Genes and Immunity (2016) 17, 179–186 © 2016 Macmillan Publishers Limited All rights reserved 1466-4879/16 www.nature.com/gene ORIGINAL ARTICLE DNA demethylation of the TIM-3 promoter is critical for its stable expression on T cells F-C Chou1, C-C Kuo1, H-Y Chen2, H-H Chen3 and H-K Sytwu1,2 The T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is selectively expressed on terminally differentiated T helper 1 (Th1) cells and acts as a negative regulator that terminates Th1 responses. The dysregulation of TIM-3 expression on T cells is associated with several autoimmune phenotypes and with chronic viral infections; however, the mechanism of this regulation is unclear. In this study, we investigated the effect of DNA methylation on the expression of TIM-3. By analyzing the sequences of TIM-3 promoter regions in human and mouse, we identified a CpG island within the TIM-3 promoter and demonstrated that the promoter activity was controlled by DNA methylation. Furthermore, treatment with 5-aza-2′-deoxycytidine enhanced TIM-3 expression on mouse primary CD4+ T cells under Th0-, Th1- or Th2-polarizing conditions. Finally, pyrosequencing analysis revealed that the methylation level of the TIM-3 promoter gradually decreased after each round of T-cell polarization, and this decrease was inversely correlated with TIM-3 expression. These data suggest that the DNA methylation of the TIM-3 promoter cooperates with lineage-specific transcription factors in the control of Th-cell development. In conclusion, DNA methylation-based regulation of TIM-3 may provide novel insights into understanding the dysregulation of TIM-3 expression under pathogenic conditions. Genes and Immunity (2016) 17, 179–186; doi:10.1038/gene.2016.6; published online 18 February 2016 INTRODUCTION polymorphism and linkage disequilibrium analysis do not provide The T-cell immunoglobulin and mucin domain-containing protein insight into the possible mechanisms of the dysregulation of TIM-3 3 (TIM-3) molecule is specifically expressed on terminally expression in these diseases, although it has been reported that 22 differentiated T helper 1 (Th1) cells and regulates Th1 cell- TIM-3 is regulated by a Th1 transcription factor, T-bet. However, –/– mediated immune responses.1 The ligand for TIM-3 was identified there is considerable expression of TIM-3 on T cells from T-bet 2 β mice, signal transducer and activator of transcription (STAT)4–/– as galectin-9, a -galactoside-binding lectin that induces T-cell – – – – apoptosis through the calcium–calpain–caspase-1 pathway.3 mice and T-bet / × STAT4 / mice, which suggests that other The roles of the galectin-9–TIM-3 pathway in the regulatory transcription factors and different mechanisms are involved in the functions of T cells have been widely studied in animal models of regulation of TIM-3 expression. Therefore, the regulation of TIM-3 autoimmune diseases, including autoimmune encephalomyelitis,2 expression on Th1 cells in different situations is not understood. autoimmune diabetes,4 rheumatoid arthritis5 and psoriasis.6 Recently, the role of epigenetic regulation in T-cell differentia- In addition, overexpression of galectin-9 has been demonstrated tion has attracted considerable attention. There is growing in several transplantation models to prolong graft survival through evidence that DNA methylation, histone modifications and the downregulation of T-cell responses,7–9 which suggests that the microRNAs are involved in the control of lineage specificity and 23 galectin-9–TIM-3 pathway could be a therapeutic target. In effector function of T-cell subsets (reviewed by Wilson et al. addition, previous reports have demonstrated that the expression and Baumjohann et al.24). During Th-cell differentiation, cell of TIM-3 is dysregulated on T cells from patients with multiple division is accompanied by epigenetic changes that guide the sclerosis,10 autoimmune hepatitis11,12 or during chronic viral production of lineage-specific cytokines and maintain the stability – infections.13–15 These reports indicated that the galectin-9–TIM-3 of the differentiated state.25 27 Because TIM-3 expression is pathway plays a pivotal role in the control of immune responses. restricted to selected cell types, the detailed mechanism of its However, the underlying mechanisms of the regulation of TIM-3 regulation are unclear. We hypothesized that epigenetic regula- expression, especially under pathogenic conditions, are not fully tion may be involved in the control of TIM-3 expression. In this understood. study, we identified a CpG island within the promoter region of Given the importance of TIM-3 in the regulation of T-cell TIM-3 and analyzed the effect of DNA methylation on the immune responses, previous reports have focused on genetic promoter activity. Interestingly, treatment with the DNA methyl- association studies that analyze the single-nucleotide polymorph- transferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine (5-Aza) sig- isms of the TIM-3 promoter in patients with allergic nificantly increased TIM-3 expression on polarized Th1 and Th2 phenotypes16–19 or childhood asthma20 and type 1 diabetes cells, but not on Th17 cells. Finally, using pyrosequencing analysis, families.21 Although researchers have put considerable effort into we demonstrated that the methylation level of the TIM-3 promoter the study of the genetics of TIM-3, the data from single-nucleotide was inversely correlated with TIM-3 expression, indicating that 1Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan; 2Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan and 3Department of Medicine, National Defense Medical Center, Taipei, Taiwan. Correspondence: Professor H-K Sytwu, Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, 161, Section 6, MinChuan East Road, Neihu, Taipei 114, Taiwan. E-mail: [email protected] Received 14 September 2015; revised 27 November 2015; accepted 4 January 2016; published online 18 February 2016 DNA methylation controls TIM-3 expression F-C Chou et al 180 DNA methylation was involved in the control of TIM-3 expression. DNA methylation on the activity of the TIM-3 promoter, we cloned These results suggest a novel regulatory mechanism for TIM-3 that different fragments of the DNA sequence from human (Figure 1b) can be applied to investigation of the dysregulation of TIM-3 and mouse (Figure 1c) TIM-3 into the pGL4.21 luciferase reporter expression under pathogenic conditions. or a CpG-free pCpGL luciferase reporter,28 and performed reporter assays. The pCpGL vector contains no CpG site; therefore, it can be used to evaluate the effect of DNA methylation on promoter RESULTS activity. The longest fragments containing the predicted CpG Identification of human and mouse TIM-3 promoter island displayed the strongest promoter activity for both human To examine the transcriptional activity in the 5′-flanking region of (Figure 1d) and mouse (Figure 1e). To test whether the CpG island- TIM-3, we obtained the DNA sequence from the UCSC Genome containing fragments had independent promoter activity or acted Bioinformatics website (http://genome.ucsc.edu/) and analyzed it as enhancers, we first performed the reporter assay and using the rVISTA program. We found that there are three demonstrated that the human − 1702 to − 899 and mouse conserved noncoding sequences near the transcription start site − 3393 to − 3210 constructs had no promoter activity (Figure 1d; of human and mouse TIM-3 (Figure 1a). By using CpG Island data not shown). We then investigated whether DNA methylation Explorer software, we identified a CpG island within the upstream regulated the promoter activity. As shown in Figures 1f–h, DNA sequence of both the human (Figure 1b) and mouse (Figure 1c) methylation greatly reduced the promoter activity in both human TIM-3 genes. To examine the transcriptional activity of the (Figure 1f) and mouse (Figure 1g) promoter constructs. We predicted TIM-3 promoter regions and evaluate the effect of noticed that the reporter activity of the promoter constructs, Figure 1. Identification of the Tim-3 promoter. (a) Comparison of human Tim-3 upstream sequence to that of mouse, rat and cattle. The sequences were obtained from the UCSC database and the interspecies conservation plot was generated using the mVISTA tool on the website. Schematic illustration of the putative promoter fragments of (b) human and (c) mouse Tim-3 used for the reporter assay. The CpG islands indicated in the figure were predicted using CpGIE software. Determination of (d) human and (e) mouse promoter activity by a luciferase reporter assay. Effect of (f) human and (g and h) mouse promoter methylation on reporter assays. To investigate the effect of CpG methylation on the activity of the Tim-3 promoter, the promoter fragments were cloned into a CpG-free vector for an in vitro methylation and reporter assay. (i) Enhancer activity assay. Putative enhancer fragments were cloned into a CpG-free EF1A-promoter/enhancer cassette and the enhancer activity was evaluated. This construct was further methylated in vitro to evaluate the effect of DNA methylation on enhancer activity. Representative results from at least three independent experiments are shown. CNS, conserved nucleotide sequence. Paired t-test; **Po0.01. Genes and Immunity (2016) 179 – 186 © 2016 Macmillan Publishers Limited DNA methylation controls TIM-3 expression F-C Chou et al 181 human − 1192 to +42 (Figure 1f) and mouse − 3238 to +67 Effects of DNMT inhibitor on transcription of TIM-3 and (Figure 1g) was reduced by DNA methylation. This phenomenon lineage-specific transcription factors in Th-cell subsets was also observed when we used the mouse minimal promoter To investigate whether DNA methylation regulates TIM-3 expres- construct for an in vitro methylation and reporter assay (Figure 1h). sion and restricts TIM-3 expression on Th1 cells, we analyzed TIM-3 Thus, we propose that the CpG island-containing fragment may expression on different Th subsets.