医学免疫学课件：NA甲基化与系统性红斑狼疮

1、NA甲基化与系统性红斑狼疮DDNA Methylation and Systemic Lupus Erythematosus(SLE)ContentsDNA Methylation and SLEDNA Methylation and SLEDNA methylationDNMTCREMTreatment01020304Systematic Lupus Erythematosus Chronic inflammatory disease of unknown Production of a number of antinuclear antibodiesWomen are affected m

2、ore frequently than menProtean clinical manifestationsAffect virtually every organVary dramatically from patient to patient. Most common: skin, musculoskeletal, mild hematologic, and serologic involvement Some patients have: predominately hematologic, renal, or central nervous system manifestationsD

3、NA MethylationWu, H., & Zhang, A. Y. (2014). Reversing dna methylation: mechanisms, genomics, and biological functions. Cell, 156(1-2), 45-68.DNMT: de novo DNA methyltransferases DNA从头甲基化转移酶TET: ten eleven translocationWu, H., & Zhang, A. Y. (2014). Reversing dna methylation: mechanisms, genomics, a

4、nd biological functions. Cell, 156(1-2), 45-68.A: PCR results showed that in CD4+ T lymphocytes from SLE patients, DNMT1 is significantly lower than that in normal population (P 0.05); B: Western Blot results showed that in CD4+ T lymphocytes from SLE patients, the protein expression of DNMT1 was si

5、gnificantly lower than that in normal population; C: The DNA total methylation level of CD4+ T lymphocyte in SLE patients were significantly lower than that in healthy people; D: In SLE patients, the expression of DNMT1 and IgG antibody titers were negatively correlated (P 0.05).Liang, J., Zhu, X. H

6、., Qin, H. H., Lin, J. R., Wang, D. Q., & Huang, L., et al. (2015). A correlation study on the effects of DNMT1 on methylation levels in CD4(+) T cells of SLE patients. International Journal of Clinical & Experimental Medicine, 8(10), 19701-19708.DNMTLiang, J., Zhu, X. H., Qin, H. H., Lin, J. R., Wa

7、ng, D. Q., & Huang, L., et al. (2015). A correlation study on the effects of DNMT1 on methylation levels in CD4(+) T cells of SLE patients. International Journal of Clinical & Experimental Medicine, 8(10), 19701-19708.A: PCR results showed that, after the transfection of plenti6.3/v5-DNMT1 viral vec

8、tor into CD4+ T lymphocytes, the mRNA level of DNMT1 were markedly elevated; B: Western Blot results showed that, after the transfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes, the protein level of DNMT1 were markedly elevated; C: Flow cytometry results showed that, after the tra

9、nsfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes, the methylation level of total DNA raised; D: ELISA results show that, compared with the control group, the production of IgG antibody was reduced after the transfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes (

10、P = 0.021); E: 5-Aza-dc cell demethylation can significantly reverse the level of IgG antibody titers in CD4+ T lymphocytes.Liang, J., Zhu, X. H., Qin, H. H., Lin, J. R., Wang, D. Q., & Huang, L., et al. (2015). A correlation study on the effects of DNMT1 on methylation levels in CD4(+) T cells of S

11、LE patients. International Journal of Clinical & Experimental Medicine, 8(10), 19701-19708.Correlation between whole methylation and DNMT1 mRNA in patients with SLECorrelation between DNA methylation status ofDNMT1 promoter and DNMT1 mRNA in patients with SLE朱小华, & 徐金华. (2016). SLE患者DNA低甲基化对DNMT1启动子

12、表观修饰负反馈调控的研究.中国免疫学杂志,32(4), 542-545.DNA methylationalterationHypomethylation or demethylation: over-expressionCD11a and CD70LFA-1CD40LHypermethylation: reduced expressionFoxp3IL-2Chen, S. H., Lv, Q. L., Hu, L., Peng, M. J., Wang, G. H., & Sun, B. (2016). Dna methylation alterations in the pathogenes

13、is of lupus.Clinical & Experimental Immunology.CREMRauen, T. (2013). Camp responsive element modulator: a critical regulator of cytokine production. Trends in Molecular Medicine, 19(4), 262-9.CREM: cAMP-responsive Element Modulator cAMP应答元件调节子 Rauen, T. (2013). Camp responsive element modulator: a c

14、ritical regulator of cytokine production. Trends in Molecular Medicine, 19(4), 262-9.CREM overexpression induces IL-17A expression bytrans-activating the humanIL17Apromoter.A, primary human T cells were transfected with an expression plasmid encoding human CREM or pcDNA3 empty vector (EV), respectiv

15、ely. BandC, pcDNA3 empty vector or CREM expression plasmid was transfected into primary human T cells, and 5 h after transfection RNA was analyzed for IL-17A and 18 S rRNA expression using regular PCR (B) and real-time qPCR (C). D, alignment of the CRE consensus sequence with the CRE site (111/104)

16、of the proximal humanIL17Apromoter. Schematic below displays theIL17Areporter plasmids used for luciferase assays.IL17Ap(195mut)-luc indicates a reporter plasmid containing a site-directed mutation at the CRE site (111/104). Primary human T cells were transfected with theIL17Areporter plasmids and e

17、ither pcDNA3 empty vector (white bars) or CREM expression plasmid (gray bars). Cells were lysed 5 h after transfection, and firefly luciferase activity was measured and normalized byRenillaluciferase activity. For each reporter pcDNA3 EV co-transfection was set to 1, and the relative effect mediated

18、 by CREM was calculated. Each experiment was performed in T cells from at least four different individuals, and values are given as mean S.D.Thomas, R., Hedrich, C. M., Yuang-Taung, J., Klaus, T., & Tsokos, G. C. (2011). Camp-responsive element modulator (crem) protein induces interleukin 17a expres

19、sion and mediates epigenetic alterations at the interleukin-17a gene locus in patients with systemic lupus erythematosus. Journal of Biological Chemistry, 286(50), 43437-46.Decreased CpG-DNA methylation in IL-17A-secreting and SLE T cells.A, CpG sites within the CNS and the proximal promoter (PP) of

20、 the humanIL17Agene are indicated.B, nave CD4+T cells from healthy blood donors that had been stimulated with anti-CD3/anti-CD28 antibodies for 72 h followed by stimulation with PMA/ionomycin for another 5 h were subjected to an IL-17A secretion assay. ChIP analyses were performed in both IL-17A-enr

21、iched T cells (black bars) and non-IL-17A-secreting T cells (gray bars), using an antibody that specifically detects methylated CpG sequences. Methylated DNA was recovered, and CNS and proximal promoter regions were amplified by real-time qPCR. Completely methylated (input, 100%) and unmethylated hu

22、man DNA samples (negative control, 0%) were included. Values are given as mean S.D. (error bars) from four independent experiments.C, total T cells from six individually matched SLE (gray bars) and healthy control individuals (CON;black bars) were subjected to CpG-DNA immunoprecipitation. The percen

23、tage of methylated DNA is given as mean S.D.Thomas, R., Hedrich, C. M., Yuang-Taung, J., Klaus, T., & Tsokos, G. C. (2011). Camp-responsive element modulator (crem) protein induces interleukin 17a expression and mediates epigenetic alterations at the interleukin-17a gene locus in patients with syste

24、mic lupus erythematosus. Journal of Biological Chemistry, 286(50), 43437-46.DNMT3a recruitment to the CRE site (111/104) is decreased in SLE T cells.A, ChIP was performed using total T cells from four matched pairs of SLE patients and healthy controls (CON) and anti-DNMT3a antibody. Immunoprecipitat

25、ed DNA was analyzed by real-time qPCR using primers detecting a portion of theIL17Apromoter harboring the novel CRE site (111/104). Ratios between anti-DNMT3a immunoprecipitated and input DNA are shown.Dotted linesassociate data from the matched control/SLE pairs.Horizontal barsrepresent the mean.B,

26、 percentage of anti-DNMT3a immunoprecipitated DNA in T cells from the control individual was set to 100%, and relative DNMT3a binding in the corresponding SLE patient was calculated. Values are given as mean S.D. (error bars).C, Jurkat T cells were transfected with pcDNA3 empty vector (EV) or an exp

27、ression plasmid for DNMT3a. Relative IL-17A mRNA expression was analyzed 5 h after transfection. Values are given as mean S.D. from three experiments.D, schematic of CpG-DNA methylation sites within the proximal 195 bp of theIL17Areporter construct.E, pGL3-Basic andIL17Ap(195)-luc reporter plasmids

28、were methylated as outlined under “Experimental Procedures.” Promoter activities of the unmethylated and the methylated reporters were assessed in primary human T cells. Values are given as mean S.D. from three experiments.Thomas, R., Hedrich, C. M., Yuang-Taung, J., Klaus, T., & Tsokos, G. C. (2011

29、). Camp-responsive element modulator (crem) protein induces interleukin 17a expression and mediates epigenetic alterations at the interleukin-17a gene locus in patients with systemic lupus erythematosus. Journal of Biological Chemistry, 286(50), 43437-46.CREM effects on IL-17A/IL-17F expression in S

30、LE T lymphocytes.Overexpression of CREM in T lymphocytes from SLE patients results in increased CREM recruitment to bothIL17AandIL17Fpromoter. As we reported previously , CREM binding results in increased expression of IL-17A in SLE T cells. CREM recruitment to theIL17Fpromoter mediates reduced IL-1

31、7F expression. These diametric mechanisms result in an “SLE-characteristic” imbalance between IL-17A and IL-17F levels and probably impaired assembly of IL-17A/IL-17F heterodimers, which have reduced proinflammatory activity as compared with IL-17A homodimers. This mechanism may have severe implicat

32、ions for the inflammatory cascades in SLE disease expression.Hedrich, C. M., Rauen, T., Kis-Toth, K., Kyttaris, V. C., & Tsokos, G. C. (2011). Camp-responsive element modulator (crem) suppresses il-17f protein expression in t lymphocytes from patients with systemic lupus erythematosus (sle). Journal

33、 of Biological Chemistry, 287(7), 4715-25.IL-2 and IL-17A mRNA expression and promoter methylation in CD4+T cells. (A) IL-2 mRNA expression of naive, central memory (CM) and effector memory (EM) CD4+T cells in response to stimulation with anti-CD3 and anti-CD28 antibodies (mean SD). (B) CpG-DNA meth

34、ylation of theIL2promoter in CD4+T cells. Methylation index (MI) as assessed relative to methylated (100%) and unmethylated (0%) control DNA are shown (mean SD). (C) IL-17A mRNA expression of naive, CM and EM CD4+T cells in response to stimulation with anti-CD3 and anti-CD28. (D) CpG-DNA methylation

35、 of theIL17Apromoter in CD4+T cells (mean SD).Hedrich, C. M., Crispin, J. C., Rauen, T., Ioannidis, C., Apostolidis, S. A., & Lo, M. S., et al. (2012). Camp response element modulator controls IL2 and IL17a expression during cd4 lineage commitment and subset distribution in lupus.CREM mRNA expressio

36、n and promoter methylation in CD4+ T cells. (A) pGL3-basic and CREM P1 reporter plasmids were methylated. Promoter activities of the unmethylated and the methylated reporters were assessed in primary human T cells (mean SD). (B) T cells were transfected with pcDNA3.1 or DNMT3a. DNMT3a resulted in re

37、duced CREM mRNA expression (P 4). T cells from SLE patients and controls were screened for CpG-DNA methylation of the proximal promoter of (B)CREM, (D)IL2, and (F)IL17A.Hedrich, C. M., Crispin, J. C., Rauen, T., Ioannidis, C., Apostolidis, S. A., & Lo, M. S., et al. (2012). Camp response element mod

38、ulator controls il2 and il17a expression during cd4 lineage commitment and subset distribution in lupus.SLE patients exhibit a higher percentage of effector CD4+T cells. (A) A representative pair of age, sex, and ethnicity matched SLE patient and the corresponding healthy control. T cells were stain

39、ed for CD3, CD4, CD45RA, and CCR7 to define naive, central memory (CM), and effector memory (EM) CD4+T cells. (B) Frequency of EM CD4+T cells in total CD4+T cells from SLE patients (SLEDAI: 48) and controls.Hedrich, C. M., Crispin, J. C., Rauen, T., Ioannidis, C., Apostolidis, S. A., & Lo, M. S., et

40、 al. (2012). Camp response element modulator controls il2 and il17a expression during cd4 lineage commitment and subset distribution in lupus.ChIP microarray analysis of H3K4me3 enrichments in SLE and control CD4+T cells.aChIP microarray panels showing relative H3K4me3 enrichments at various gene pr

41、omoters 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 micr

42、oarray panels. Each individualdotshows the Cy3/Cy5 ratio representing relative H3K4me3 enrichment at a specific gene promoter. The CREM promoter dot (indicated by ablue line) is located in the sixteenth column, seventh row.bRelative H3K4me3 enrichment at the CREM promoter in SLE and control CD4+T ce

43、lls, quantified from the results shown in (a)H3K4me3 enrichment at the CREM promoter in SLE and control CD4+T cells.aRelative H3K4me3 enrichment within the CREM promoter in SLE and healthy CD4+T cells was assessed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin).bPos

44、itive correlation between the levels of H3K4me3 and CREM mRNA in SLE CD4+T cells. All reactions were run in triplicateZhang et al.(2016). Increased set1 binding at the promoter induces aberrant epigenetic alterations and up-regulates cyclic adenosine 5-monophosphate response element modulator alpha

45、in systemic lupus erythematosus.Clinical Epigenetics,8.Set1 and MLL1 binding at the CREM promoter in SLE and control CD4+T cells.a,bRelative levels of Set1 (a) and MLL1 (b) binding within the CREM promoter region in SLE and healthy CD4+T cells were analyzed by ChIP and real-time PCR. Results were no

46、rmalized to input DNA (total chromatin).cPositive correlation between Set1 promoter binding and H3K4me3 level in SLE CD4+T cells.dPositive correlation between Set1 promoter binding and CREM mRNA level in SLE CD4+T cells. All experiments were repeated three timesZhang et al.(2016). Increased set1 bin

47、ding at the promoter induces aberrant epigenetic alterations and up-regulates cyclic adenosine 5-monophosphate response element modulator alpha in systemic lupus erythematosus.Clinical Epigenetics,8.Effects of Set1 down-regulation on CD4+ T cells from SLE patients. a, b Relative Set1 and CREM protei

48、n levels were evaluated by western blotting analysis of SLE CD4+ T cells 72 h after transfection with Set1-siRNA or control-siRNA. -actin served as an endogenous control. c, d Relative Set1 (c) and H3K4me3 (d) levels within the CREM promoter in SLE CD4+ T cells transfected with Set1-siRNA or control

49、-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-siRN

50、A. g Relative DNA methylation level at the CREM 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 CREM promoter region in SLE CD4+ T ce

51、lls 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 triplicateZhang et al.(2016). Increased set1 binding at the promoter induces aberrant epigenetic alterations a

52、nd up-regulates cyclic adenosine 5-monophosphate response element modulator alpha in systemic lupus erythematosus.Clinical Epigenetics,8.Relationships between H3K4me3, DNA methylation, and DNMT3a.aNegative correlation between H3K4me3 enrichment and DNA methylation level at the CREM promoter in SLE C

53、D4+T cells.bRelative level of DNMT3a binding within the CREM 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.cNe

54、gative correlation between H3K4me3 enrichment and DNMT3a binding at the CREM promoter in SLE CD4+T cellsZhang et al.(2016). Increased set1 binding at the promoter induces aberrant epigenetic alterations and up-regulates cyclic adenosine 5-monophosphate response element modulator alpha in systemic lu

55、pus erythematosus.Clinical Epigenetics,8.TreatmentsiRNA-CREMIL-2：reverse effects of CREMHigh dose(25ng/mouse)Restores Treg cellsReverses enhanced IgG levelsReferencesWu, H., & Zhang, A. Y. (2014). Reversing dna methylation: mechanisms, genomics, and biological functions. Cell, 156(1-2), 45-68.Liang,

56、 J., Zhu, X. H., Qin, H. H., Lin, J. R., Wang, D. Q., & Huang, L., et al. (2015). A correlation study on the effects of DNMT1 on methylation levels in CD4(+) T cells of SLE patients. International Journal of Clinical & Experimental Medicine, 8(10), 19701-19708.Liang, J., Zhu, X. H., Qin, H. H., Lin,

57、 J. R., Wang, D. Q., & Huang, L., et al. (2015). A correlation study on the effects of DNMT1 on methylation levels in CD4(+) T cells of SLE patients. International Journal of Clinical & Experimental Medicine, 8(10), 19701-19708.朱小华, & 徐金华. (2016). SLE患者DNA低甲基化对DNMT1启动子表观修饰负反馈调控的研究.中国免疫学杂志,32(4), 542-545.Chen, S. H., Lv, Q. L., Hu, L., Peng, M. J., Wang, G. H.,