protein kinase. It belongs to the germinal center kinase (GCK) family, and it can negatively regulate TCR signaling and T cell-mediated immune responses.Our group previously used ChIP-on-chip to compare H3K27me3enrichment at gene promoters of CD4+T cells from5patients with SLE and5healthy controls. Our data demonstrated that H3K27me3level within the HPK1promoter region of SLE CD4+T cells was markedly higher than that of healthy controls. As we have mentioned, H3K27me3can inhibit gene transcription, therefore, we speculated that increased H3K27me3level within the HPK1promoter may repress HPKl expression in SLE CD4+T cells. Since HPK1is a negative regulator of TCR signaling and T cell-mediated immune responses, we hypothesized one of the pathogenesises of SLE as follows:at the HPK1promoter, H3K27methyltransferase overexpresses, or H3K27demethylase is reduced, subsequently H3K27me3within the HPK1promoter is up-regulated. This inhibits HPK1expression, leading to abnormal T cell reactivity and ultimately contributing to autoimmunity in SLE. To prove the hypothesis, we used the following approaches:the first part, to research the role that HPK1plays in SLE pathogenesis:(1) To measure the HPK1expression in CD4+T cells from normal controls and SLE patients;(2) to examine the effects on cell proliferation and productions of IFNy and IgG which are brought by down-regulating HPK1in normal CD4+T cells, and up-regulating HPK1in SLE CD4+T cells. The second part, to investigate the mechanism of aberrant histone methylation that down-regulates HPK1expression in SLE CD4+T cells:(1) To detect H3K27me3enrichment at the HPK1promoter in CD4+T cells from normal controls and SLE patients;(2) to test the levels of JMJD3and EZH2at the HPK1promoter in normal and SLE CD4+T cells;(3) to examine the effect on HPK1expression that is brought by oppositely regulating EZH2or/and JMJD3binding at the HPK1promoter in normal and SLE CD4+T cells. These studies would further reveal the role of HPK1plays and the mechanism of abnormal epigenetic alterations that regulates HPK1in SLE, thereby providing a theoretical basis for more effective therapy of SLE.Part I Expression of HPK1in CD4+T cells from SLE patients and the role of HPK1in autoimmune responsesSection I Expression of HPKl in CD4+T cells from SLE patientsObjective:To measure the HPK1expression in SLE CD4+T cells and the relationship between HPK1expression and SLE disease activity.Methods:Peripheral blood mononuclear cells (PBMCs) from15healthy controls and15SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. HPK1mRNA levels were determined by one step real-time RT-PCR, and HPK1protein levels were examined by western blotting.Results:Compared to normal controls, HPK1mRNA levels were significantly decreased in CD4+T cells from SLE patients (P=0.002); relative to normal CD4+T cells, HPK1mRNA levels were significantly reduced both in CD4+T cells from untreated patients and drug-treated patients (P=0.002; P=0.004); and there was no significant difference in HPK1mRNA levels between untreated patients and drug-treated patients (P=0.482); in addition, HPK1mRNA levels were negatively correlated with disease activity in SLE patients as measured by SLEDAI (R=-0.731, P=0.002); and HPK1protein levels were also significantly lower in SLE CD4+T cells (P<0.001); compared to normal CD4+T cells, HPK1protein levels were significantly reduced both in CD4+T cells from untreated patients and drug-treated patients (P<0.001; P<0.001); and there was no significant difference in HPK1protein levels between untreated patients and drug-treated patients (P=0.712).Conclusion:HPK1expressions were significantly decreased in CD4+T cells from SLE patients, which negatively correlate with disease activity. These data suggest that HPK1is involved in the development of SLE. Section Ⅱ Inducing norma1CD4+T cells autoreactivity by down-regulating HPK1expressionObjective:To examine whether down-regulating HPK1can inducing normal CD4+T cells autoimmune responses.Methods:PBMCs from3healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells and CD19+B cells were then isolated by positive selection using magnetic beads. HPK1-siRNA or control siRNA was transfected into normal CD4+T cells by transient electroporation. HPK1protein levels were examined by western blotting. Cell proliferations were evaluated with MTT assay. Detection of IFNy and IgG levels were performed by ELISA.Results:The HPK1protein levels were significantly down-regulated in normal CD4+T cells transfected with HPK1-siRNA (P=0.001), and cell proliferation (P=0.012), productions of IFNy (P=0.008) and IgG (P=0.005) all significantly increased。Conclusion:Inhibiting the expression of HPK1can induce autoreactivity in normal CD4+T cells. Section Ⅲ Inhibiting SLE CD4+T cells autoreactivity by overexpressing HPK1Objective:To examine whether overexpressing HPK1can correct or reverse SLE CD4+T cells autoimmune responses.Methods:PBMCs from3SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells and CD19+B cells were then isolated by positive selection using magnetic beads. HPK1-expressing plasmid or control plasmid was transfected into SLE CD4+T cells by transient electroporation. HPK1protein levels were examined by western blotting. Cell proliferations were evaluated with MTT assay. Detection of IFNy and IgG levels were performed by ELISA.Results:The HPK1protein levels were significantly up-regulated in SLE CD4+T cells transfected with HPK1-expressing plasmid (P=0.007), and cell proliferation (P=0.009), productions of IFNy (P=0.005) and IgG (P=0.003) all significantly decreased。Conclusion:Overexpression of HPK1can reduce autoreactivity in SLE CD4+T cells. Part Ⅱ The mechanism of aberrant histone methylation that down-regulates HPK1expression in SLE CD4+T cellsSection I H3K27me3enrichment at the HPK1promoter in CD4+T cells from SLE patientsObjective:To measure H3K27me3enrichment at the HPK1promoter in CD4+T cells from SLE patients and the relationship between H3K27me3enrichment and HPK1expression.Methods:Used the cells from section I of part I. Amounts of H3K27me3within the HPK1promoter were analyzed by chromatin immunoprecipitation (ChIP) and real-time PCR.Results:Relative to healthy controls, H3K27me3enrichment at the HPK1promoter was significantly increased in CD4+T cells from SLE patients (P<0.001); moreover, H3K27me3enrichment at the promoter was negatively correlated with HPK1mRNA level in SLE CD4+T cells (R=-0.775, P=0.001).Conclusion:H3K27me3enrichment at the HPK1promoter in SLE CD4+T cells was increased, which was negatively correlated with HPK1expression. These data suggest that the up-regulation in H3K27me3 enrichment at the promoter may contribute to the reduction of HPK1in SLE CD4+T cells. Section II EZH2and JMJD3levels at the HPK1promoter in CD4+T cells from SLE patientsObjective:To measure EZH2and JMJD3levels at the HPK1promoter in CD4+T cells from SLE patients and the relationships between their levels, H3K27me3enrichment, and HPK1expression.Methods:Used the cells from section I of part I. EZH2and JMJD3levels at the HPK1promoter were evaluated by ChIP and real-time PCR.Results:Compared to normal controls, JMJD3binding at the HPK1promoter was significantly decreased in CD4+T cells from SLE patients (P<0.001); however, EZH2binding at the HPK1promoter did not differ significantly between SLE and control groups (P=0.223); JMJD3binding at the HPK1promoter in SLE CD4+T cells was negatively correlated with H3K27me3enrichment at the promoter (R=-0.756, P=0.001), and was positively correlated with HPK1mRNA level (R=0.645, P=0.009).Conclusion:The decreased JMJD3binding at the HPK1promote results in H3K27me3within the promote of HPK1up-regulating in SLE CD4+T cells. It may be the critical cause of HPK1expression down-regulation. Section Ⅲ The effect on HPK1expression in normal CD4+T cells by down-regulating JMJD3binding at the HPKl promoterObjective:To examine the effects on HPK1expression and H3K27me3enrichment at the HPK1promoter which are brought by down-regulating JMJD3binding within the HPK1promoter in normal CD4+T cells.Methods:PBMCs from3healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. JMJD3-siRNA or control siRNA was transfected into normal CD4+T cells by transient electroporation. JMJD3and HPK1protein levels were examined by western blotting. JMJD3and H3K27me3levels at the HPK1promoter were evaluated by ChIP and real-time PCR.Results:The JMJD3and HPK1protein levels were significantly down-regulated in normal CD4+T cells transfected with JMJD3-siRNA (P=0.002; P=0.002). JMJD3binding at the HPK1promoter also decreased markedly (P=0.012), and H3K27me3enrichment at the HPK1promoter significantly increased (P=0.019). Conclusion:Down-regulating JMJD3in normal CD4+T cells can inhibit HPK1expression, and this regulation is accomplished at least in part via decreasing JMJD3binding at the HPK1promoter, therefore increasing H3K27me3enrichment at the region. Section IV The effect on HPK1expression in SLE CD4+T cells by up-regulating JMJD3binding at the HPK1promoterObjective:To examine the effects on HPK1expression and H3K27me3enrichment at the HPK1promoter which are brought by up-regulating JMJD3binding within the HPK1promoter in SLE CD4+T cells.Methods:PBMCs from3SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. JMJD3-expressing plasmid or control plasmid was transfected into SLE CD4+T cells by transient electroporation. JMJD3and HPK1protein levels were examined by western blotting. JMJD3and H3K27me3levels at the HPK1promoter were evaluated by ChIP and real-time PCR.Results:The JMJD3and HPK1protein levels were significantly up-regulated in SLE CD4+T cells transfected with JMJD3-expressing plasmid (P=0.003; P=0.006). JMJD3binding at the HPK1promoter also increased markedly (P=0.003), and H3K27me3enrichment at the HPK1promoter significantly decreased (P=0.009).Conclusion:Up-regulating JMJD3in SLE CD4+T cells can induce HPK1expression, and this regulation is accomplished at least in part via increasing JMJD3binding at the HPK1promoter, therefore decreasing H3K27me3enrichment at the region.