Genetic and environmental factors regulate the type 1 diabetes gene CTSH via differential DNA methylation

Abstract

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. 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. 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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 + + IFN-γ for revealed a reduction in CTSH expression by of proinflammatory cytokine such as and these cytokines regulate CTSH transcription by its DNA we for open chromatin in CTSH for epigenetic the of C. A. W. J. M. A. A. P. Zhang Z. Wang J. M.D. et analysis of human 2015; PubMed Scopus Google we found that in human islets, the CTSH promoter and intron 1 with and open chromatin for active transcriptional The CTSH promoter and intron 1 are and was to cytokine of islets CTSH DNA methylation in We found that cytokine the methylation of the CTSH promoter to of the transcription was whereas a region in intron 1 containing to of the CpG of is as was with the methylation increased by to and open chromatin region in intron 1 of CTSH in human islets IL-1β + + IFN-γ for using the the of and identified an open chromatin the intron 1 of of human islets a the of the proinflammatory cytokine for the and seven for the DNA were and were in with for which DNA methylation was the cytosine–phosphate–guanine cathepsin interferon IL-1β, interleukin TNF-α, tumor necrosis factor DNA methylation of the open chromatin region in intron 1 mediates transcriptional were a luciferase of an factor promoter Subsequently, the were either in by M. or of was by using in human embryonic kidney 293 cells showed that in methylation of the promoter with the or the the CpG intron 1 that are responsible for regulating CTSH transcription, or containing and were for the effect of methylation on promoter activity. showed that the methylation of attenuated the promoter by the luciferase by was by methylation. these that methylation of the in intron 1 is responsible for promoter DNA methylation is a is to the by DNA and is by to and Zhang Y. active DNA function and Genet. 2017; PubMed Scopus Google Scholar). and are by DNA to Zhang Y. active DNA function and Genet. 2017; PubMed Scopus Google Scholar). hypermethylation can either of or downregulation of or we the expression of in human we found that the of were and the of for The expression of was by studies using human islets as beta cells A. S. P. R. R. J.L. Wang J.P. and transcriptional of human beta cells 2020; Scopus Google Scholar, A. A. P. S. A. M. M.K. Smith M. C. R. of human pancreatic islets in and type 2016; 24: Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. L. A. Z. Zhang W. Tomer Y. of beta cells by via triggers autoimmune 2019; PubMed Scopus Google Scholar). a proinflammatory cytokine the expression of and the expression of and was and cytokine of in cytokine was using These suggest that CTSH hypermethylation was caused by attenuated CpG demethylation by further we by a lowered CTSH transcription R. K. S. Z. M. M. A. E. H. H. P. DNA methylation in 2016; PubMed Scopus Google Scholar). a of and and and we to human islets with a of for where in were found under with caused a reduction in with in a of CTSH transcription the that cytokines CTSH transcription by in hypermethylation of the CpG region in intron 1 we can CTSH intron 1 we a the human with the in that the functional the can the expression of the the risk variant of CTSH the DNA methylation of we analysis using the We found that a T1D in the is in with and that are the cytokine-induced region in intron 1. we is in with the between genotype and CTSH intron 1 methylation in we a assay that methylation in the of DNA we found that the protective variant of correlated with a higher methylation variability with the A + in islets for to be a lower mean methylation in individuals with the than with the A We methylation in of or with of showed in methylation which were found in the and + of correlated with increased methylation variability at individuals the and individuals the + were by methylation were by the methylation The correlates with increased methylation variability than the A for as in the of or with cytokines were by methylation of showed in methylation cytokine addition, as by were in and cytosine–phosphate–guanine variability was between the in that we for and + However, of the human showed that the correlated with higher transcriptional variability than the A the we transcription in of islets or with cytokines + In islets, the mean expression in islets the was lower than that of the A A and which was with the expression locus of the proinflammatory cytokines IL-1β + + IFN-γ CTSH expression was in individuals with the still correlated with a lower expression than with the A In we DNA methylation mediates the genetic and environmental as proinflammatory of T1D risk at the CTSH We identified a mechanism where the genetic risk and proinflammatory cytokines regulate CTSH expression by differential DNA methylation of the CpG residues in intron 1. CTSH cathepsin which is a of the that are involved in of D. The J. PubMed Scopus Google as as complex class II M. C.E. U. Roth W. C. T. of the cathepsin H in 2011; PubMed Scopus Google CTSH was associated with autoimmunity in and autoimmune R. Zhang Y. Z. H. Cathepsin H in and of of receptor Res. 2018; PubMed Scopus Google and T1D J.C. P. Cooper J.D. C. G. J. C. H. Stevens H. et association study and meta-analysis that loci risk of type 1 Genet. PubMed Scopus Google Scholar). CTSH is in pancreatic beta cells and cells in cells J. L. G. M. P. Y. M. S. P. G. et study to cells in Genet. PubMed Scopus Google Scholar). is a of on to the of T1D. Studies found that downregulation of CTSH in the of proinflammatory cytokines beta-cell apoptosis via the (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, T. S. C. R. J. The to cathepsin cytokine-induced apoptosis in 2020; PubMed Scopus Google effect correlated with the T1D protective the risk (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 by DNA hypermethylation of three key CpG residues of an open chromatin cytokine which was in with the that DNA hypermethylation gene The of findings is that the T1D risk at the CTSH locus correlated with less methylation variability at these CpG residues, whereas the protective correlated with higher methylation methylation variability was found in of T1D and D.S. R. S. H. S. A.R. A. S. S. K. M. et DNA methylation variability in type 1 diabetes three immune Commun. 2016; PubMed Scopus Google Scholar, A.P. D. S. J.T. A. D.S. S. A. J. DNA methylation variability in 2018; 10: PubMed Scopus Google implying that epigenetic potentially mediates the environmental methylation and transcriptional variability were identified in than and cells are the to S. Chen L. E. D. A.L. S. A. et analysis of differential transcriptional and epigenetic variability human immune 2017; PubMed Scopus Google Scholar). cells a more rapid and response to environmental cells and cells to a of and findings the that individuals with the protective variant of CTSH were more sensitive to proinflammatory beta-cell (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 individuals with the risk variant may T1D by a genetic effect via a mechanism C. Cutler A.J. M.L. Burren Cooper J.D. A.R. Guo H. Walker N.M. S. S.J. M. et of a complex disease susceptibility region using a to Genet. 2015; PubMed Scopus Google Scholar). a variant of the and receptor gene was associated with autoimmunity in children K. Wong N.J. M. Toppari J. A. et analyses in children at increased genetic risk for type 1 2019; PubMed Scopus Google variant was identified in the T1D with these that individuals with risk may to environmental to beta-cell We found that CTSH intron 1 hypermethylation is on the loss of Tet1/3 a cytokine loss of is to has been shown to and DNA and as to function, where has effect Guo H. DNA and as molecular for active DNA PubMed Google Scholar, T. Nguyen C. DNA methylation in PubMed Scopus Google Scholar, S. U. M. L. C. A. and with 2015; PubMed Scopus Google Scholar). is in beta cells A. A. P. S. A. M. M.K. Smith M. C. R. of human pancreatic islets in and type 2016; 24: Full Text Full Text PDF PubMed Scopus Google further studies are to the involvement of and in to the CTSH locus to its transcriptional are than by et J. S. J. S. of insulin DNA in response to proinflammatory cytokines the progression of autoimmune diabetes in 2016; PubMed Google Scholar, J. S. G. R. T. M. A.L. M.L. D. J. cells responses to in type 1 2020; Scopus Google who found that cytokines IL-1β + + IFN-γ increased the transcription of and in human islets, whereas we The be to the cytokine in A of using is that the of cytokine of islets that in the in gene transcription in may a and which be on the cytokine of the C. of beta cells to of insulin secretion through and remodeling and of gene 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). study has to the mean and of methylation and gene expression was of the of human In we observed that individuals with the genotype a higher methylation variability and a lower mean methylation of variability, we have to an mean in We a transcriptional variability between the and + in were in the a to in transcriptional of human islets, and of the may have to of the C. T. in human and type 2019; Full Text Full Text PDF PubMed Scopus Google Scholar, E. E. The of DNA methylation with and genotype in and PubMed Scopus Google Scholar). we to the effect of CTSH intron 1 methylation. is a of the potential In revealed a molecular mechanism differential DNA methylation mediates the genetic and environmental influences at the CTSH that CTSH genetic risk was associated with early diabetes onset (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 and beta-cell function of the human effect 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 the of CTSH gene dysregulation in the of T1D understanding may the for studies CTSH in T1D. islets were the of were as The was by the of as and by the of were either in containing with or in with proinflammatory cytokines, IL-1β and IFN-γ were in the and of cytokines for was in and to the at were with for and was cells were in with and We to the effect of CTSH intron 1 methylation is a human to and human transcription factors to study CTSH gene is to study the associated with CTSH transcriptional which be to beta cells were in with for islets were by and DNA was using the DNA blood to the was using the of were DNA using the to the DNA was using the with was on a system using the were at an of for a of was using the of gene expression was the for was using the assay was to the genotype for of DNA were using the DNA the DNA was using on a for were using the and cells were DNA was using the and using to DNA were of methylation was by DNA methylation of the A DNA an intron 1 region of CTSH was the intron of the which was as intron The expression of the gene was by a human The promoter in the was of CpG and was for to the CTSH intron 1 In the CTSH promoter is enriched with CpG CTSH promoter be the is in and be for were using the was using the the intron were the E. cells were for DNA the intron than DNA was using CpG M. in a for the of DNA was for a in a for was the of M. the of in and were using the or the and intron were cells using the for at a of containing the CTSH promoter were as a either the active or the was with either the or the intron as as the was using the assay luciferase for was by to activity. for the and were to the that the expression of the was to were islets using assay with for three at for and for to was using the assay was using the the and were human islets using the and and were using the and to was using was using the and was using the and the was using the system the CTSH methylation and were using the was using the was at the of the and was at the of the of the the most the assay methylation of and the at the of were the and were using the and methylation of the as a gene for and was and for the CTSH and assay were in A was to the was at the for to promote to the and at for to the CTSH methylation was using the for was using the of the the of a DNA as of CTSH of of CTSH of was to a were as mean was to gene expression and between and was to the between individuals with was to the with was to A was and the study are in the and The that have of with the of J. Y. and Y. T. the J. Y. the J. M. and Y. T. and the J. Y. the J. M. and Y. T. and the was in by of and and the of to Y. the and to J. and the J. J. Y. is the of as to the in the and for the of the and of the The is the of the and the of the of

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