Study On Abnormal Histone Modification Patterns In SLE CD4+ T Cells And The Interference With Duplex SIRT1-siRNA On MRL-lpr/lpr Mouse Model Of Lupus

Background Systemic lupus erythematosus(SLE)is a chronic and potentially fatal autoimmune disorder,characterized by auto-antibody overproduction.Multiple studies have demonstrated the role of epigenetic alterations in SLE.A growing body of literature indicates that failure to maintain DNA methylation levels and patterns in mature T cells may contribute to the development of SLE.Changes in DNA methylation and histone modifications are a hallmark of genes undergoing epigenetic deregulation in disease.Since DNA is compacted into a highly condensed and ordered structure,considerable interest has focused on chromatin modifications,such as reversible acetylation and methylation of histone tails.However,limited information exists on the global pattern of histone modifications in SLE.Global site-specific hypermethylation(except H3K4 methylation)and hypoacetylation in histone H3 and H4 has been observed in MRL-lpr/lpr mice compared with control MRL/MPJ mice.MRL-lpr/lpr mice treated with histone deactylase(HDAC)inhibitors(HDIs),such as trichostatin(TSA), demonstrate an increased accumulation of acetylated histone H3 and H4 in the total cellular chromatin and an improvement in the kidney disease of these mice.However,there are no reports in alterations in histone H3/H4 methylation/acetylation status,the interplay between DNA methylation and histone acetylation,and how these modifications affect the chromatin structure,in SLE patients.The discovery of a series of enzymes that regulate histone acetylation and methylation status provides us an approach to the insight of these aberrance histone modifications.As for in mammals,P300, CREBBP and PCAF are well informed histone acetyltransferases(HATs) that promote the reaction of histone acetylation.In contrast,HDAC1, HDAC2,HDAC4,HDAC5,HDAC7A,SIRT1 are histone deacetylases (HDAC)that catalyze the removal of acetyl groups from acetylated histones.Histone methyltransferases(HMTs)in mammals mainly include SUV39H1,SUV39H2 and EZH2 that assist the methylation of specific loci of histones.Aberrances of histone acetylation and methylation level may closely relate to lupus.We aimed to investigate the alterations in global histone H3/H4 methylation/acetylation status and the expression of twelve members of three classes of chromatin modifier genes in SLE CD4+ T cells,including: the histone acetyltransferases(HATs)-P300,CREBBP and PCAF;the histone deacetylases(HDACs)-HDAC1,HDAC2,HDAC4,HDAC5, HDAC7,and SIRT1;and the histone methyltransferases (HMTs)-SUV39H1,SUV39H2 and EZH2.As such,we aimed to assess the presence of aberrant global post-translational modifications of histones in SLE patients. Histone deacetylase inhibitors(HDIs),a promising therapeutic approach to cancer and autoimmune diseases,are characteristic of causing accumulation of acetylated histones and other transcriptional regulators.Histone deacetylase inhibitor trichostatin A(TSA)corrected the site-specific hypoacetylation states on H3 and H4 in MRL-lpr/lpr mice with improvement of disease phenotype.However,by using small interfering RNA(siRNA)to knocking out or down the HDACs gene in SLE has not been shown.In recent years,the gene intervention technique has been boomed and become one of the main technique in molecular biology,RNA interference(RNAi)was induced by small interfering ribonucleic acid (siRNA),and then silenced the target gene expression at the post-transcription mRNA level.At present,RNAi,demonstrated great potential in the gene functional research and gene therapy and has been applied in leukemia,autoimmune diseases and so on.Therefore,in this study we also attempted to correct the aberrant histone code of lupus in vivo by small interfering RNA(siRNA)duplexes targeting on SIRT1. ChapterⅠAbnormal Histone Modification Patterns in Lupus CD4~+ T cellsPARTⅠAbnormal histone modification patterns in SLE CD4+ T cellsPurpose Epigenetic factors are important in the onset of human disease such as cancer,aging and autoimmunity.Changes in DNA methylation and histone modifications,the major epigenetic marks,are a hallmark in genes that undergo epigenetic deregulation in disease.We have recently demonstrated that global and gene-specific DNA demethylation involved in systemic lupus erythematosus(SLE).The aim of this study to to investigate whether alterations in histone modifications occur in SLE patients.Methods 20 SLE patients were recruited from the out-patient of Second Xiangya Hospital of Central South University.All patients fulfilled at least 4 of the SLE classification criteria of the American College of Rheumatology;disease activity was assessed using the SLE Disease Activity Index(SLEDAI).Ten out of 20 SLE patients had active disease with mean SLEDAI value[mean±SD:9.800±4.940]compare to 10 patients with lower disease activity[SLEDAI:1.000±1.700].PBMC from 20 SLE patients and 10 healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation,CD4+ subsets were isolated by positive selection using Miltenyi beads and protocols provided by the manufacturer.Histone extraction and detection of global histone H3/H4 acetylation and H3K4/H3K9 methylation were performed following the protocol of Epigentek Assay Kit.Furthermore,the mRNA levels of twelve members of three classes of chromatin modifier genes were measured by real-time quantitative polymerase chain reaction.Results Global histone H3 and H4 hypoacetylation was observed in active lupus CD4+ T cells compared with controls(P=0.002 and P= 0.009,respectively).The degree of histone H3 acetylation negatively correlated with increased disease activity in lupus patients as measured by SLEDAI(R=-0.889,P=0.044).In addition,we found global histone H3K9 hypomethylation in both active and inactive lupus CD4+ T cells compared with controls(P=0.001,P=0.003,respectively).However, global levels of H3K4 methylation were no different between patients and controls.SIRT1 mRNA levels were significantly increased in active lupus CD4+ T cells compared with healthy controls(P＜0.001),while mRNA levels of CREBBP,P300,HDAC2,HDAC7,SUV39H2 and EZH2 were significantly down-regulated in active lupus patients(P＜0.001,P＜0.001, P=0.01,P＜0.001,P=0.003,P=0.001,respectively).Furthermore, HDAC2,HDAC7,SUV39H2 and EZH2 mRNA levels were also significantly down-regulated in inactive lupus CD4~+ T cells,compared with the healthy controls(P=0.009,P＜0.001,P=0.04,P=0.001, respectively). Conclusion Histone modifications appear abnormal in CD4~+ T cells in SLE. PARTⅡAlterations in expression of selected histone modifier genes in MRL/lpr micePurpose MRL-lpr/lpr mice develop to autoimmune disease similar to human systemic erythematosus(SLE).To further understand the interplay between various histone modifications,including acetylation and methylation,and lupus,we analyze the expression of selected histone modifier genes in CD4+ T cells from the MRL-lpr/lpr mouse model of lupus.Methods 18-week-old female MRL-lpr/lpr mice(n=10)and MRL/MPJ wild-type mice(n=10)were involved in the study and were sacrificed by cervical vertebra dislocation.Spleen were removed and CD4+ T cell were isolated by positive selection using Miltenyi beads and protocols provided by the manufacturer.Real-time quantitative RT-PCR was used to detect the expression of 12 members of 3 classes of chromatin modifier genes including:histone acetyltransferases (HATs)-P300,CREBBP and PCAF;histone deacetylases(HDACs)HDAC1, HDAC2,HDAC4,HDAC5,HDAC7 and SIRT1;and histone methyltransferases(HMTs)-SUV39H1,SUV39H2 and EZH2.SIRT1 protein was analysed by western-blot.Results The expression of P300,PCAF,HDAC7,SUV39H2 and EZH2 were significantly decreased in CD4+ T cells from MRL-lpr/lpr mice compared with MRL/MPJ mice(P=0.04,P=0.04,P=0.04,P= 0.03,P=0.01,respectively).However,SIRT1 mRNA levels were significantly increased in lupus mice CD4+ T cells compared with controls(P=0.04).Furthermore,SIRT1 protein was higher expressed in MRL-lpr/lpr mice CD4+ T cells compared with controls(P=0.01).The results were mostly consistent with the changes in human lupus.Conclusion It seems that aberrant histone code occurs in lupus mice. ChapterⅡThe Potential Effects of SIRT1 siRNA on Correction of Aberrant Histone Code in Lupus Mouse ModelPurpose Histone deacetylase inhibitors(HDACI),a promising therapeutic approach to lupus,are characteristic of causing accumulation of acetylated histones and other transcriptional regulators.Histone deacetylase inhibitor trichostatin A(TSA)correct the site-specific hypoacetylation states on H3 and H4 in MRL-lpr/lpr mice with improvement of disease phenotype.We have found that the expression of histone deacetylases(HDACs)-SIRT1 was increased in CD4+ T cells from SLE patients and MRL-lpr/lpr routine model of lupus.Therefore, this study is to investigate whether intravenous duplex siRNA targeting SIRT1 could inhibit SIRT1 expression on lupus mouse CD4+ T cells and correct aberrant histone code in vivo.Methods 18-week-old female MRL-lpr/lpr mice(n=18)were randomized into Four groups:(1)groupⅠ(SIRT1-siRNA)(n=9); (2)groupⅡ(Negative control siRNA)(n=3);(3)groupⅢ(Saline)(n=3); (4)groupⅣ(Blank control group)(n=3).Synthetic siRNAs were delivered in vivo using a modified "hydrodynamic transfection method", by which 50μg siRNA dissolved in 1 ml PBS was rapidly injected into the tail vein.The injection was repeated at 8 and 24 h later.Control mice were injected with an equal volume of normal saline or negative control siRNA.We detected SIRT1 mRNA and protein in CD4+ T cells from MRL-lpr/lpr mice at 24 h after saline,control siRNA or SIRT1 siRNA injection,and at days 5 and 10 after SIRT1 siRNA injection.The level of serum anti ds-DNA antibody and antinuclear antibodies(ANA)was also assayed by ELISA.Global histone H3/H4 acetylation were performed following the protocol of Epigentek Assay Kit.The sediment of IgG immune complex in kidney was determined by direct immunofluorescence,and pathological damage of kidney was determined by HE staining method.Results1)The expression of SIRT1 in both mRNA and protein level was significantly reduced in MRL-lpr/lpr mice CD4+ T cells compare with blank control group at twenty-four hours after 3 injections of SIRTI-siRNA(P=0.04,P=0.001,respectively).2)Compared to blank control group,the global histone H3 and H4 acetylation level were also significantly increased in SIRT1-siRNA group(P=0.02,P=0.01,respectively),and the effects persisted without diminution for 5 days(P=0.04,P=0.04),H4 acetylation was still increasing at days 10(P=0.001).3)The level of anti-dsDNA antibodies was significantly reduced at days 10 post injection in SIRT1-siRNA group compared with blank control group(P=0.04).However,the titer of ANA had no significant difference at days 10 post injection between two groups(P＞0.05).4)Injection with SIRT1-siRNA could improve the damage of vessels and nephric tubules,and decrease immune complex deposit in glomerulus as well as reduce the amount of urine protein excretion at days 10 post injection.5)In contrast,compared with untreated mice,there were no significant changes noted in global histone H3/H4 acetylation,proteinuria and pathological damage of kidney in MRL-lpr/lpr mice treated with negative control siRNA or saline.Conclusion Silencing SIRT1 expression with RNAi holds promise as a lupus therapeutic agent.