Histone Methylation In Patients With Hashimoto Thyroiditis And The Mechanism Of Iodine

Background:Hashimoto thyroiditis?HT?,also known as Autoimmune thyroiditis?AIT?,is a clinically common endocrine disease.The precise pathogenesis of Hashimoto's thyroiditis is not fully elucidated.It's believed that genetics and environmental factors together contribute to the development of disease,and iodine is viewed as the one of the most important environmental factors.Epigenetic modification can regulate gene transcription by altering chromatin structure,recruiting transcription factors,and regulating various enzymes without affecting DNA sequences.It is a bridge connecting environmental factors and genetic background.Histone methylation and methyl modifying enzymes have been reported to be associated with many autoimmune diseases.However,no systematic studies of histone methylation and HT have been conducted.In this study,we mainly investigated the genome-wide Tri-methylated Histone H3 Lysine4?H3K4me3?patterns and global H3K4me3 protein levels in primary thyrocytes and thyroid glands from HT patients,analyzed the effects of high iodine on thyroid cells through analyzing the genome-wide distribution and global protein expression of H3K4me3 and Tri-methylated Histone H3 Lysine 27?H3K27me3?and subsequently explored the possible mechanism in HT.Methods:1)Primary thyrocytes and tissues from HT patients:The distribution of genome-wide H3K4me3 pattern in primary thyrocytes from HT patients and controls?3HT vs 3control?was analyzed by immunoprecipitation sequencing?chip-seq?.The biological functions and enrichment pathways of related genes with differential H3K4me3 enrichment in Transcription starting Sites?TSSs?were analyzed by KEGG and GO analysis.RT-PCR was used to verify the transcription levels of important genes?8HT vs 8control?with increased H3K4me3 enrichments in the promoter.Western blotting?WB?was used to detect the global H3K4me3 levels in primary thyrocytes?4HT vs 4control?and thyroid tissue?6HT vs 6control?.RT-PCR was used to screen for abnormal expression of histone methyltransferase in thyroid tissues?30HT vs 30control?.Immunohistochemistry and Western blotting were used to verify the protein expression of histone methyltransferase in thyroid tissues.2)Iodine on thyroid cells:normal human thyroid cell line N-Thy-ori-3-was stimulated with 10^-3M and 10^-6M sodium iodide for 6hours and 24 hours,respectively.Chip-seq was used to study the genome-wide distribution of H3K4me3 and H3K27me3 to explore the differential enrichment sites of H3K4me3 and H3K27me3.RT-PCR and WB were used to verify the expression level of genes with significant differential enrichments in the promoter.After stimulation with high iodine,histone methyltransferases which may be abnormally expressed were detected by RT-PCR in primary thyrocytes and N-thy-ori3-1.After iodine stimulation with gradient rise for 6 and 24 hours,Western blotting was used to detect global protein levels of H3K4me3 and H3K27me3 in N-thy-ori3-1,and immunofluorescence was used to analyze cell localization of methyltransferases.Enzyme-linked immunosorbent assay?ELISA?was used for the detection of H3K4 overall methylation level.3)GSK-J4 in vitro study:Thyroid primary thyrocytes and N-thy-ori3-1 were stimulated with high iodine,TNF?,and then co-stimulated with JMJD3 inhibitor GSK-J4 or MLL1 inhibitor MM102.The expression of MCP1 and CXCL10 was detected by RT-PCR and Western blotting.The overall protein level of H3K27me3 was examined.Flow cytometry was used to detect oxidative stress levels and apoptosis in thyroid cells.Results:1)Changes of H3K4me3 in primary thyrocytes and tissues:We finally identified an average of 1458 differential H3k4me3 peak regions between HT and control group and got 1187 differential peaks-related genes.according to GO analysis,the differential peaks-related genes were mainly involved in the biological process of immune system process,response to stimulus,regulation of cell adhesion,positive regulation of leukocyte activation.KEGG pathway analysis suggested these genes were remarkably enriched in Cell adhesion molecules?CAMs?,Autoimmune thyroid disease,Pathways in cancer.The most important differential genes with higher H3K4me3 peaks signal in HT group enriched in the autoimmune thyroid disease pathway include:TG,TSHR,MHCII,CTLA,CD80/86 and Fas/Fasl.It was noted that the TG promoter of thyroid follicular cells from HT patients had 2.45 times enrichment signal of H3K4me3on average compared with the control group.In addition,we also found chemokines including CCL2,CXCL8,ICAM1,CXCL10,IL18,ITGA4 and apoptosis factors including FASLG and TRAIL showed increased H3K4me3 in the promoter around TSSs.The increased mRNA levels of CCL2,IL8,and ICAM1 were confirmed in primary thyrocytes from HT patients.The global protein expression of H3K4me3 in thyroid tissues from HT patients was significantly higher compared with controls while there was no difference in the global protein expression of H3K4me3 from the primary thyrocytes between the two groups.Besides,the mRNA and protein levels of MLL1 in thyroid tissue of patients with HT were significantly elevated.2)Effect of iodine on thyroid cells:After iodine stimulation of N-thy-ori3-1 with gradient rise for 6 and 24 hours,the global level of histone H3K4me3 increased gradually with the increase of sodium iodide concentration,but after 24 hours the trend decreased,showing a transient increase.The overall methylation of H3K4 has no reaction to iodine stimulation.Iodine stimulation can cause elevated levels of histone methylation transferase MLL1 in N-thy-ori3-1 and primary cultured thyroid cells.Immunofluorescence showed that MLL1 was localized in the nucleus.Chip-seq suggested that H3K4me3 enrichment in the promoter region of JMJD3 was increased after iodine stimulation.When thyroid cells were stimulated with10^-3M sodium iodide for 24 hours,the mRNA and protein levels of JMJD3 also increased,accompanied by a decrease in the overall protein level of H3K27me3.The expression of JMJD3 in thyroid glands also enhanced from HT patients.KEGG and GO analysis showed that genes with differential enrichment of H3K4me3 were mainly concentrated in ubiquitination proteolysis,cell junction,cAMP signaling pathway,lysine degradation pathway,and the genes with differential enrichment of H3K27me3 were mainly concentrated in cell junctions,calcium signaling pathways,and glutamine synapses.3)Study of JMJD3 inhibitor GSK-J4 in vitro:Stimulation with high iodine were found to increase CCL2 expression in HT thyrocytes,while no reaction was found in N-thy-ori3-1 and thyrocytes from control group.The mRNA and protein levels of CCL2 and CXCL10 in thyroid tissue from patients with HT were significantly increasd compared to the controls.After TNF?stimulation,the mRNA and protein levels of CCL2and CXCL10 were significantly increased.When GSK-J4 was co-stimulated,the transcriptional expression levels and protein levels of CCL2 and CXCL10 in N-thy-ori3-1 were significantly decreased,accompanied by the increased protein level of H3K27me3.TNF?was also found to increase the number of apoptotic cells and the level of oxidative stress.After co-stimulation with GSK-J4,thyroid cell apoptosis and the reactive oxygen species?ROS?in cells were markedly decreased.Conclusions:1.Increased H3K4me3 enrichment was found in TSSs of genes including TG,CCL2,CXCL10,IL8,ICAM1,IL18,ITGA4,FASLG,TRAIL in thyrocytes from HT patients and may be associated with increased transcriptional expression of CCL2,IL8,ICAM1,providing a possible explanation from the epigenetic perspective for the mechanism of increased expression of inflammation-related molecules associated with HT.2.Genes with H3K4me3 differential enrichment in HT patients are mainly enriched in cell adhesion molecules?CAMs?,autoimmune thyroid disease?Autoimmune thyroid disease?cancer pathway?Pathways in cancer?,indicating that differential changes in H3K4me3 in thyrocytes are involved in the development of HT.3.The global protein level of H3K4me in thyroid tissues from HT patients was significantly higher and the increased expression of MLL1 in tissues may be involved.4.Iodine stimulation can promote the transient increase of the whole protein level of H3K4me3 in thyroid cells,and also induce the high expression of MLL1.5.High Iodine stimulation can promote the increase of JMJD3 expression by affecting the increase of H3K4me3 enrichment in JMJD3 promoter.Increased JMJD3 expression was found in thyroid tissue from patients with HT,suggesting that JMJD3 may become an important factor regulated by iodine to affect HT 6.Thyroid cells treated with high iodine showed great similarity with the genome-wide pattern of H3K4me3 and H3K27me3.The genes with differential enrichment were mainly concentrated in cell junction,proteolysis and calcium signaling pathways.7.Iodine can induce the high expression of chemokines CCL2 and CXCL10 in thyroid cells with genetic background or in inflammatory environment to participate in the pathological process of HT.8.JMJD3 inhibitor GSK-J4 can significantly inhibit the expression of CCL2 and CXCL10 in thyroid cells by increasing H3K27me3 level,suggesting that it may delay or block inflammatory cell infiltration in early development of HT.9.GSK-J4 can reduce the oxidative stress level and apoptosis of thyroid cells,suggesting a potential protective role for prevention or treatment of HT through various mechanisms.