H3K27 Methylation Dynamics During CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3
Total Page：16
File Type：pdf, Size：1020Kb
H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3 This information is current as Sarah A. LaMere, Ryan C. Thompson, Xiangzhi Meng, H. of September 24, 2021. Kiyomi Komori, Adam Mark and Daniel R. Salomon J Immunol published online 25 September 2017 http://www.jimmunol.org/content/early/2017/09/23/jimmun ol.1700475 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/09/23/jimmunol.170047 Material 5.DCSupplemental 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 • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 25, 2017, doi:10.4049/jimmunol.1700475 The Journal of Immunology H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3 Sarah A. LaMere,1 Ryan C. Thompson, Xiangzhi Meng, H. Kiyomi Komori,2 Adam Mark,1 and Daniel R. Salomon3 The changes to the epigenetic landscape in response to Ag during CD4 T cell activation have not been well characterized. Although CD4 T cell subsets have been mapped globally for numerous epigenetic marks, little has been done to study their dynamics early after activation. We have studied changes to promoter H3K27me3 during activation of human naive and memory CD4 T cells. Our results show that these changes occur relatively early (1 d) after activation of naive and memory cells and that demethylation is the predominant change to H3K27me3 at this time point, reinforcing high expression of target genes. Additionally, inhibition of the H3K27 demethylase JMJD3 in naive CD4 T cells demonstrates how critically important molecules required for T cell differen- tiation, such as JAK2 and IL12RB2, are regulated by H3K27me3. Our results show that H3K27me3 is a dynamic and important Downloaded from epigenetic modification during CD4 T cell activation and that JMJD3-driven H3K27 demethylation is critical for CD4 T cell function. The Journal of Immunology, 2017, 199: 000–000. D4 T cells are an integral component of the adaptive which confer another layer of complexity to this already enigmatic immune response, facilitating Ag-specific “memory” via system. Epigenetic studies in T cells have been more limited and http://www.jimmunol.org/ C adaptation following exposure to pathogens and other in this early phase of investigation are still largely descriptive, foreign invaders. The molecular mechanisms responsible for the although clear correlations between epigenetic modifications and transition to memory are still poorly understood and likely stem T cell differentiation have been illustrated with genome-wide from multiple epigenetic processes in the cell following activation studies (6–13). (1). Numerous epigenetic marks have been explored in cancer and H3K27me3, in particular, is a conventionally “repressive” his- embryonic stem (ES) cell differentiation, including DNA methyl- tone modification (9, 14) that plays a role in CD4 T cell differ- ation, histone modifications, and chromatin regulators, all of which entiation (15, 16). However, its dynamics during CD4 T cell appear to play roles in modulating gene expression (2–5). These activation and early differentiation has not been explored, and marks can target multiple regulatory regions, including promoters, its role in early differentiation is still poorly characterized. One by guest on September 24, 2021 enhancers, superenhancers, gene bodies, and intergenic regions, study performed global mapping of H3K27me3 after in vitro polarization of murine CD4 T cell differentiation to reveal that the presence of H3K27me3 in Th-related genes corresponded to Department of Molecular and Experimental Medicine, The Scripps Research Insti- tute, La Jolla, CA 92037 silencing of those genes in their opposing lineages (7). A study of 1Current address: University of California, San Diego, La Jolla, CA. murine CD8 T cell dynamics after viral infection also demon- 2Current address: Synthetic Genomics, La Jolla, CA. strated a profound loss of H3K27me3 following activation, sup- porting the role of repressive H3K27me3 marks in naive CD8 3Daniel R. Salomon is deceased. T cells to maintain a state of restraint during rest (8). ORCIDs: 0000-0002-4612-0425 (S.A.L.); 0000-0002-0450-8181 (R.C.T.); 0000- 0002-4743-9972 (X.M.). Two demethylases, JMJD3 and UTX, are known to catalyze Received for publication March 31, 2017. Accepted for publication August 21, 2017. H3K27me3 demethylation. Recently an exploration into the role of This work was supported by National Institutes of Health Grants U19 AI063603 (to Jmjd3 in mice upon the regulation of CD4 T cell differentiation D.R.S.), 1TL1 TR001113-01 (to S.A.L.), and T32 DK007022-30 (to H.K.K.). H.K.K. found that a conditional knockout of Jmjd3 resulted in skewing to was the recipient of a Juvenile Diabetes Research Foundation Postdoctoral Fellow- Th2 and Th17 differentiation (15). Both demethylases are required ship, X.M. received support from the Predoctoral Mendez National Institute of Trans- plantation’s Fund, and Verna Harrah Research Funds provided support to the for in vivo thymocyte differentiation in mice (17). In ES cells, Salomon laboratory. JMJD3 appears to delocalize polycomb repressive complex (PRC) The raw data for ChIP and RNA sequencing presented in this article have been proteins, which is essential for further development. Additionally, submitted to the Gene Expression Omnibus under accession number GSE73214 UTX is a component of the MLL complex, strongly suggesting (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73214). that H3K27 demethylation can be coupled with the “activating” Address correspondence and reprint requests to Dr. Sarah A. LaMere at the current address: Department of Pathology, University of California, San Diego, 9500 Gilman methylation of H3K4 by MLL (18). UTX is ubiquitously Drive, La Jolla, CA 92093. E-mail address: [email protected] expressed in tissues and is also important for embryonic cell de- The online version of this article contains supplemental material. velopment (19). In contrast, JMJD3 is commonly induced during Abbreviations used in this article: B2M, b-2 microglobulin; CGI, CpG island; ChIP, inflammation or upon exposure to antigenic or oncogenic stimuli chromatin immunoprecipitation; ChIP-seq, ChIP sequencing; CPM, counts per mil- (18, 20, 21). JMJD3 inhibits somatic cell reprogramming in in- lion; ES, embryonic stem; FDR, false discovery rate; FPKM, fragments per kilobase per million fragments sequenced; IDR, Irreproducible Discovery Rate; IPA, Ingenu- ducible pluripotent stem cells, whereas UTX is essential for it, ity Pathway Analysis; PRC, polycomb repressive complex; qPCR, quantitative PCR; suggesting contrasting roles for these two enzymes (22, 23). The qRT-PCR, quantitative real time PCR; RNA-seq, RNA sequencing; siRNA, small two enzymes also play contrasting roles in acute lymphoblastic interfering RNA; TSS, transcription start site. leukemia, with JMJD3 inducing the neoplastic process and UTX Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 acting as a tumor suppressor (24). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700475 2 H3K27 METHYLATION OF CD4 T CELLS In the current study, we have examined the dynamics of added to each ChIP assay and incubated at 4˚C with rotation for 2 h. Beads promoter-associated H3K27me3 upon activation of human naive were washed three times in low-salt wash buffer and then two times with and memory CD4 T cells. We find that, in both subsets, profound high-salt wash buffer (0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl) with 5 min of rotation at 4˚C for demethylation of H3K27 is observed by 1 d after activation, which each wash. Beads were resuspended in 150 ml of elution buffer (1% SDS, is in contrast to H3K4 methylation, for which changes are not ob- 0.1 M NaHCO3) and incubated in a ThermoMixer at 65˚C for 30 min at served until days later (25). Mapping specific states of H3K27me3 1200 rpm to reverse cross-linking. Two microliters of Proteinase K to known immune pathways demonstrates that loss of H3K27me3 (Invitrogen) was added to each sample, and 6 ml of 5 M NaCl was added for a total concentration of 200 mM. Samples were then incubated in a early in activation corresponds to pathways crucial to T cell func- ThermoMixer at 65˚C overnight at 1200 rpm. Eluted samples were re- tion, including T cell activation and the JAK/STAT pathways. moved from the beads and purified using the QIAGEN QIAquick PCR Mechanistic studies by perturbation of H3K27 demethylation with a purification kit, per the manufacturer’s instructions. small-molecule inhibitor (GSK-J4) and small interfering RNA Preparation of sequencing libraries for ChIP-seq and deep (siRNA) knockdown of the two H3K27 demethylases confirms that RNA-seq H3K27 demethylation by JMJD3 is important for key members of early differentiation–related pathways. Altogether, these data con- RNA for RNA-seq was isolated from purified cells using an AllPrep kit firm that H3K27 is a highly dynamic epigenetic modification in (QIAGEN), following the manufacturer’s instructions, and purified total CD4 T cells during early activation, and the nature of these dynamic RNA was converted to cDNA using Ovation RNA-seq (NuGEN), followed by S1 endonuclease digestion (Promega), as previously described (26).