Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Bendelac, A., Savage, P.B. & Teyton, L. The biology of NKT cells. Annu. Rev. Immunol. 25, 297–336 (2007).
Rossjohn, J., Pellicci, D.G., Patel, O., Gapin, L. & Godfrey, D.I. Recognition of CD1d-restricted antigens by natural killer T cells. Nat. Rev. Immunol. 12, 845–857 (2012).
Kawano, T. et al. CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides. Science 278, 1626–1629 (1997).
Cui, J. et al. Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors. Science 278, 1623–1626 (1997).
Bendelac, A. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J. Exp. Med. 182, 2091–2096 (1995).
Lazarevic, V. et al. The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells. Nat. Immunol. 10, 306–313 (2009).
Kovalovsky, D. et al. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nat. Immunol. 9, 1055–1064 (2008).
Savage, A.K. et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29, 391–403 (2008).
Seiler, M.P. et al. Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT cell lineage differentiation in response to TCR signaling. Nat. Immunol. 13, 264–271 (2012).
Matsuda, J.L. et al. Homeostasis of Vα14i NKT cells. Nat. Immunol. 3, 966–974 (2002).
Townsend, M.J. et al. T-bet regulates the terminal maturation and homeostasis of NK and Vα14i NKT cells. Immunity 20, 477–494 (2004).
Engel, I. & Kronenberg, M. Transcriptional control of the development and function of Vα14i NKT cells. Curr. Top. Microbiol. Immunol. 381, 51–81 (2014).
Cohen, N.R. et al. Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells. Nat. Immunol. 14, 90–99 (2013).
Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).
Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).
Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).
Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).
Simon, J.A. & Kingston, R.E. Mechanisms of polycomb gene silencing: knowns and unknowns. Nat. Rev. Mol. Cell Biol. 10, 697–708 (2009).
Bernstein, B.E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).
Agger, K. et al. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449, 731–734 (2007).
Lan, F. et al. A histone H3 lysine 27 demethylase regulates animal posterior development. Nature 449, 689–694 (2007).
Lee, M.G. et al. Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science 318, 447–450 (2007).
De Santa, F. et al. The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell 130, 1083–1094 (2007).
Heintzman, N.D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).
Welstead, G.G. et al. X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner. Proc. Natl. Acad. Sci. USA 109, 13004–13009 (2012).
Mansour, A.A. et al. The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming. Nature 488, 409–413 (2012).
Li, Q. et al. Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T-cell differentiation. Nat. Commun. 5, 5780 (2014).
Lee, S., Lee, J.W. & Lee, S.K. UTX, a histone H3-lysine 27 demethylase, acts as a critical switch to activate the cardiac developmental program. Dev. Cell 22, 25–37 (2012).
Thieme, S. et al. The histone demethylase UTX regulates stem cell migration and hematopoiesis. Blood 121, 2462–2473 (2013).
Ntziachristos, P. et al. Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia. Nature 514, 513–517 (2014).
van Haaften, G. et al. Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer. Nat. Genet. 41, 521–523 (2009).
Cook, K.D. et al. T follicular helper cell-dependent clearance of a persistent virus infection requires T cell expression of the histone demethylase UTX. Immunity 43, 703–714 (2015).
Manna, S. et al. Histone H3 lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation. Nat. Commun. 6, 8152 (2015).
Dobenecker, M.W. et al. Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation. J. Exp. Med. 212, 297–306 (2015).
Benlagha, K., Wei, D.G., Veiga, J., Teyton, L. & Bendelac, A. Characterization of the early stages of thymic NKT cell development. J. Exp. Med. 202, 485–492 (2005).
Lee, Y.J., Holzapfel, K.L., Zhu, J., Jameson, S.C. & Hogquist, K.A. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat. Immunol. 14, 1146–1154 (2013).
Gapin, L., Matsuda, J.L., Surh, C.D. & Kronenberg, M. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat. Immunol. 2, 971–978 (2001).
Mao, A.P. et al. Multiple layers of transcriptional regulation by PLZF in NKT cell-cell development. Proc. Natl. Acad. Sci. USA 113, 7602–7607 (2016).
Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
Pinello, L., Xu, J., Orkin, S.H. & Yuan, G.C. Analysis of chromatin-state plasticity identifies cell-type-specific regulators of H3K27me3 patterns. Proc. Natl. Acad. Sci. USA 111, E344–E353 (2014).
Kanda, M. et al. Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells. Proc. Natl. Acad. Sci. USA 113, E3394–E3402 (2016).
Engel, I. et al. Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nat. Immunol. 17, 728–739 (2016).
Williams, K.L. et al. BATF transgenic mice reveal a role for activator protein-1 in NKT cell development. J. Immunol. 170, 2417–2426 (2003).
Jordan-Williams, K.L., Poston, S. & Taparowsky, E.J. BATF regulates the development and function of IL-17 producing iNKT cells. BMC Immunol. 14, 16 (2013).
Lawson, V.J., Maurice, D., Silk, J.D., Cerundolo, V. & Weston, K. Aberrant selection and function of invariant NKT cells in the absence of AP-1 transcription factor Fra-2. J. Immunol. 183, 2575–2584 (2009).
Thomsen, M.K. et al. JUNB/AP-1 controls IFN-γ during inflammatory liver disease. J. Clin. Invest. 123, 5258–5268 (2013).
Miller, S.A., Mohn, S.E. & Weinmann, A.S. Jmjd3 and UTX play a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression. Mol. Cell 40, 594–605 (2010).
Pereira, R.M. et al. Jarid2 is induced by TCR signalling and controls iNKT cell maturation. Nat. Commun. 5, 4540 (2014).
Vahedi, G. et al. Super-enhancers delineate disease-associated regulatory nodes in T cells. Nature 520, 558–562 (2015).
Passegué, E., Wagner, E.F. & Weissman, I.L. JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell 119, 431–443 (2004).
Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).
Johnson, W.E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).
Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).
McLean, C.Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).
Kurachi, M. et al. The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells. Nat. Immunol. 15, 373–383 (2014).
Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Rousseeuw, P.J. Silhouettes: A graphical aid to the interpretation and validation of cluster analysis. J. Comput. Appl. Math. 20, 53–65 (1987).
Mathelier, A. et al. JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles. Nucleic Acids Res. 42, D142–D147 (2014).
We thank the US National Institutes of Health Tetramer Facility for CD1d-tetramers, the Center for Cancer Computational Biology sequencing facility at the Dana-Farber Cancer Institute for Illumina HiSeq2000 sequencing, the Microarray core facility at the Dana-Farber Cancer Institute Molecular Biology Core Facilities for microarray analysis, and the Dana-Farber Cancer Institute Jimmy Fund FACS core facility for cell sorting. Supported by the Howard Hughes Medical Institute (S.H.O.), the US National Institutes of Health (P30DK0492 to S.H.O., and RO1 AI083426 to F.W.), the Cancer Research Institute Predoctoral Emphasis Pathway in Tumor Immunology (S.B.), the National Research Foundation of Korea (2012R1A6A3A03040248 to J.H.K.) and the National Human Genome Research Institute of the US National Institutes of Health (Career Development Award K99HG008399 to L.P.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Utx locus in UTX-targeted embryonic stem cells (ESCs) that were provided from EUCOMM. (b) PCR analysis verifying loxP sites targeting exon 3 of Utx in ESC clones. (c) Targeting strategy for Jmjd3 locus to delete exons 17-19. (d) Southern blot analysis of Jmjd3-targeted ESC clones. (e) Representative genotyping of conditional UTX-or JMJD3-KO mice. (f) Analysis of Utx or Jmjd3 mRNA levels in blood, DP thymocytes, or iNKT cells by qRT-PCR using deletion-specific primers. Data are mean ± s.d. from three independent experiments.
(a) Conditional UTX-deficient (UTX KO) mice or wild-type (WT) littermates were injected intraperitoneally with 2 μg α-GalCer. Two hours later, lymphocytes from thymus and liver were collected for intracellular staining of IFN-γ and IL-4, prior to analysis by flow cytometry. Additionally, thymic iNKT cells were restimulated with PMA and Ionomycin for 4 hours before analysis. (b) Flow cytometry analysis of WT or UTX KO thymic iNKT cells by measurement of transcription factors T-bet, PLZF, and RORγt, gated on CD1d-tetramer+ cells. Shown are the absolute cell counts of NKT1, NKT2, or NKT17 cells among thymic iNKT cells from WT or UTX KO (n = 3). (c) Analysis of CD1d expression on DP thymocytes from WT or UTX KO mice using flow cytometry. (d) DP thymocytes were isolated from WT or UTX KO mice. Subsequently, mRNA transcripts of Vα14-Jα18 TCR were analyzed by qRT-PCR and normalized to constant alpha chain (Cα) amplification. Y-axis depicts relative expression (n = 5). Results in (b-d) are representative of three independent experiments. Data are mean ± s.e.m. **P < 0.01 as analyzed by unpaired t-test.
Supplementary Figure 3 Characterization of gene-expression alterations in control and UTX-KO thymic iNKT cells.
(a) Heat map of gene expression microarray (WT; n=5, KO; n=4). (b) GSEA of downregulated or (c) upregulated genes in UTX-deficient (KO) iNKT cells compared to wild-type (WT). ES, enrichment score; NES, normalized ES; FDR, false discovery rate. (d) iNKT cells in stage 0 and stage 1-3 have reduced signature gene expression in UTX KO as assessed by qRT-PCR. (e) Confirming the loss of UTX transcripts in stage 0 and stage 1-3 iNKT cells in UTX KO as assessed by qRT-PCR. Data are mean ± s.d. from three independent experiments.
(a,b) Chromosomal distribution of WT-specific (a) or UTX-KO-specific (b) H3K27me3 peaks in iNKT cells. (c,d) Distribution of WT-specific (c) or UTX-KO-specific (d) H3K27me3 peaks within the genome of iNKT cells.
Supplementary Figure 5 Characterization of regions with H3K27me3 peaks in control and UTX-KO iNKT cells.
(a) GREAT analysis of WT-specific H3K27me3 peaks (n = 4,250). (b) GREAT analysis of UTX KO-specific H3K27me3 peaks (n = 9,645). (c,d) Assessing statistical significance of H3K27me3 distribution around the promoters of downregulated (c) and upregulated (d) genes between WT and UTX-deficient (KO) iNKT cells based on permutation test where 100,000 permutations were used to calculate the distribution of the difference between two average profiles. Black line depicts empirical null model, dotted red line indicates the observed value for the measured profiles. (e,f) GREAT analysis of all downregulated (e) or upregulated (f) genes in UTX KO iNKT cells compared to WT controls.
Supplementary Figure 6 Cluster analysis of the chromatin landscape around gene promoters in control or UTX-KO iNKT cells.
Heat maps represent the abundance of H3K4me3 and H3K27me3 marks around the promoters of downregulated (a) or upregulated genes (b) in UTX-deficient (KO) iNKT cells compared to wild-type (WT). Genes are subdivided into different clusters dependent on their histone mark pattern. C1-4 = clusters 1 to 4. (c) Integrating gene expression with chromatin state for upregulated genes. Average abundance of H3K27me3 and H3K4me3 around upregulated gene promoters is depicted as z-score in reads per million (rpm). Average profiles are grouped in three clusters of upregulated genes with WT in blue and KO in green. The upper panel shows expression of upregulated genes as logarithmic fold change (log2FC). (d) GREAT analysis of downregulated genes in UTX-deficient iNKT cells in cluster 3 and cluster 4.
Mixed bone marrow-chimeric mice were generated using a 1:1 ratio of wild-type (WT) and conditional UTX-deficient (KO) bone marrow cells for transfer into Rag2-/- hosts (n = 5). Twelve weeks later, WT cells were detected by anti-CD45.1, and frequencies of thymic iNKT cells were analyzed by flow cytometry. (a) Depicted are the ratios of WT to KO iNKT cells gated on the different maturational stages. (b) Bar graph depicting the results from (a). (c) In the same experiment as described in (a), lymphocytes from liver were analyzed for iNKT cells (CD3+tetramer+) as well as conventional T cells (CD3+tetramer-). Depicted are the ratios of WT to KO T lymphocytes. Results are representative of two independent experiments.
(a,b) Epigenetic landscape of the super-enhancers for Tbx21 (a) and Il2rb (b) that exhibit UTX-dependent accessibility. Depicted are the ChIP-Seq tracks of H3K27ac and heat maps of ATAC-Seq, H3K27me3, and H3K4me3 in WT or UTX-deficient (KO) iNKT cells around the super-enhancer (SE) for Tbx21 (a) and Il2rb (b). Zoomed in tracks in Fig. 8f and Fig. 8g are shown in black rectangle and highlighted with a star. (c) GREAT analysis of the super-enhancer elements in iNKT cells. (d) GREAT analysis of the SE elements in iNKT cells that exhibit UTX-dependent accessibility. (e) Transcription factor target motif analysis using Haystack in iNKT cell SE elements that exhibit UTX-dependent accessibility. Depicted are the binding motifs of RELA and BHLHE40.
Supplementary Figures 1–8 (PDF 1767 kb)
List of genes that exhibit UTX-dependent chromatin regulation in cluster 3 and cluster 4 (XLS 47 kb)
List of JunB target genes in cluster 3 (XLSX 12 kb)
List of iNKT super enhancers (XLSX 59 kb)
List of super-enhancer regions that lost accessibility in UTX-KO (WT-specific) or gained accessibility in UTX-KO (KO-specific) (XLS 34 kb)
About this article
Cite this article
Beyaz, S., Kim, J., Pinello, L. et al. The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells. Nat Immunol 18, 184–195 (2017). https://doi.org/10.1038/ni.3644
Cell Reports (2021)
Deciphering the multifaceted roles of TET proteins in T‐cell lineage specification and malignant transformation
Immunological Reviews (2021)
Biochemical Society Transactions (2021)
Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells
The EMBO Journal (2020)
Nature Reviews Immunology (2020)