Global DNA Methylation Remodeling Accompanies CD8 T Cell Effector Function Christopher D. Scharer, Benjamin G. Barwick, Benjamin A. Youngblood, Rafi Ahmed and Jeremy M. Boss This information is current as of October 1, 2021. J Immunol 2013; 191:3419-3429; Prepublished online 16 August 2013; doi: 10.4049/jimmunol.1301395 http://www.jimmunol.org/content/191/6/3419 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/08/20/jimmunol.130139 Material 5.DC1 References This article cites 81 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/191/6/3419.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Global DNA Methylation Remodeling Accompanies CD8 T Cell Effector Function Christopher D. Scharer,* Benjamin G. Barwick,* Benjamin A. Youngblood,*,† Rafi Ahmed,*,† and Jeremy M. Boss*,† The differentiation of CD8 T cells in response to acute infection results in the acquisition of hallmark phenotypic effector functions; however, the epigenetic mechanisms that program this differentiation process on a genome-wide scale are largely unknown. In this article, we report the DNA methylomes of Ag-specific naive and day-8 effector CD8 T cells following acute lymphocytic chorio- meningitis virus infection. During effector CD8 T cell differentiation, DNA methylation was remodeled such that changes in DNA methylation at gene promoter regions correlated negatively with gene expression. Importantly, differentially methylated regions were enriched at cis-elements, including enhancers active in naive T cells. Differentially methylated regions were associated with cell type–specific transcription factor binding sites, and these transcription factors clustered into modules that define networks targeted Downloaded from by epigenetic regulation and control of effector CD8 T cell function. Changes in the DNA methylation profile following CD8 T cell activation revealed numerous cellular processes, cis-elements, and transcription factor networks targeted by DNA methylation. Together, the results demonstrated that DNA methylation remodeling accompanies the acquisition of the CD8 T cell effector phenotype and repression of the naive cell state. Therefore, these data provide the framework for an epigenetic mechanism that is required for effector CD8 T cell differentiation and adaptive immune responses. The Journal of Immunology, 2013, 191: 3419–3429. n response to acute infection, naive CD8 T cells differentiate Epigenetic mechanisms ensure the maintenance and inheritance http://www.jimmunol.org/ into effector cells capable of killing infected cells and clearing of gene-expression programs through cell division and include DNA I the infection. Effector CD8 T cell function is characterized by methylation and histone modifications (8, 9). Mammalian DNA the induction of a specific transcriptional program that drives rapid methylation primarily involves the methylation of CpG dinucleo- proliferation, expression of key cytokines and effector proteins tides and is associated with a repressed epigenetic state when found necessary for cell killing, and the capacity to migrate into infected in gene promoters (8, 10–12). DNA methylation is maintained or tissue (1–4). Upon Ag clearance, 90% of effector cells undergo deposited de novo by one of three DNA methyltransferases (DNMT1, apoptosis, whereas the remaining cells complete their differenti- DNMT3A, or DNMT3B). Methylated CpG DNA is recognized/ interpreted by a family of methyl-CpG–binding proteins (10, 13, ation into a pool of memory CD8 T cells (5). A number of the by guest on October 1, 2021 critical transcription factors that drive this differentiation program, 14). DNA methylation readers and writers are vital components of such as Blimp-1 (Prdm1), Tbet (Tbx21), and Eomesodermin (Eomes) the adaptive immune response. Deletion of the maintenance meth- have been identified and characterized (2, 6, 7). However, little is yltransferase DNMT1 during T cell development resulted in normal known about the epigenetic programs that enforce the induced gene lineage formation but led to homeostatic defects and the inability expression changes and permit faithful inheritance of effector to silence lineage-specific genes in CD4 T cell differentiation (15). function during the proliferative phase of infection. Similarly, deletion of the de novo methyltransferase DNMT3A in CD4 T cells did not affect lineage specification but permitted ectopic cytokine expression and increased lineage plasticity (16). *Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322; Interestingly, conditional deletion of DNMT1 at the time of CD8 † and Emory Vaccine Center, Emory University, Atlanta, GA 30322 T cell activation resulted in a diminished effector pool, fewer Received for publication May 24, 2013. Accepted for publication July 22, 2013. memory CD8 T cells, and a reduced ability to clear Ag (17). In This work was supported by National Institutes of Health Grants PO1AI 080192-05 contrast, deletion of the DNA methylation reader MBD2 had no and U19 AI05726-08 to (J.M.B. and R.A.), as well as by an American Cancer Society effect on proliferation but inhibited the formation of a functional Postdoctoral Fellowship (PF-09-134-01-MPC to B.A.Y.). memory compartment following viral challenge (18). These studies The sequences presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number demonstrate the importance of DNA methylation in maintaining GSE44638. phenotypic programs during the rapid proliferation of effector CD8 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Emory Univer- T cells and indicate that correct interpretation of methylated se- sity, 1510 Clifton Road, Room 3001, Atlanta, GA 30322. E-mail address: jmboss@ quences is functionally important for the adaptive immune response. emory.edu The dynamic nature of DNA methylation in hematopoietic cell The online version of this article contains supplemental material. types has been cataloged during early differentiation of the myeloid Abbreviations used in this article: cBS, clonal bisulfite sequencing; ChIP-seq, chro- and lymphoid lineages (19), mouse erythropoiesis (20), and between matin immunoprecipitation sequencing; D8, day 8; DMR, differentially methylated region; ENCODE, Encyclopedia of DNA Elements; ES, embryonic stem; FDR, human regulatory T cells and naive CD4 cells (21). However, none false discovery rate; GO, Gene Ontology; H3K27ac, histone H3 lysine 27 acetylated; of these studies profiled a clonal or Ag-specific population of cells. H3K4me, histone H3 lysine 4 methyl; H3K27me, histone H3 lysine 27 methyl; LCMV, lymphocytic choriomeningitis virus; MeDIP, methyl DNA immunoprecipitation; A recent study in CD4 T cells demonstrated that developmental MeDIP-seq, methyl DNA immunoprecipitation sequencing; PDE, putative distal en- TCR-specific signaling can establish a pre-existing methylation hancer; RefSeq, NCBI Reference Sequence Database; rpm, reads per million; TSS, profile, such that only CD4 T cells that upregulated Foxp3 and transcription start site. exhibited a specific methylation epitype were able to differentiate Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 into the regulatory T cell lineage (22). These data suggest epi- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301395 3420 EFFECTOR CD8 T CELL EPIGENETICS genetic heterogeneity in the naive CD4 T cell pool and highlight to the manufacturer’s recommended protocol (Illumina). Following agarose the importance and value of profiling the linear differentiation of gel size selection, naive and D8 effector CD8 T cell samples were im- m cells that possess a single TCR. munoprecipitated with 1 g of an anti-5-methylcytosine Ab (Eurogentec) at 4˚C overnight. Methylated DNA was purified, and one third of the Dynamic DNA methylation in CD8 T cells has been studied only sample was amplified for 12 cycles by PCR along with the input fraction at the single gene level. The effector cytokine Ifng and inhibitory to generate sequencing libraries. Libraries were quantitated by Agilent receptor Pdcd1 genes are methylated and silenced in naive CD8 BioAnalyzer, and each library was sequenced using a single-end, 50-bp T cells, demethylated and expressed at the effector stage, and protocol on a single lane of a HiSeq 2000 instrument at the Southern California Genotyping Consortium. All sequencing data are available at remethylated when expression is silenced in memory CD8 T cells the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/ (23, 24). Methylation