Genetic and environmental factors regulate the type 1 diabetes gene CTSH via differential DNA methylation
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100774
ISSN1083-351X
AutoresYi Ye, Mihaela Stefan–Lifshitz, Yaron Tomer,
Tópico(s)Immune Cell Function and Interaction
ResumoCathepsin H (CTSH) is a type 1 diabetes (T1D) risk gene; large-scale genetic and epidemiological studies found that T1D genetic risk correlates with high CTSH expression, rapid decline of beta-cell function, and early onset T1D. Counterintuitively, transcriptional downregulation of CTSH by proinflammatory cytokines has been shown to promote beta-cell apoptosis. Here, we potentially explain these observed contrasting effects, describing a new mechanism where proinflammatory cytokines and T1D genetic risk variants regulate CTSH transcription via differential DNA methylation. We show that, in human islets, CTSH downregulation by the proinflammatory cytokine cocktail interleukin 1β + tumor necrosis factor α + interferon γ was coupled with DNA hypermethylation in an open chromatin region in CTSH intron 1. A luciferase assay in human embryonic kidney 293 cells revealed that methylation of three key cytosine–phosphate–guanine dinucleotide (CpG) residues in intron 1 was responsible for the reduction of promoter activity. We further found that cytokine-induced intron 1 hypermethylation is caused by lowered Tet1/3 activities, suggesting that attenuated active demethylation lowered CTSH transcription. Importantly, individuals who carry the T1D risk variant showed lower methylation variability at the intron 1 CpG residues, presumably making them less sensitive to cytokines, whereas individuals who carry the protective variant showed higher methylation variability, presumably making them more sensitive to cytokines and implying differential responses to environment between the two patient populations. These findings suggest that genetic and environmental influences on a T1D locus are mediated by differential variability and mean of DNA methylation. Cathepsin H (CTSH) is a type 1 diabetes (T1D) risk gene; large-scale genetic and epidemiological studies found that T1D genetic risk correlates with high CTSH expression, rapid decline of beta-cell function, and early onset T1D. Counterintuitively, transcriptional downregulation of CTSH by proinflammatory cytokines has been shown to promote beta-cell apoptosis. Here, we potentially explain these observed contrasting effects, describing a new mechanism where proinflammatory cytokines and T1D genetic risk variants regulate CTSH transcription via differential DNA methylation. We show that, in human islets, CTSH downregulation by the proinflammatory cytokine cocktail interleukin 1β + tumor necrosis factor α + interferon γ was coupled with DNA hypermethylation in an open chromatin region in CTSH intron 1. A luciferase assay in human embryonic kidney 293 cells revealed that methylation of three key cytosine–phosphate–guanine dinucleotide (CpG) residues in intron 1 was responsible for the reduction of promoter activity. We further found that cytokine-induced intron 1 hypermethylation is caused by lowered Tet1/3 activities, suggesting that attenuated active demethylation lowered CTSH transcription. Importantly, individuals who carry the T1D risk variant showed lower methylation variability at the intron 1 CpG residues, presumably making them less sensitive to cytokines, whereas individuals who carry the protective variant showed higher methylation variability, presumably making them more sensitive to cytokines and implying differential responses to environment between the two patient populations. These findings suggest that genetic and environmental influences on a T1D locus are mediated by differential variability and mean of DNA methylation. Type 1 diabetes (T1D) is caused by loss of immune tolerance to insulin-producing beta cells in the pancreas. Beta-cell autoimmune destruction is believed to be triggered by certain environmental influences, such as microbial infections or dietary components (1Rewers M. Ludvigsson J. Environmental risk factors for type 1 diabetes.Lancet. 2016; 387: 2340-2348Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Subsequently, immune cells infiltrate the islets, causing beta-cell destruction, either directly or through secretion of proinflammatory cytokines, such as interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). The extent and rate of progression of beta-cell destruction are modulated by the individual's genetic background. It was shown that genes that frequently interact with environmental factors were enriched in disease risk loci (2Czamara D. Eraslan G. Page C.M. Lahti J. Lahti-Pulkkinen M. Hamalainen E. Kajantie E. Laivuori H. Villa P.M. Reynolds R.M. Nystad W. Haberg S.E. London S.J. O'Donnell K.J. Garg E. et al.Integrated analysis of environmental and genetic influences on cord blood DNA methylation in new-borns.Nat. Commun. 2019; 10: 2548Crossref PubMed Scopus (32) Google Scholar, 3Teh A.L. Pan H. Chen L. Ong M.L. Dogra S. Wong J. MacIsaac J.L. Mah S.M. McEwen L.M. Saw S.M. Godfrey K.M. Chong Y.S. Kwek K. Kwoh C.K. Soh S.E. et al.The effect of genotype and in utero environment on interindividual variation in neonate DNA methylomes.Genome Res. 2014; 24: 1064-1074Crossref PubMed Scopus (214) Google Scholar). In addition, these interactions can be mediated by epigenetic mechanisms, such as histone remodeling and cytosine–phosphate–guanine dinucleotide (CpG) methylation (4Stefan M. Wei C. Lombardi A. Li C.W. Concepcion E.S. Inabnet 3rd, W.B. Owen R. Zhang W. Tomer Y. Genetic-epigenetic dysregulation of thymic TSH receptor gene expression triggers thyroid autoimmunity.Proc. Natl. Acad. Sci. U. S. 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Epidemiol. 2003; 32: 1-22Crossref PubMed Scopus (1973) Google Scholar, 7Davies N.M. Holmes M.V. Davey Smith G. Reading Mendelian randomisation studies: A guide, glossary, and checklist for clinicians.BMJ. 2018; 362: k601Crossref PubMed Scopus (372) Google Scholar). We identified several susceptibility loci where DNA methylation likely mediates the risk of T1D; one of these susceptibility loci is the cathepsin H (CTSH) locus (8Ye J. Richardson T.G. McArdle W.L. Relton C.L. Gillespie K.M. Suderman M. Hemani G. Identification of loci where DNA methylation potentially mediates genetic risk of type 1 diabetes.J. Autoimmun. 2018; 93: 66-75Crossref PubMed Scopus (11) Google Scholar). CTSH has been associated with T1D by Genome-Wide Association Studies (GWAS) (9Onengut-Gumuscu S. Chen W.M. Burren O. Cooper N.J. Quinlan A.R. Mychaleckyj J.C. Farber E. Bonnie J.K. Szpak M. Schofield E. Achuthan P. Guo H. Fortune M.D. Stevens H. 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Genetic variants predisposing most strongly to type 1 diabetes diagnosed under age 7 years lie near candidate genes that function in the immune system and in pancreatic beta-cells.Diabetes Care. 2020; 43: 169-177Crossref PubMed Scopus (20) Google Scholar). Patients with the T1D risk variant correlated with increased CTSH transcription, early onset T1D (younger than 7 years), and rapid decline of beta-cell function (12Inshaw J.R.J. Cutler A.J. Crouch D.J.M. Wicker L.S. Todd J.A. Genetic variants predisposing most strongly to type 1 diabetes diagnosed under age 7 years lie near candidate genes that function in the immune system and in pancreatic beta-cells.Diabetes Care. 2020; 43: 169-177Crossref PubMed Scopus (20) Google Scholar, 13Koskinen M.K. Mikk M.L. Laine A.P. Lempainen J. Loyttyniemi E. Vahasalo P. Hekkala A. Harkonen T. Kiviniemi M. Simell O. Knip M. Veijola R. Ilonen J. Toppari J. Longitudinal pattern of first-phase insulin response is associated with genetic variants outside the class II HLA region in children with multiple autoantibodies.Diabetes. 2020; 69: 12-19Crossref PubMed Scopus (6) Google Scholar). Counterintuitively, functional studies demonstrated that the transcriptional downregulation of CTSH promoted beta-cell apoptosis (14Floyel T. Brorsson C. Nielsen L.B. Miani M. Bang-Berthelsen C.H. Friedrichsen M. Overgaard A.J. Berchtold L.A. Wiberg A. Poulsen P. Hansen L. Rosinger S. Boehm B.O. Ram R. Nguyen Q. et al.CTSH regulates beta-cell function and disease progression in newly diagnosed type 1 diabetes patients.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10305-10310Crossref PubMed Scopus (57) Google Scholar). CTSH is silenced by proinflammatory cytokines, such as IL-1β, TNF-α, and IFN-γ (14Floyel T. Brorsson C. Nielsen L.B. Miani M. Bang-Berthelsen C.H. Friedrichsen M. Overgaard A.J. Berchtold L.A. Wiberg A. Poulsen P. Hansen L. Rosinger S. Boehm B.O. Ram R. Nguyen Q. et al.CTSH regulates beta-cell function and disease progression in newly diagnosed type 1 diabetes patients.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10305-10310Crossref PubMed Scopus (57) Google Scholar). In addition, patients who carry the protectively variant exhibited lower CTSH expression, higher HbA1c, and less diabetes remission (14Floyel T. Brorsson C. Nielsen L.B. Miani M. Bang-Berthelsen C.H. Friedrichsen M. Overgaard A.J. Berchtold L.A. Wiberg A. Poulsen P. Hansen L. Rosinger S. Boehm B.O. Ram R. Nguyen Q. et al.CTSH regulates beta-cell function and disease progression in newly diagnosed type 1 diabetes patients.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10305-10310Crossref PubMed Scopus (57) Google Scholar). We hypothesized that DNA methylation is involved in regulating the genetic and environmental influences of CTSH expression and that understanding its molecular mechanism will potentially unravel the observed paradox in T1D. Purified pancreatic islets incubated with a cytokine cocktail containing IL-1β + TNF-α + IFN-γ for 24 h revealed a marked reduction in CTSH mRNA expression levels (p < 0.0001, n = 10) accompanied by upregulation of proinflammatory cytokine signature genes, such as IRF-1 (p = 0.0286, n = 4), ICE (p = 0.0286, n = 4), FAS (p = 0.0286, n = 4), and BBC3 (p = 0.0286, n = 4) (Fig. 1). To test whether these cytokines regulate CTSH transcription by modifying its DNA methylation, we first searched for open chromatin in CTSH suggestive for epigenetic regulation. Using reference data from the National Institutes of Health Roadmap Epigenomics Mapping Consortium (15Roadmap Epigenomics C. Kundaje A. Meuleman W. Ernst J. Bilenky M. Yen A. Heravi-Moussavi A. Kheradpour P. Zhang Z. Wang J. Ziller M.J. Amin V. Whitaker J.W. Schultz M.D. Ward L.D. et al.Integrative analysis of 111 reference human epigenomes.Nature. 2015; 518: 317-330Crossref PubMed Scopus (2917) Google Scholar), we found that in normal human islets, the CTSH promoter and intron 1 regions overlapped with H3K4me3 and H3K9ac peaks (Fig. 2A), indicating open chromatin for active transcriptional regulation. The CTSH promoter and intron 1 regions are CG rich, and therefore, bisulfite sequencing was performed to examine whether cytokine exposure of islets modified CTSH DNA methylation in this region. We found that upon cytokine exposure, the methylation status of the CTSH promoter (−272 bp to −1 bp upstream of the transcription start site [TSS]) was unchanged, whereas a region in intron 1 containing CpGs 21–36 (+371 bp to +807 bp downstream of TSS, the first CpG site downstream of TSS is denoted as CpG1) was hypermethylated with the average methylation increased by 11% to 46% (n = 3, Figs. 2B and S1).Figure 2An open chromatin region in intron 1 of CTSH became hypermethylated in human islets after IL-1β + TNF-α + IFN-γ exposure for 24 h. A, using the reference data from the National Institutes of Health Roadmap Epigenomics Mapping Consortium, H3K4me3 and H3K9ac peaks identified an open chromatin overlapping the intron 1 of CTSH. B, bisulfite sequencing of human islets derived from a nondiabetic individual with/without the exposure of the proinflammatory cytokine cocktail; ten clones for the control and seven clones for the cytokine-treated DNA sample were sequenced. Black squares represent methylated CpGs, and white squares represent unmethylated CpGs. SNP-CpGs were highlighted in gray. SNP rs11072817 overlaps with CpG29, for which DNA methylation status was excluded from the analysis. CpG, cytosine–phosphate–guanine dinucleotide; CTSH, cathepsin H; IFN-γ, interferon γ; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor α.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate whether DNA methylation of the open chromatin region in intron 1 mediates transcriptional downregulation, CpG21–36 were cloned into a CpG-free luciferase vector (pCpG-lucia), downstream of an elongation factor 1α (EF-1α) promoter (Fig. 3A). Subsequently, the plasmids were either in vitro methylated by M. SssI or mock methylated. Methylation status of CpG21–36 was confirmed by digesting plasmids using methylation-sensitive restriction enzymes (data not shown). Transient transfection in human embryonic kidney 293 (HEK293) cells showed that in vitro methylation of CpG21–36 significantly downregulated the EF-1α promoter activity compared with the mock-methylated plasmid or the unmethylated control (p < 0.0001, n = 3; Fig. 3B). To pinpoint the exact CpG sites within intron 1 that are responsible for regulating CTSH transcription, unmethylated, methylated, or mock-methylated plasmids containing CpG21–24, CpG25–29, CpG30–36, and CpG30–33 were analyzed separately for the effect of their methylation on promoter activity. Our results showed that only the methylation of CpG30–36 attenuated the generic promoter activity by reducing the luciferase signal by approximately 50% (p < 0.0001, n = 3; Fig. 3B). Promoter activity was however not affected by CpG30–33 methylation. Taken together, these data suggested that methylation of the CpG34–36 site in intron 1 is responsible for promoter downregulation. DNA methylation is a dynamic process. Methyl group is added to the 5-cytosine by DNA methyltransferases (DNMTs), and 5-mC is oxidized by 10–11 translocation (Tet) proteins to generate 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (16Wu X. Zhang Y. TET-mediated active DNA demethylation: Mechanism, function and beyond.Nat. Rev. Genet. 2017; 18: 517-534Crossref PubMed Scopus (542) Google Scholar). 5-formylcytosine and 5-carboxylcytosine are subsequently excised by thymine DNA glycosylase (TDG) back to 5-cytosine (16Wu X. Zhang Y. TET-mediated active DNA demethylation: Mechanism, function and beyond.Nat. Rev. Genet. 2017; 18: 517-534Crossref PubMed Scopus (542) Google Scholar). Therefore, hypermethylation can result from either upregulation of DNMTs or downregulation of Tet or TDG enzymes. To investigate this, first, we verified the expression of Tet in purified human islets. Using quantitative RT–PCR, we found that the Ct values of Tet1, 2, 3 were approximately 25, 23, and 26, respectively, against the Ct value of approximately 15 for β-ACT (data not shown). The expression of Tet was also supported by other studies using primary human islets as well stem cell–differentiated beta cells (17Nyalwidhe J.O. Jurczyk A. Satish B. Redick S. Qaisar N. Trombly M.I. Vangala P. Racicot R. Bortell R. Harlan D.M. Greiner D.L. Brehm M.A. Nadler J.L. Wang J.P. Proteomic and transcriptional profiles of human stem cell-derived beta cells following enteroviral challenge.Microorganisms. 2020; 8295Crossref Scopus (4) Google Scholar, 18Segerstolpe A. Palasantza A. Eliasson P. Andersson E.M. Andreasson A.C. Sun X. Picelli S. Sabirsh A. Clausen M. Bjursell M.K. Smith D.M. Kasper M. Ammala C. Sandberg R. Single-cell transcriptome profiling of human pancreatic islets in health and type 2 diabetes.Cell Metab. 2016; 24: 593-607Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 19Stefan-Lifshitz M. Karakose E. Cui L. Ettela A. Yi Z. Zhang W. Tomer Y. Epigenetic modulation of beta cells by interferon-alpha via PNPT1/mir-26a/TET2 triggers autoimmune diabetes.JCI Insight. 2019; 4e126663Crossref PubMed Scopus (21) Google Scholar). Following a 24-h proinflammatory cytokine exposure, the expression of DNMTs DNMT1 (p = 0.22, n = 9), DNMT3a (p = 0.057, n = 11), DNMT3b (p = 0.18, n = 7), Tet2 (p = 0.4796, n = 12), and TDG (p = 0.36, n = 6) did not change, but the expression of Tet1 and Tet3 was significantly reduced (p = 0.0006, n = 10 and p = 0.0006, n = 10, respectively, Fig. 4A). Functionally, cytokine exposure significantly reduced Tet hydroxylase activities (p = 0.0079, n = 5, Fig. 4B). Reduction of Tet1 in nuclear lysate after cytokine exposure was seen using Western blotting (Fig. S2). These suggest that CTSH hypermethylation was caused by attenuated CpG demethylation by Tet proteins. To further test this hypothesis, we examined whether Tet protein inhibition by a broad dioxygenase inhibitor dimethyloxalylglycine (DMOG) lowered CTSH transcription (20Amouroux R. Nashun B. Shirane K. Nakagawa S. Hill P.W. D'Souza Z. Nakayama M. Matsuda M. Turp A. Ndjetehe E. Encheva V. Kudo N.R. Koseki H. Sasaki H. Hajkova P. De novo DNA methylation drives 5hmC accumulation in mouse zygotes.Nat. Cell Biol. 2016; 18: 225-233Crossref PubMed Scopus (122) Google Scholar). After testing a range of DMOG concentrations (100, 300, and 600 μM) and incubation times (24 and 48 h), we decided to incubate human islets with a DMOG concentration of 600 μM for 48 h, where no apparent changes in cell morphology were found under microscopic examination. Treatment with DMOG caused a significant reduction in Tet enzyme activity (p = 0.0476, n = 6; Fig. 4C). Treatment with DMOG (600 μM × 48 h) resulted in a significant suppression of CTSH transcription (p = 0.0022, n = 6; Fig. 4D) supporting the hypothesis that cytokines downregulate CTSH transcription by suppressing Tet activity resulting in hypermethylation of the CpG region in intron 1 we identified. To test whether overexpressing Tet1 can demethylate CTSH intron 1 CpGs, we cotransfected a plasmid expressing the human Tet1 catalytic domain together with the in vitro methylated CpG21–36 lucia plasmid into HEK293 cells. Figure 4E shows that only the Tet1 functional catalytic domain (p = 0.009, Welch's test) but not the mutant Tet1 catalytic domain can reactivate the expression of the methylated CpG21–36 plasmid. To investigate whether the risk variant of CTSH affects the DNA methylation of CpG34–36, we first conducted linkage disequilibrium (LD) analysis using the 1000 Genomes phase 3 data. We found that SNP rs3825932, a GWAS tagged T1D SNP in the Northern European population (r2 = 0.84; Fig. 5), is in tight LD with SNPs rs11072817 and rs11072818 that are located within the cytokine-induced hypermethylated region in intron 1. From hereon, we analyzed only rs11072817 because it is in complete LD with rs11072818. To investigate the relationship between genotype and CTSH intron 1 methylation in humans, we developed a Taqman methylation-specific quantitative PCR (MS-qPCR) assay that selectively quantifies CpG34 methylation in the presence of unmethylated DNA background (Fig. S3). Using MS-qPCR, we found that the protective variant G allele of rs11072817 (GG; n = 15) correlated with a significantly higher methylation variability compared with the A allele (GA + AA; n = 10) in normal islets (F test for variances, p < 0.0001; Fig. 6A). There tends to be a lower mean methylation in individuals with the G allele than those with the A allele (Welch's t test, p = 0.016). We also examined CpG34 methylation profile in nine pairs of islet samples treated or not treated with cytokines. Five of nine pairs showed increase in CpG34 methylation (Fig. 6B), which were found in both the GG and GA + AA genotypes.Figure 6The G allele of SNP rs11072817 correlated with increased methylation variability at CpG34. A, fifteen individuals from the GG group and ten individuals from the GA + AA group were quantified by MS-qPCR. CpG34 methylation levels were divided by rs11072817 genotypes. Boxplots represent interquartile ranges; lines within the boxplots represent medians, whiskers represent minimum–maximum methylation levels. The G allele correlates with increased methylation variability than the A allele (F test for variances, p < 0.0001, denoted as ∗∗∗ in the graph). B, nine pairs of islet samples treated or not treated with cytokines were analyzed by MS-qPCR. Arrows represent paired methylation change. Five of nine pairs showed increase in CpG34 methylation after cytokine addition, as marked by asterisks. Genotypes were highlighted in red and blue. CpG, cytosine–phosphate–guanine dinucleotide; MS-qPCR, methylation-specific quantitative PCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transcriptional variability was not detected between the rs11072817 genotypes in samples that we analyzed (F test for variances, p = 0.68; GG, n = 13; and GA + AA, n = 6; Fig. 7A). However, extraction of the Genotype-Tissue Expression (GTEx) consortium data from 305 human pancreases showed that the G allele correlated with higher transcriptional variability (larger interquartile range) than the A allele (Fig. S4). Of the 19 samples collected, we compared transcription in 18 pairs of islets treated or not treated with cytokines (GG, n = 12; GA + AA, 6). In normal islets, the mean expression in islets carrying the G allele was lower than that of the A allele (p = 0.04, Welch's test; Fig. 7, A and B), which was consistent with the pancreas expression quantitative trait locus data of the GTEx consortium (Fig. S4). Under proinflammatory cytokines IL-1β + TNF-α + IFN-γ treatment, CTSH expression was reduced in both genotypes, but individuals with the G allele still correlated with a significantly lower expression than those with the A allele (p = 0.02, Welch's test; Fig. 7B). In this study, we investigated whether DNA methylation mediates the genetic and environmental influence (act as proinflammatory cytokines) of T1D risk at the CTSH locus. We identified a novel mechanism where the genetic risk and proinflammatory cytokines regulate CTSH expression by differential DNA methylation of the same CpG residues in intron 1. CTSH encodes cathepsin H, which is a member of the papain-like cysteine proteases that are primarily involved in endolysosomal protein degradation, activation of other proteases (21Turk B. Turk D. Turk V. Protease signalling: The cutting edge.EMBO J. 2012; 31: 1630-1643Crossref PubMed Scopus (202) Google Scholar), as well as major histocompatibility complex class II antigen presentation. Although CTSH-null mice do not demonstrate gross developmental defects (22Buhling F. Kouadio M. Chwieralski C.E. Kern U. Hohlfeld J.M. Klemm N. Friedrichs N. Roth W. Deussing J.M. Peters C. Reinheckel T. Gene targeting of the cysteine peptidase cathepsin H impairs lung surfactant in mice.PLoS One. 2011; 6e26247Crossref PubMed Scopus (28) Google Scholar), CTSH was associated with autoimmunity in mouse models and humans including experimental autoimmune encephalomyelitis (23Okada R. Zhang X. Harada Y. Wu Z. Nakanishi H. Cathepsin H deficiency in mice induces excess Th1 cell activation and early-onset of EAE though impairment of toll-like receptor 3 cascade.Inflamm. Res. 2018; 67: 371-374Crossref PubMed Scopus (5) Google Scholar) and T1D (24Barrett J.C. Clayton D.G. Concannon P. Akolkar B. Cooper J.D. Erlich H.A. Julier C. Morahan G. Nerup J. Nierras C. Plagnol V. Pociot F. Schuilenburg H. Smyth D.J. Stevens H. et al.Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes.Nat. Genet. 2009; 41: 703-707Crossref PubMed Scopus (1209) Google Scholar). CTSH is expressed in pancreatic beta cells and antigen-presenting cells but not in T cells (25Faraco J. Lin L. Kornum B.R. Kenny E.E. Trynka G. Einen M. Rico T.J. Lichtner P. Dauvilliers Y. Arnulf I. Lecendreux M. Javidi S. Geisler P. Mayer G. Pizza F. et al.ImmunoChip study implicates antigen presentation to T cells in narcolepsy.PLoS Genet. 2013; 9e1003270Crossref PubMed Scopus (143) Google Scholar). There is a lack of consensus on how it contributes to the pathogenesis of T1D. Studies found that downregulation of CTSH in the presence of proinflammatory cytokines increases beta-cell apoptosis via the small GTPase Rac2 pathways (14Floyel T. Brorsson C. Nielsen L.B. Miani M. Bang-Berthelsen C.H. Friedrichsen M. Overgaard A.J. Berchtold L.A. Wiberg A. Poulsen P. Hansen L. Rosinger S. Boehm B.O. Ram R. Nguyen Q. et al.CTSH regulates beta-cell function and disease progression in newly diagnosed type 1 diabetes patients.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10305-10310Crossref PubMed Scopus (57) Google Scholar, 26Floyel T. Mirza A.H. Kaur S. Frorup C. Yarani R. Storling J. Pociot F. The Rac2 GTPase contributes to cathepsin H-mediated protection against cytokine-induced apoptosis in insulin-secreting cells.Mol. Cell Endocrinol. 2020; 518: 110993Crossref PubMed Scopus (1) Google Scholar), but this effect correlated with the T1D protective allele not the risk allele (14Floyel T. Brorsson C. Nielsen L.B. Miani M. Bang-Berthelsen C.H. Friedrichsen M. Overgaard A.J. Berchtold L.A. Wiberg A. Poulsen P. Hansen L. Rosinger S. Boehm B.O. Ram R. Nguyen Q. et al.CTSH regulates beta-cell function and disease progression in newly diagnosed type 1 diabetes patients.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10305-10310Crossref PubMed Scopus (57) Google Scholar). Here, we showed that CTSH downregulation is induced by DNA hypermethylation of three key CpG residues of an open chromatin upon inflammatory cytokine treatment, which was in line with the general consensus that DNA hypermethylation downregulates gene expression. The uniqueness of our findings is that the T1D risk allele at the CTSH locus correlated with less methylation variability at these CpG residues, whereas the protective allele correlated with higher methylation variability. Increased methylation variability was found in affected siblings of T1D and rheumatoid arthritis discordant-monozygotic twin pairs (27Paul D.S. Teschendorff A.E. Dang M.A. Lowe R. Hawa M.I. Ecker S. Beyan H. Cunningham S. Fouts A.R. Ramelius A. Burden F. Farrow S. Rowlston S. Rehnstrom K. Frontini M. et al.Increased DNA methylation variability in type 1 diabetes across three immune effector cell types.Nat. Commun. 2016; 7: 13555Crossref PubMed Scopus (79) Google Scholar, 28Webster A.P. Plant D. Ecker S. Zufferey F. Bell J.T. Feber A. Paul D.S. Beck S. Barton A. Williams F.M.K. Worthington J. Increased DNA methylation variability in rheumatoid arthritis-discordant monozygotic twins.Genome Med. 2018; 10: 64Crossref PubMed Scopus (35) Google Scholar), implying that epigenetic plasticity potentially mediates the disease-causing environmental stimuli. Similarly, substantial methylation and transcriptional variability were identified in neutrophils rather than monocytes and T cells because neutrophils are the first responders to inflammatory stimuli (29Ecker S. Chen L. Pancaldi V. Bagger F.O. Fernandez J.M. Carrillo de Santa Pau E. Juan D. Mann A.L. Watt S. Casale F.P. Sidiropoulos N. Rapin N. Merkel A. Consortium B. Stunnenberg H.G. et al.Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types.Genome Biol. 2017; 18: 18Crossref PubMed Scopus (54) Google Scholar). Higher phenotypic plasticity renders cells a more rapid and effective response to environmental cues. For example, B cells and T cells use recombination to generate a highly diverse repertoire of immunoglobulins and T-cell receptors. Thus, our findings support the notion that individuals with the protective variant of CTSH were more sensitive to proinflammatory cytokines–induced beta-cell damages (14Floyel T. Brorsson C. Nielsen L.B. Miani M. Bang-Berthelsen C.H. Friedrichsen M. Overgaard A.J. Berchtold L.A. Wiberg A. Poulsen P. Hansen L. Rosinger S. Boehm B.O. Ram R. Nguyen Q. et al.CTSH regulates beta-cell function and disease progression in newly diagnosed type 1 diabetes patients.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10305-10310Crossref PubMed Scopus (57) Google Scholar). In contrast, individuals with the risk variant may develop T1D driven by a strong genetic effect via a dif
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