Epigenetic Mechanism Of Abnormal Expression Of HERVs In CD4~+ T Cells Of Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE)is a chronic autoimmune disease of complex etiology,often involving multiple organs throughout the body.The course of disease is prolonged and repeated,which seriously endangers human health.SLE is characterized by lacking of immune tolerance and abnormal activation of the immune system,in which T lymphocytes play a key role.Current studies report that a variety of factors including genetic,hormonal and environmental factors are involved in the development of SLE.Human endogenous retroviruses(HERVs)are the remnants of exogenous retroviruses that parasitized the human genome millions of years ago.Most HERVs are silenced due to the self-protection of the human genome,but in recent years HERVs has been found to be de-suppressed in tumors and autoimmune diseases,leading to disease progression.Our previous findings revealed that HERVs,including ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1,which were aberrantly expressed in CD4~+T cells of SLE patients,were transcribed to form double-stranded RNAs to activate the type I interferon pathway and enhance the immune inflammatory response,involving in the pathological mechanism of SLE disease.However,the mechanism of aberrant transcriptional activation of HERVs in SLE CD4~+T is still unclear.Previous studies have shown that the expression of HERVs is epigenetically regulated mainly at the chromatin level through DNA methylation and histone modifications,and at the post-transcriptional level through RNA editing and RNA interference.Extensive DNA hypomethylation and aberrant histone modifications have been identified in SLE disease,and whether these epigenetic modifications have a regulatory role in the expression of HERVs in SLE CD4~+T cells has not been reported.Therefore,we hypothesize that aberrant epigenetic modifications in SLE disease are involved in the regulation of transcriptional activation of HERVs.To test the above hypothesis,the epigenetic mechanism of aberrantly activated HERVs in SLE CD4~+T cells was investigated in two parts.In the first part,we investigated whether the H3K4me2 modification mediated by histone demethylase LSD1 regulates the expression of multiple HERVs in CD4~+T cells of SLE patients.In the second part,the involvement of DNA methylation in regulating the expression of HERVs in CD4~+T cells of SLE patients was explored.The results showed that H3K4me2 modification and DNA demethylation were involved in regulating aberrant activation of HERVs in SLE CD4~+T cells.This study reveals the epigenetic mechanism of abnormal expression of HERVs in CD4~+T cells of SLE patients,providing an important basis for further elucidation of the role of HERVs in SLE disease and potential new targets for the treatment of SLE.Objective:To investigate the mechanism of histone demethylase LSD1-mediated histone H3K4me2 modifications,DNA methylation modifications in regulating the expression of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1 in CD4~+T cells of SLE.Methods:1.Chromatin immunoprecipitation-quantitative polymerase chain reaction(Ch IP-q PCR)was used to detect the H3K4me2modification levels of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1 themselves and their upstream and downstream2000bp sequences in CD4~+T cells of SLE patients and healthy controls(n=5).2.Real-time quantitative polymerase chain reaction(RT-q PCR)and Western blot(WB)were used to detect m RNA and protein expression of LSD1 in CD4~+T cells of SLE patients and healthy controls.3.RT-q PCR was used to detect the m RNA expression levels of ERV3-16A3＿I-int、LTR40C、LTR16A、THE1B、MER21C and MLT1F1in Jurkat cells activated by anti-CD3 and anti-CD28 antibodies.And Ch IP-q PCR was used to detect the H3K4me2 modification levels of HERVs themselves and their upstream and downstream 2000bp sequences in activated Jurkat cells.4.RT-q PCR and WB were used to detect the m RNA and protein expression of LSD1 in Jurkat cells activated by anti-CD3 and anti-CD28antibodies.5.The expression levels of ERV3-16A3＿I-int、LTR40C、LTR16A、THE1B、MER21C and MLT1F1s were detected by RT-q PCR after treatment of Jurkat cells with GSK-LSD1(LSD1 inhibitor).Ch IP-q PCR was used to detect the H3K4me2 modification levels of HERVs themselves and their upstream and downstream 2000bp sequences in GSK-LSD1-treated Jurkat cells.6.Bisulfite sequencing PCR(BSP)was used to detect DNA methylation levels of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1 in CD4~+T cells of SLE patients and healthy controls.7.The expression levels of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1 were detected by RT-q PCR after treatment of Jurkat cells with 5-AZA(DNA methyltransferase inhibitor).The DNA methylation levels of HERVs were detected by BSP assay in5-AZA-treated Jurkat cells.Results:1.The H3K4me2 modification levels of ERV3-16A3＿I-int itself sequence and its upstream and downstream 2000bp sequences(P<0.05),the upstream 2000bp sequence of LTR40C(P<0.05),the downstream 2000bp sequence of LTR16A(P<0.05),THE1B itself sequence(P<0.01),and the upstream 2000bp sequence of MER21C(P<0.05)in CD4~+T of SLE patients were significantly increased than in healthy controls.The H3K4me2 modification level of MLT1F1 itself sequence was increased in CD4~+T cells of SLE patients,but without statistically significant.2.Compared with healthy controls,the m RNA(P<0.0001)and protein(P<0.01)expression levels of LSD1 in CD4~+T cells of SLE patients were significantly reduced.3.Compared with the control group,the m RNA expression levels of ERV3-16A3＿I-int(P<0.01),LTR40C(P<0.01),LTR16A(P<0.01),THE1B(P<0.01),MER21C(P<0.05),and MLT1F1(P<0.01)were significantly increased in activated Jurkat cells.The H3K4me2modification levels of the upstream and downstream 2000bp sequences of ERV3-16A3＿I-int(P<0.01),LTR40C itself sequence and its upstream2000bp sequence(P<0.01),LTR16A itself sequence and its upstream and downstream 2000bp sequences(P<0.01),the upstream 2000bp sequence of MER21C(P<0.05)were significantly increased in the activated group than in the control group.The H3K4me2 levels of THE1B and MLT1F1themselves sequence and their upstream and downstream 2000bp sequences were increased in the activated group than in the control group,but without statistically significant.4.The m RNA(P<0.01)and protein(P<0.01)expression levels of LSD1 were significantly reduced in activated Jurkat cells compared to the control group.5.The m RNA expression levels of LTR40C(P<0.05),LTR16A(P<0.05),THE1B(P<0.01),MER21C(P<0.05)and MLT1F1(P<0.05)were significantly increased in 100μM GSK-LSD1-treated Jurkat cells compared with the control group.The m RNA expression level of ERV3-16A3＿I-int was increased in the GSK-LSD1-treated groups,but without statistically significant.The H3K4me2 modification level of the upstream 2000bp sequence of LTR40C(P<0.01)was significantly increased in the GSK-LSD1-treated group than in the control group.The H3K4me2 levels of ERV3-16A3＿I-int,LTR16A,THE1B,MER21C and MLT1F1 themselves sequence and their upstream and downstream2000bp sequences were increased in the GSK-LSD1-treated group,but without statistically significant.6.Compared with healthy controls,the DNA methylation levels of ERV3-16A3＿I-int(P<0.001),LTR40C(P<0.05),LTR16A(P<0.001),THE1B(P<0.001),MER21C(P<0.01)and MLT1F1(P<0.01)in CD4~+T cells of SLE patients were significantly reduced.7.In 0.3μM 5-AZA-treated Jurkat cells,the relative m RNA expression levels of ERV3-16A3＿I-int(P<0.0001),LTR40C(P<0.0001),LTR16A(P<0.0001),THE1B(P<0.0001),MER21C(P<0.001)and MLT1F1(P<0.001)were significantly increased than in the control group.And the DNA methylation levels of ERV3-16A3＿I-int(P<0.01),LTR40C(P<0.0001),LTR16A(P<0.001),THE1B(P<0.01),MER21C(P<0.05)and MLT1F1(P<0.01)were significantly reduced compared to the control group.Conclusion:1.The expression of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1 in CD4~+T cells of SLE patients and activated Jurkat cells may be regulated by histone H3K4me2modification.The function inhibition of LSD1 can increase the levels of active histone H3K4me2 modification of HERVs themselves and their upstream and downstream 2000bp sequences,which leads to aberrant expression of HERVs.2.DNA methylation is involved in regulating the expression of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1.DNA hypomethylation of ERV3-16A3＿I-int,LTR40C,LTR16A,THE1B,MER21C and MLT1F1 in CD4~+T cells of SLE patients may lead to aberrant expression of HERVs.