Regulation of Gene Expression through Gut Microbiota-Dependent DNA Methylation in Colonic Epithelial Cells Downloaded from Kyoko Takahashi, Yutaka Sugi, Kou Nakano, Tetsuro Kobayakawa, Yusuke Nakanishi, Masato Tsuda, Akira Hosono and Shuichi Kaminogawa ImmunoHorizons 2020, 4 (4) 178-190 http://www.immunohorizons.org/ doi: https://doi.org/10.4049/immunohorizons.1900086 http://www.immunohorizons.org/ http://www.immunohorizons.org/content/4/4/178 This information is current as of September 30, 2021. Supplementary http://www.immunohorizons.org/content/suppl/2020/04/14/4.4.178.DCSupp Material lemental References This article cites 40 articles, 11 of which you can access for free at: by guest on September 30, 2021 http://www.immunohorizons.org/content/4/4/178.full#ref-list-1 by guest on September 30, 2021 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE Innate Immunity Regulation of Gene Expression through Gut Microbiota- Dependent DNA Methylation in Colonic Epithelial Cells Kyoko Takahashi, Yutaka Sugi, Kou Nakano, Tetsuro Kobayakawa, Yusuke Nakanishi, Masato Tsuda, Akira Hosono, and Shuichi Kaminogawa College of Bioresource Sciences, Nihon University, Fujisawa-shi, Kanagawa 252-0880, Japan Downloaded from ABSTRACT A huge number of commensal bacteria inhabit the intestine, which is equipped with the largest immune system in the body. Recently, http://www.immunohorizons.org/ the regulation of various physiological functions of the host by these bacteria has attracted attention. In this study, the effects of commensal bacteria on gene expression in colonic epithelial cells (CoECs) were investigated with focus on regulation of DNA methylation. RNA sequencing analyses of CoECs from conventional, germ-free, and MyD882/2 mice indicated that, out of the genes affected by commensal bacteria, those downregulated in a MyD88-independent manner were most frequently observed. Furthermore, when the 59 regions of genes downregulated by commensal bacteria in CoECs were captured using a customized array and immunoprecipitated with the anti-methyl cytosine Ab, a certain population of these genes was found to be highly methylated. Comprehensive analysis of DNA methylation in the 59 regions of genes in CoECs from conventional and germ-free mice upon pull- down assay with methyl-CpG–binding domain protein 2 directly demonstrated that DNA methylation in these regions was influenced by commensal bacteria. Actually, commensal bacteria were shown to control expression of Aldh1a1, which encodes a retinoic acid–producing enzyme and plays an important role in the maintenance of intestinal homeostasis via DNA methylation in the by guest on September 30, 2021 overlapping 59 region of Tmem267 and 3110070M22Rik genes in CoECs. Collectively, it can be concluded that regulation of DNA methylation in the 59 regions of a specific population of genes in CoECs acts as a mechanism by which commensal bacteria have physiological effects on the host. ImmunoHorizons, 2020, 4: 178–190. INTRODUCTION it is difficult to determine whether such differences in gut microbiota are the cause or the result of these diseases, studies The significance of gut microbiota in the maintenance of health have demonstrated that change in microbiota or specificbacteria has recently attracted considerable attention, as increasing evidence is actually involved in the onset, pathogenesis, and prevention of demonstrates that the gut microbiota regulates various physiolog- the diseases (10–12). Therefore, many efforts have been made to ical functions of the host (1–4). In accordance with this, it has been prevent the onset or alleviate the symptoms of the diseases by shown that dysbiosis of the intestinal ecosystem is correlated with intervention to the gut microbiota (13–17). a wide variety of diseases, including inflammatory bowel disease, As the intestine is equipped with the largest immune system allergy, cancer, autism, and metabolic syndrome (3, 5–9). Although in the body, it is considered that commensal bacteria affect the Received for publication October 11, 2019. Accepted for publication March 17, 2020. Address correspondence and reprint requests to: Dr. Kyoko Takahashi, Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-0880, Japan. E-mail address: [email protected] The sequences presented in this article have been submitted to DNA Data Bank of Japan Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra) under accession numbers DRA008905, DRA008906, and DRA008907. This study was supported in part by grants from the Japan Society for the Promotion of Science (KAKENHI 17K07801) and Nagase Science and Technology Foundation (to K.T.). Abbreviations used in this article: CoEC, colonic epithelial cell; CV, conventional; DDBJ, DNA Data Bank of Japan; GF, germ-free; IEC, intestinal epithelial cell; IP, immunoprecipitation; LMD, laser microdissection; MBD2, methyl-CpG–binding domain protein 2; NGS, next-generation sequencing; qRT-PCR, quantitative RT-PCR; Reg3, regenerating islet-derived 3; RNAi, RNA interference; RNA-seq, RNA sequencing; SCFA, short chain fatty acid; SIEC, small IEC; TE, Tris–EDTA; WT, wild-type. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY 4.0 Unported license. Copyright © 2020 The Authors 178 https://doi.org/10.4049/immunohorizons.1900086 ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons EPIGENETIC EFFECT OF COMMENSALS ON COLONIC EPITHELIAL CELLS 179 development and function of the intestinal immune system, par- by the Nihon University Animal Care and Use Committee and ticularly in the period immediately after birth when the immune conducted in accordance with their guidelines. system is in the process of maturation (18–20). Moreover, specific mechanisms might be required to establish andmaintainsymbiosis IEC preparation with commensal bacteria in the intestine considering that com- Small IECs (SIECs) and CoECs were prepared from the whole mensal bacteria are immunologically recognized as foreign Ags but smallintestine and the whole colonof mice, respectively.Epithelial are not excluded completely. Although induction of immune reac- cell preparation was performed as described previously (25). After tionsinvolvesinflammation, inflammatory reactions are strictly removing Peyer patches, the tissues were cut into 2–3-mm pieces controlled at low physiological levels in the intestine despite such a andwashedinHBSSsupplementedwith1mMDTTand0.5mM large amount of bacterial Ags. Actually, excessive inflammation is EDTA accompanied by shaking. The tissues were then treated often observed in diseases associated with dysbiosis. Interestingly, with dispase (BD Biosciences, Franklin Lakes, NJ) to collect intestinal bacteria themselves contribute to this regulation by single-cell suspensions. Lymphocytes were depleted by MACS affecting host cells; however, the precise molecular mechanisms using Dynabeads M-450 Streptavidin (Invitrogen, Thermo Fisher remain to be elucidated. Scientific) and biotin-conjugated anti-CD45 Ab (eBioscience, San Based on this context, elucidation of the physiological effects of Diego, CA). Downloaded from commensal bacteria on host cells becomes key evidence to clarify the role of commensal bacteria in intestinal homeostasis. For this Cell culture purpose, the effects of commensal bacteria on gene expression in The mouse IEC line CMT-93 developed from rectal carcinoma intestinal epithelial cells (IECs) covering the intestinal mucosa and was purchased from DS Pharma Biomedical (Osaka, Japan) and the underlying regulatory mechanisms were investigated in this cultured in DMEM supplemented with 10% (v/v) FBS (Biowest, http://www.immunohorizons.org/ study. IECs form a front line of defense by separating the intestinal Nuaillé, France), 100 U/ml penicillin, 100 mg/ml streptomycin, tract from the internal milieu and are usually exposed to commen- and 5 3 1025 M 2-ME at 37°C in a humidified incubator with sal bacteria inhabiting the intestinal tract. Therefore, commensal 5% CO2. bacteria have a great impact on gene expression in IECs. In addition, it has been shown that commensal bacteria confer RNA sequencing epigenetic effects to specific genes in IECs (21–23). DNA meth- Total RNA was prepared from SIECs and CoECs of WT-CV, ylation, as well as posttranslational histone modifications and MyD882/2-CV, and WT-GF mice by pooling from six to eight mice noncoding RNA, are important mechanisms mediating epigenetic per group using the High Pure RNA Isolation Kit (Roche, Basel, regulation of gene expression. We previously reported that DNA Switzerland). RNA sequencing (RNA-seq) was performed by methylation of the gene encoding TLR4, an innate immune INFOBIO (Tokyo, Japan). Briefly, mRNA was extracted, reverse by guest on September 30, 2021 receptor that recognizes LPS of Gram-negative bacteria, in colonic transcribed, treated with restriction enzyme NlaIII, and ligated epithelial cells (CoECs) is induced by commensal bacteria (23). As with adaptor sequences. The library was prepared by processing stimulation with LPS through TLR4 induces strong inflammatory the tag with EcoP15I and analyzed using Genome Analyzer