Multiple layers of transcriptional regulation by PLZF in NKT-cell development Ai-Ping Maoa,b, Michael G. Constantinidesa,b, Rebecca Mathewa,b, Zhixiang Zuoc, Xiaoting Chend,e,f,g, Matthew T. Weirauchd,e,f,g, and Albert Bendelaca,b,1 aCommittee on Immunology, University of Chicago, Chicago, IL 60637; bDepartment of Pathology, University of Chicago, Chicago, IL 60637; cState Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China; dCenter for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; eDivision of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; fDivision of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and gDepartment of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 Edited by Christophe Benoist, Harvard Medical School, Boston, MA, and approved May 20, 2016 (received for review January 30, 2016) The transcription factor PLZF [promyelocytic leukemia zinc finger, effector cells capable of producing both type 1 and type 2 cytokines encoded by zinc finger BTB domain containing 16 (Zbtb16)] is induced (9, 10), suggesting that PLZF is not only required, but also suffi- during the development of innate and innate-like lymphocytes to cient, for acquisition of the innate T-cell effector program. Notably, direct their acquisition of a T-helper effector program, but the molec- PLZF was recently found to be transiently expressed during the ular mechanisms involved are poorly understood. Using biotinylation- development of innate lymphoid cells (ILCs), defining a common based ChIP-seq and microarray analysis of both natural killer T (NKT) dedicated precursor to ILCs, the ILCP,andtosignificantlyimpact cells and PLZF-transgenic thymocytes, we identified several layers of the development and function of ILC lineages (11, 12). Taken to- regulation of the innate-like NKT effector program. First, PLZF bound gether, these findings indicate a broad defining role of PLZF in the and regulated genes encoding cytokine receptors as well as homing differentiation of several innate and innate-like lymphocytes. and adhesion receptors; second, PLZF bound and activated T-helper– The molecular mechanisms underlying these remarkable specific transcription factor genes that in turn control T-helper–specific properties of PLZF are only partially understood. In a γδTCR programs; finally, PLZF bound and suppressed the transcription of transgenic mouse model, the genetic inactivation of PLZF was Bach2, a potent general repressor of effector differentiation in naive found to compromise expression of the transcription factors Id2 T cells. These findings reveal the multilayered architecture of the and c-Maf, which are important for the survival of NKT cells in transcriptional program recruited by PLZF and elucidate how a single the liver and the production of IL-4, respectively (13). PLZF is transcription factor can drive the developmental acquisition of a essential for the expression of genes encoding cell surface receptors broad effector program. that shape responses to external stimuli, including costimulatory receptor Icos and cytokine receptors Il12rb1 and Il18r1, as well as PLZF | NKT | lymphocyte | development Cd44. In addition, PLZF down-regulates Sell, which encodes the homing receptor CD62L. The precise molecular mechanisms un- derlying these regulatory properties, and in particular the question atural killer (NK) T (NKT) cells are a conserved population of αβ of whether PLZF directly or indirectly controls the expression of Ninnate-like T cells that express CD1d-restricted semi-invariant effector genes, have not yet been elucidated, however. α α T-cell receptors (TCRs), using mostly the V 14-J 18 chain in mouse Here, using biotinylation-based ChIP-seq and microarray α α β β β (V 24-J 18 in human) combined with variable V 8, V 7, and V 2 analysis, we identified the PLZF target genes in NKT cells. We β (V 11 in human) chains, which confer recognition of conserved self found that many of the genes involvedintheacquisitionofaninnate and foreign lipids (1–3). NKT cell precursors arise during thymic – + + effector program, including T-helper specific transcription factors, development at the CD4 CD8 double-positive stage and undergo massive expansion on interaction with CD1d ligands expressed on Significance cortical thymocytes. They also characteristically acquire an effector CD44hiCD62Llo phenotype along with receptors of the NK cell lineage. The innate-like effector functions of NKT cells are illustrated Natural killer T (NKT) cells are a specialized population of in- by the cells’ ability to promptly secrete large quantities of both nate-like T cells that acquire their effector program during IL-4 and IFN-γ either after TCR activation or on exposure to development under the control of the transcription factor PLZF tissue- and antigen-presenting cell-derived cytokines, such as IL- (promyelocytic leukemia zinc finger, encoded by Zbtb16). To 25+IL-33 and IL-12+IL-18, respectively. Whereas C57BL/6 mice elucidate the mechanisms underlying this unique property of produce predominantly NKT cells with a T-bet–dependent type PLZF, we performed ChIP-seq and microarray analysis of NKT 1 helper phenotype (NKT1), other strains, including BALB/c, cells and PLZF-transgenic T cells, which revealed direct regula- also express substantial populations of so-called NKT2 and tion of effector genes and of T-helper–specific transcription NKT17 cells with polarized type 2 and type 17 helper programs factors. Notably, PLZF also bound and repressed Bach2, a global controlled by GATA3 and RORγt, respectively. repressor of effector differentiation. Thus, multiple layers of The BTB-zinc finger transcription factor PLZF (promyelocytic positive and negative regulation coordinate the induction of leukemia zinc finger, encoded by Zbtb16), is specifically expressed the innate effector program by PLZF. in NKT cells, but not in conventional T cells or NK cells, and di- rects the acquisition of several key components of the NKT cell Author contributions: A.-P.M., M.G.C., R.M., and A.B. designed research; A.-P.M., M.G.C., effector program during development, including cytokine and mi- R.M., X.C., and M.T.W. performed research; A.-P.M., M.G.C., R.M., Z.Z., X.C., M.T.W., and gration properties (4–7). Mutations or deletion of Zbtb16 abrogate A.B. analyzed data; and A.-P.M. and A.B. wrote the paper. both the expansion and the effector-memory differentiation of The authors declare no conflict of interest. NKT cells, resulting in reversal to a naive phenotype and re- This article is a PNAS Direct Submission. distribution to the lymph nodes and circulating blood. PLZF ex- Data deposition: The ChIP-seq and microarray data reported in this paper have been pression is also essential for the development of other innate-like T deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo cells, including MR1-specific semi-invariant αβ T cells and γδ T (accession no. GSE81772). cells expressing the Vγ1.1-Vδ6.3 TCR (4, 8). Importantly, ectopic 1To whom correspondence should be addressed. Email: [email protected]. expression of PLZF in the CD4 lineage during thymic development This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + hi lo converts conventional naïve CD4 thymocytes into CD44 CD62L 1073/pnas.1601504113/-/DCSupplemental. 7602–7607 | PNAS | July 5, 2016 | vol. 113 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1601504113 Downloaded by guest on September 28, 2021 cytokine receptors, and other key surface receptors, are directly A B n = 6865, R = 0.80 bound by PLZF. Notably, we also found that PLZF binds and n = 2579, R = 0.92 suppresses Bach2, a broad and potent regulator of effector pro- grams. These results elucidate how a single transcription factor A/F/PLZF tg thy NKT can act at multiple levels to regulate the developmental acquisi- tion of the innate effector gene program. 2619 2579 1667 -2 0 2 4 6 Results Tg thy log2(RPM) PLZF Predominantly Binds DNA at Regulatory Sites Carrying Consensus -4 -4 0-2 2 4 6 Sequences for ETS, RUNX, and E Proteins. We identified the direct NKT log2(RPM) targets of PLZF in NKT cells through ChIP-seq analysis. Because ChIP-seq with various anti-PLZF antibodies was not successful in C Exons D NKT cells, we generated a transgenic strain expressing a biotin 5’ UTR acceptor peptide and a 3×Flag at the N terminus of PLZF (A/F/ Introns Upstream PLZF tg) under control of the Cd4 promoter, and crossed it to mice expressing a bacterial biotin ligase BirA transgene. In these mice, PLZF could be specifically biotinylated in vivo without al- Density tering its interaction with major binding partners, such as CUL3 and HDAC1, or its effector-promoting function (Fig. S1). We used 3’ UTR Downstream 0.00 0.01 0.02 0.03 magnetic streptavidin bead pull-down of chromatin from A/F/PLZF Intergenic -100 10 20304050 tg; BirA tg thymocytes (referred to as Tg thy) or from purified Vα14- Distance to nearest TSS (Kb) Jα18 tg; A/F/PLZF tg; BirA tg NKT thymocytes (referred to as NKT cells) before DNA sequencing. Using the model-based analysis of E F ChIP-seq (MACS) peak calling software with a P value threshold of Motif No. P E protein 1e-5, we identified 5,198 peaks in the Tg thy and 4,246 peaks in the ETS 1034 7e-79 ETS NKT cells, of which 2,579 peaks were shared (Fig. 1 A and B). RUNX Visual comparison of peaks in the Tg thy and NKT cells con- ETS & E protein firmed that the binding landscape of PLZF was largely overlapping E protein 662 1e-34 in both mouse models.