The Role Of Histone Acetylation And Methylation In Thyroid Of Autoimmune Thyroiditis

Background:Autoimmune thyroiditis?AIT?,also termed Hashimoto thyroiditis?HT?,is a typical organ-specific autoimmune thyroid disease.The pathogenesis of HT is not fully understood.It is believed that the interaction between genetic susceptibility and environmental triggers contribute to the impairment of immune tolerance to thyroid autoantigens,thus leading to the development of this disease.Epigenetics provide a plausible explanation for the links between environmental factors and genetic background.Epigenetic modifications,defined as stable and heritable alterations in gene expression without the changes of original DNA sequence,mainly include DNA methylation,histone modifications?usually acetylation and methylation?,and microRNA?miRNA?profile and play an important role in gene transcriptional activation,differentiation and genomic stability.Histone acetylation is an important marker of transcriptional activation,and both the amino acid residues and amount of methylation jointly determine the effect of histone methylation on gene regulation.Histone H3 lysine4 methylation?H3K4me?is believed to be the marker of gene activation,while histone H3 lysine 27 trimethylation?H3K27me3?is believed to be associated with chromatin silencing.Histone acetylation and methylation are widely involved in the biological processes of apoptosis,metabolism,inflammation and immune response.It is reported that abnormal histone modifications and modifying enzymes are closely related to the occurrence and development of various autoimmune diseases,including rheumatoid arthritis?RA?,systemic lupus erythematosus?SLE?,Type 1 diabetic mellitus?T1DM?and Graves'disease.Recent studies on autoimmune thyroid disease mainly focus on peripheral blood of Graves'patients.It was reproted that global histone H4 acetylation level in peripheral blood mononuclear cells?PBMCs?of patients with Graves'disease was significantly lower than those of control,while mRNA expression of histone deacetylases HDAC1 and HDAC2 were significantly higher than those of control.Limbach et al.found decreased levels of H3K4me3 and H3K27ac marks at several T cell signaling genes in ChIP-seq analysis in CD4+T cells and CD8+T cells of patients with Graves'disease.A recent study found that the expression of SIRT1 in CD4+T cells of HT patients was significantly increased.However,there has been no systematic studies of the relationship between histone acetylation and methylation and HT.In this study,we investigated the role of histone acetylation and methylation in HT through clinical patients with HT and animal model with spontaneous autoimmune thyroiditis?SAT?.Methods:Part ?:According to the postoperative pathological report and thyroid function,thyroid tissues adjacent to the lesions or in contralateral lobe of patients undergoing surgery for nodular goiter or thyroid micropapillary carcinoma were collected and divided into HT group?n=30?and Control group?n=30?.Serum levels of free T3,free T4,thyrotropin,thyroid peroxidase antibody?TPOAb?and thyroglobulin antibody?TgAb?were measured by electrochemiluminescent immunoassays.Total RNA was extracted from thyroid tissues,and relative mRNA expression of several histone modifying enzymes including HDAC1-6,SIRT1,P300,CREBBP,KMT2A-D and JMJD3 were detected by real-time PCR.Immunohistochemical staining was used to investigate the protein expression and cellular localization of HDAC2,HDAC5,CREBBP and JMJD3in thyroid tissues.Total histone was extracted from thyroid tissues,and the levels of global histone H3 acetylation?H3ac?,H3K4me1/3 and H3K27me3 in thyroid tissues were detected by ELISA.Part ?:Five-week-old NOD.H-2^h44 mice were randomly divided into SAT group,Control group,GSK J4?selective inhibitor of JMJD3?group,GSK J4 control group,PF CBP1?selective inhibitor of CREBBP?group and PF CBP1 control group.The mice in six group were given 0.05%sodium iodide?NAI?in drinking water,sterile water,0.05%NAI in drinking water+intraperitoneal?i.p.?injections of GSK J4,0.05%NAI in drinking water+i.p.injections of 0.9%normal saline?NS?,0.05%NAI in drinking water+i.p.injections of PF CBP1,and 0.05%NAI in drinking water+i.p.injections of 4%dmso in 0.9%NS,respectively.The mice of six groups were killed after 8 weeks.Plasma,spleen and thyroid tissues were collected.HE staining in frozen secctions of thyroid tissues and serum TGAb level were used to evaluate the inflammatory status of mice.Total RNA was extracted from the thyroid tissues of mice in SAT and SAT-control group,and relative mRNA expression of HDAC2,HDAC5,CREBBP and JMJD3 were detected by real-time PCR.Immunohistochemical staining was used to investigate histone H3 lysine 9 and 14 acetylation?H3K9-14ac?and H3K27me3 levels in the thyroid tissues of mice.Splenocytes were stained by flow cytometry to detect the alteration of the proportion of Th1 and Th17 cells after treatment with GSK J4 and PF CBP1 in mice.Results:1.The expression of HDAC2 and HDAC5 in the thyroid tissues of HT patients were significantly decreased than that of the control group,and the expression of CREBBP and global H3ac level were significantly increased than that of the control group.There were no statistically significant differences in the expression of KMT2A-D mRNA between two groups,while H3K4me1 and H3K4me3 levels in HT patients were significantly higher than those in the control group.The expression of JMJD3 in the thyroid tissues of HT patients was significantly higher than that of the control group,while H3K27me3 levels was significantly lower than that of the control group.The results of bivariate correlation analysis showed that the levels of HDAC2 mRNA,HDAC5 mRNA and H3K27me3 were negatively correlated with serum TPOAb and TgAb,while the levels of CREBBP mRNA,JMJD3 mRNA and global H3ac were positively correlated with TPOAb and TgAb.2.The levels of CREBBP,H3K9-14ac and JMJD3 in the thyroid tissues of SAT mice were significantly higher than those of SAT-control mice,while H3K27me3 levels were significantly lower than those of SAT-control mice.Compared with the mice in PF CBP1 control group,mice in SAT+PF CBP1 group had significantly decreased relative thyroid weight,thyroidal inflammatory infiltration,serum TgAb level,thyroidal H3K9-14ac level and proportion of Th1 cells in splenocytes.There were no statistically significant differences in the proportion of Th17cells in splenocytes.Compared with the mice in GSK J4 control group,mice in SAT+GSK J4 group had significantly decreased relative thyroid weight,thyroidal inflammatory infiltration,serum TgAb level,proportion of Th1 cells in splenocytes and increased H3K27me3 level in thyroid tissues.Conclusion:1.Compared with those of control group,H3ac and H3K4me1/3 levels were significantly increased in the thyroid tissues of HT patients,and H3K27me3 level was significantly decreased.The levels of histone modifications and modifying enzymes differentially expressed between groups were significantly correlated with serum TPOAb and TGAb levels.Overall transcriptional activation of chromatin regulated by elevated H3ac and H3K4me1/3 levels,as well as decreased H3K27me3 level is closely related to the development of HT.2.SAT mice had significantly elevated CREBBP mRNA and H3K9-14ac level,elevated JMJD3 mRNA and decreased H3K27me3 level in thyroid tissues.NOD.H-2^h4 mice i.p.injected with PF CBP1 and GSK J4 had significantly decreased H3K9-14ac level and increased H3K27me3 level in thyroid tissues,mild inflammatory infiltration under high iodine intake,significantly decreased serum TgAb titer and proportion of Th1 cells in splenocytes.