Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation

This information is current as Keito Inaba, Takuya Yashiro, Ikumi Hiroki, Ryosuke of October 3, 2021. Watanabe, Kazumi Kasakura and Chiharu Nishiyama J Immunol published online 1 July 2020 http://www.jimmunol.org/content/early/2020/06/30/jimmun ol.1901008 Downloaded from

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 1, 2020, doi:10.4049/jimmunol.1901008 The Journal of Immunology

Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation

Keito Inaba,*,†,1 Takuya Yashiro,*,1 Ikumi Hiroki,* Ryosuke Watanabe,* Kazumi Kasakura,* and Chiharu Nishiyama*

PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and . The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of immunology. To reveal the mechanism by which Pdcd1lg2 expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell–specific transcription factors PU.1 and IRF4 de- creased PD-L2 expression in GM-CSF–induced mouse bone marrow–derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene Downloaded from via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 , which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow–derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic http://www.jimmunol.org/ regulator in DCs. The Journal of Immunology, 2020, 205: 000–000.

he Pdcd1lg2 gene encodes PD-L2 (CD273, -DC), that PD-L2 also binds to RGMb (DORAGON) (1, 2), and the which has been identified as the second ligand of pro- binding of PD-L2 to RGMb or PD-1 suppresses T cell activation T grammed cell death-1 (PD-1). Although numerous studies (3), induces Th1 responses (4), and regulates Ab production by have demonstrated the anticancer effects of checkpoint inhibitors B-1 cells (5). Furthermore, the involvement of PD-L2 in malaria targeting PD-1 and/or PD- (another ligand of PD-1), the bio- and asthma was also demonstrated in studies using mouse models logical significance of PD-L2 and the relationship between PD-L2 (6, 7). These findings suggest the possibility that PD-L2, which and its target molecules is still unclear. Recently, it was reported contributes to several events in adaptive immunity, may be a by guest on October 3, 2021 therapeutic target for immune-related diseases. When PD-L2 and PD-L1 are compared, their gene expression *Department of Biological Science and Technology, Faculty of Industrial Science profiles are shown to be strikingly different. PD-L1 is ubiquitously and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; and †Department of Computational Biology and Medical Science, expressed, whereas the expression of PD-L2 is highly restricted Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8568, to APCs, especially monocytes (8). In previous reports of the Japan transcriptional regulation of the Pdcd1lg2 gene, it was revealed 1K.I. and T.Y. contributed equally to this work. that IL-4 and IL-13 transactivate the Pdcd1lg2 gene via STAT6 in ORCIDs: 0000-0002-9319-3467 (T.Y.); 0000-0002-9604-3632 (R.W.); 0000-0002- activated macrophages and dendritic cells (DCs) (9, 10). Because 4082-9097 (C.N.). STAT6 is a ubiquitous molecule, the mechanism by which the Received for publication August 21, 2019. Accepted for publication June 1, 2020. Pdcd1lg2 gene is expressed in a monocyte-specific manner is still This work was supported by Japan Society for the Promotion of Science (JSPS) unknown. Therefore, it is necessary to identify the transcription Grant-in-Aid for Scientific Research (B) 20H02939 (to C.N.), JSPS Grant-in-Aid for Scientific Research (C) 19K05884 (toT.Y.)and19K08920(toK.K.),the factor(s) involved in cell type-specific Pdcd1lg2 gene expression. Ministry of Education, Culture, Sports, Science and Technology-Supported Pro- The hematopoietic cell–specific transcription factor PU.1, gram for the Strategic Research Foundation at Private Universities (Translational which is encoded by the Spi1 gene, belongs to the Ets-family and Research Center, Tokyo University of Science), the Tokyo University of Science Grant for President’s Research Promotion (to C.N.), the Tojuro Iijima Foundation is essential for gene expression and the development of lymphoid for Food Science and Technology (to C.N. and T.Y.), and the Takeda Science and myeloid cells. A Spi1 gene knockout mouse exhibited severe Foundation (to C.N.). immunodeficiency, including the incomplete development and K.I. performed experiments, analyzed data, and wrote the paper; T.Y. designed re- production of DCs, macrophages, B cells, T cells, NK cells, and search, performed experiments, and analyzed data; I.H., R.W., and K.K. performed experiments; C.N. designed research and wrote the paper. neutrophils (11–16). PU.1 transactivates its target not only Address correspondence and reprint requests to Prof. Chiharu Nishiyama, Depart- as a monomer that binds to Ets motifs but also as a dimer with ment of Biological Science and Technology, Faculty of Industrial Science and Tech- IRF4 or IRF8 that binds to Ets-IRF composite element (EICE) nology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, sequences. Previously, studies, including ours, showed that PU.1 Japan. E-mail address: [email protected] plays a critical role in the expression of several specific genes Abbreviations used in this article: BMDC, bone marrow–derived DC; cDC, conven- tional DC; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP-sequencing; DC, important for the function of DCs (17–22). Based on these ob- ; EICE, Ets-IRF composite element; EMSA, electrophoretic mobility servations, we hypothesized that PU.1 is involved in the cell type- shift assay; HAT, histone acetyltransferase; MHCII, MHC class II; PD-1, pro- specific expression of the Pdcd1lg2 gene and investigated the grammed cell death-1; siRNA, small interfering RNA. molecular mechanism by which PU.1 regulates the Pdcd1lg2 gene Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 in the current study.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901008 2 PU.1/IRF4 TRANSACTIVATES THE PDCD1LG2 GENE IN DCs

Epigenetic regulation is involved in the determination of pCMV-Myc-IRF4, and the BglII/SalI–digested Myc-IRF4 cDNA frag- cell type-specific gene expression. In particular, conformational ment from pCMV-Myc-IRF4 was inserted into pIRES2-AcGFP1 to obtain changes in chromatin resulting from histone acetylation mediated pIRES2-AcGFP-Myc-IRF4. by histone acetyltransferase (HAT) and histone deacetylation Chromatin immunoprecipitation assay mediated by histone deacetylase, resulting in transactivation and Chromatin immunoprecipitation (ChIP) assays were performed as previ- suppression, respectively, are often observed in various cell types. ously described (22). Anti-PU.1 Ab (D-19), anti-IRF4 Ab (M-17) (Santa Although PU.1 is known to interact with p300 and GCN5, which Cruz Biotechnology), and anti-acetyl histone H3(K27) Ab (MABI0309; are typical HATs (23, 24), whether PU.1 affects histone acetylation MEDICAL & BIOLOGICAL LABORATORIES) were used. Goat IgG in the Pdcd1lg2 gene in DCs has not been clarified so far. (02-6202; Invitrogen) and mouse IgG1 (02-6100; Invitrogen) were used as isotype control Abs. The amount of precipitated DNA was determined In the current study, we identified PU.1 as a transcription factor by quantitative PCR using an Applied Biosystems StepOne Real-Time that regulates the Pdcd1lg2 gene via cis-enhancing elements in a PCR System. The nucleotide sequences of the primer sets used for the cooperative manner with IRF4 in DCs, and we also found that PCR of acetyl histone H3(K27) were as follows: mouse Pdcd1lg2 gene PU.1 modulates the p300-mediated histone acetylation of the CNS0 (forward primer, 59-GGCAGCTGACAAGAACAATGG-39; reverse 9 9 Pdcd1lg2 gene. primer, 5 -AGGCTCTCTCAAGCCGCTTA-3 ), CNS1 (forward primer, 59-TGTGGGAGGCAGGAGGAA-39; reverse primer, 59-CTTGGACCTT- CAAACCAATGG-39), CNS2 (forward primer, 59-GTCCTAATGACTC- CATCCCTAAGC-39;reverseprimer,59-ACACCTGTAGGACATTGCTGACA- Materials and Methods 39), CNS3 (forward primer, 59-CGCAGAGTGGATTTGAAACAAA-39; Mice and cells reverse primer, 59-CAGGGAGAAAAGTGACTAAATCAGAA-39), and Bone marrow–derived DCs (BMDCs) were generated from BALB/c mice CNS4 (forward primer, 59-AGAAACCTGGAGGCAGAAGCT-39; reverse purchased from Japan SLC (Hamamatsu, Japan) as previously described primer, 59-CAGCGGGCCAGTGAACAG-39). The nucleotide sequences of Downloaded from (17, 19). CD11c+ DCs were isolated by using a MACS separation system the primer sets used for the PCR of PU.1 and IRF4 were as follows: CNS2 with anti-mouse CD11c MicroBeads and an autoMACS (all from Miltenyi forward primer, 59-CGGCCTTACTTCCTAATTTCAAAC-39; reverse primer, Biotech). All animal experiments were performed in accordance with 59-CTCTGCCCCCATCACATAGTG-39) and CNS3 (forward primer, 59- the approved guidelines of the Institutional Review Board of Tokyo Uni- TGACAGCCAGCCCTGAGAA-39; reverse primer, 59-TCACAGAGGAA- versity of Science, Tokyo, Japan. The human embryonic kidney cell line ATGAAACCACTTG-39). The primer sets for CNS0, CNS1, and CNS4 were HEK293T was maintained as previously described (21). same as those used for acetyl histone H3(K27).

Knockdown by small interfering RNA introduction Electrophoretic mobility shift assay http://www.jimmunol.org/ Small interfering RNAs (siRNAs) for mouse Spi1 (Spi1-MSS257676), Irf4 The probe DNA and transcription factor were prepared as previ- (MSS205500, MSS205501), Irf8 (MSS236847), Ep300 (MSS220766), and ously described (17, 21). The fluorescence of the electrophoresis gels was control siRNA (Stealth RNAi siRNA Negative Universal Control) from detected using a Typhoon FLA 7000 image analyzer (GE Healthcare). Invitrogen was introduced into DCs with Nucleofector 2b (Lonza) using an Statistical analysis Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza), as previously described (21). A two-tailed Student t test was used to perform the statistical analysis. The p values ,0.05 were considered to be significant. Quantitative RT-PCR

The purification of total RNA and the synthesis of cDNA were performed Results by guest on October 3, 2021 using a ReliaPrep RNA Cell Miniprep System (Promega) and ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan), respectively. The mRNA Involvement of PU.1 and IRF4 in the expression of PD-L2 levels of Pdcd1lg2, Spi1, Irf4, Ep300 and Gapdh were determined with a in BMDCs StepOne Real-Time PCR System (Applied Biosystems) using THUN- DERBIRD Probe qPCR Mix (Toyobo) and the TaqMan Gene Expression To evaluate the involvement of PU.1 and IRF4 in PD-L2 expression Assays (Mm00488142_m1 for mouse Spi1 and 4352329E for rodent in BMDCs, we introduced Spi1 siRNA or Irf4 siRNA into Gapdh; Applied Biosystems); mRNA levels were also determined us- BMDCs. First, we confirmed that the transfection of siRNA for ing THUNDERBIRD SYBR qPCR Mix (Toyobo) with the following Spi1 and Irf4 significantly decreased the mRNA levels of Spi1 and synthesized oligonucleotides as primer sets: mouse Pdcd1lg2 (forward Irf4, respectively, and markedly reduced the amount of the target primer, 59-GAACCTGAGCTTACAACTTCATCCT-39; reverse primer, 59-ACGTCTACGGTGTACACTTCTTTAGG-39), mouse Irf4 (forward proteins in BMDCs (Fig. 1A, 1B). In these experimental condi- primer, 59-CCCCATTGAGCCAAGCATAA-39; reverse primer, 59-G- tions, the Pdcd1lg2 mRNA levels were significantly decreased by CAGCCGGCAGTCTGAGA-39), and mouse Ep300 (forward primer, the knockdown of PU.1 and IRF4 (Fig. 1A, 1B). In contrast, the 59-CCAAGCGCCTGCAAGAA-39; reverse primer, 59-TATCCTTGT- knockdown of IRF8, another typical partner of PU.1, did not affect AGTCATGGACAATACGTT-39). the Pdcd1lg2 mRNA level (Fig. 1C), whereas the mRNA level of Flow cytometry Il12b, which is a positive control gene that is cooperatively Flow cytometry for the detection of cell surface MHC class II (MHCII), transactivated by PU.1 and IRF8 (27), was significantly down- CD11b, CD11c, and PDCA-1 was performed as described in our previous regulated by the Irf8 siRNA transfection. We also performed flow report (21, 22). An FITC-conjugated anti-mouse PD-L2 Ab (MIH37; cytometry to define the cell surface expression level of PD-L2 and Miltenyi Biotech) was used to stain the BMDCs and Flt3L-BMDCs. The found that PD-L2 expression was mainly detected on MHCIIhigh/ intracellular staining was performed as previously described (25). CD11bint cells (so-called DC-like cells) rather than MHCIIint/ Western blotting analysis CD11bhigh cells (-like cells) (siNega in Fig. 1D, 1E). When PU.1 was knocked down, the number of DC-like cells and Western blotting analyses using anti-PU.1 Ab (D-19; Santa Cruz Bio- technology), anti-IRF4 Ab (M-17; Santa Cruz Biotechnology), and b-actin the PD-L2 expression level on DC-like cells were markedly de- (AC-15; Sigma-Aldrich) were performed as previously described (22). creased (Fig. 1D, bottom). A similar tendency was observed in IRF4 knockdown cells (Fig. 1E bottom) but not in IRF8 knock- Overexpression of PU.1 and IRF4 down cells (Fig. 1F). Furthermore, the intracellular staining of Expression plasmids, PU.1 (26), pIRES2-AcGFP1-Myc-IRF4 (generated as permeabilized cells showed that the cellular PD-L2 protein levels follows), and its control plasmid pIRES2-AcGFP1 (Clontech Laborato- in the total BMDCs were markedly and slightly reduced by the ries), were introduced into BMDCs by an electroporation with Nucleo- fector 2b. GFP+ cells were judged to be the plasmid-carrying cells. knockdown of PU.1 and IRF4, respectively (Fig. 1G). The above Mouse IRF4 cDNA isolated from pCR3.1-IRF4 (19) was inserted into knockdown experiments demonstrate that suppression of PU.1 and the EcoRI/KpnI site of pCMV-Myc-N (Clontech Laboratories) to generate IRF4 downregulated the expression of PD-L2 in BMDCs. Then, to The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Effects of PU.1 and IRF4 knockdown by siRNAs on the mRNA levels and cell surface expression levels in BMDCs. The mRNA expression of Pdcd1lg2 and the target gene in Spi1 siRNA-treated (A), Irf4 siRNA-treated (B), or Irf8 siRNA-treated (C) BMDCs at 48 h after siRNA transfection. The data represent the mean 6 SD of three independent assays performed in triplicate samples. The protein levels of PU.1 and IRF4 in siRNA-transfected cells at 48 h after transfection were determined by Western blotting analysis (A and B). *p , 0.05. Surface expression level of PD-L2 in DC-like cells and macrophage-like cells in Spi1 siRNA- (D), Irf4 siRNA- (E), or Irf8 siRNA-treated (F) BMDCs at 48 h after transfection. Shadow represents isotype control; line represents anti-PD-L2 Ab. Intracellular expression level of PD-L2 in the total cells in siRNA-treated BMDCs (G) and expression plasmid-transfected BMDCs (H). A representative result of three independent experiments is shown (D–H). evaluate the effect of the excess amount of PU.1 and IRF4 on that PU.1 and IRF4 bind to these EICE sequences in CNS1, 3, and PD-L2 expression, we overexpressed PU.1 or IRF4 in BMDCs. As 4, we performed ChIP assays using BMDCs. Quantitative PCR shown in Fig. 1H, the cellular PD-L2 level was apparently up- targeting the Pdcd1lg2 gene showed that PU.1 significantly binds regulated in IRF4-overexpressed cells. These results indicate that to all CNS regions, and the amount of PU.1 binding to CNS1 and PU.1 and IRF4 are involved in the transcription and subsequent CNS3 was relatively higher than that found in other regions protein expression of PD-L2 on BMDCs and the development of (Fig. 2C), whereas such binding was not detected in a control the PD-L2–expressing DC population. region (21.5 kb of the Gata3) that does not contain binding sites for PU.1 and/or IRF4 (29). Although the amount of DNA Binding of PU.1 and IRF4 to the Pdcd1lg2 gene in DCs immunoprecipitated with anti-IRF4 Ab was small, which was To clarify whether PU.1 and IRF4 directly bind to the Pdcd1lg2 likely because of the low affinity of the Ab used, significant gene, we used publicly available ChIP-sequencing (ChIP-seq) data binding of IRF4 to CNS1 and CNS3 was detected (Fig. 2D). and found four highly H3K27-acetylated loci in BMDCs (termed Based on these results, we demonstrated that both PU.1 and CNS1, 2, 3, and 4) (Fig. 2A). Interestingly, stronger binding sig- IRF4 bind to the Pdcd1lg2 gene around CNS1 and CNS3, nals of PU.1 and IRF4 were also detected at the sites corre- which carry typical EICE sequences, and only PU.1 but not sponding to the H3K27 signals, whereas such strong signals were IRF4 binds to the Pdcd1lg2 gene via Ets motifs in other CNSs not observed in the promoter region (CNS0), suggesting that PU.1 in PD-L2–expressing DCs. and IRF4 specifically bind to CNS1, 2, 3, and 4 rather than the promoter region of the Pdcd1lg2 gene. When we searched for PU.1 and IRF4 transactivate the Pdcd1lg2 gene through direct motifs recognizable by PU.1 and IRF4, we found that a typical binding to an EICE sequence in intron 2 EICE sequence, which is the binding site for the PU.1/IRF4 het- To confirm that the PU.1/IRF4 heterodimer directly binds to EICE erodimer (28), is located in CNS1, 3, and 4 (Fig. 2B). To confirm sequences in the Pdcd1lg2 gene, we performed an electrophoretic 4 PU.1/IRF4 TRANSACTIVATES THE PDCD1LG2 GENE IN DCs

To evaluate the effects of PU.1 and IRF4 on the transcriptional activity of the Pdcd1lg2 promoter and to clarify the involvement of CNS3-EICE as a cis-enhancing element in the Pdcd1lg2 gene, we performed a luciferase assay with coexpression plasmids for PU.1 and IRF4 (Fig. 3D). Luciferase activity driven by the CNS3 region of the Pdcd1lg2 gene was markedly increased by the coexpression of PU.1 and IRF4, as observed in a reporter plasmid carrying a typical EICE motif that was previously revealed to be transactivated by PU.1 and IRF4 (22). The transactivation effect of IRF4, which significantly upregulated the luciferase activity driven by CNS3, may be nonspecific because a similar tendency was observed for the promoterless reporter plasmid. When the EICE sequence in CNS3 was mutated, the synergistic effect of PU.1 and IRF4 on transactivation activity was not observed, suggesting that PU.1 and IRF4 cooperatively transactivate the Pdcd1lg2 gene through the EICE in CNS3. In the luciferase assays using reporter plasmids carrying the CNS1 region and the CNS4 region, cooperative transactivation by PU.1 and IRF4 was not observed (data not shown). PU.1 bound to the CNS3 probe even when the IRF4 binding Downloaded from motif was mutated (Fig. 3C), probably because the remaining Ets motif in the mutated probe can be bound with monomeric PU.1. To clarify whether PU.1 binds to CNS3 by itself in living cells, we evaluated the effect of IRF4 knockdown on the binding degree of PU.1 to the EICE in CNS3 by a ChIP assay. As shown in Fig. 3E, the significant amount of PU.1 binding to CNS3 was detected in http://www.jimmunol.org/ IRF4 knockdown BMDCs. In contrast, the coexpression of PU.1 without IRF4 did not increase the luciferase activity driven by the CNS3 (CNS3WT with PU.1 in Fig. 3D). These results suggest that both PU.1 and IRF4 are required for the EICE-mediated trans- activation, although PU.1 can bind to the EICE without IRF4. FIGURE 2. Binding of PU.1 and IRF4 to the Pdcd1lg2 gene in BMDCs. Overall, we demonstrated that PU.1 and IRF4 transactivate the ChIP-seq profiles showing PU.1 and IRF4 occupancies and histone modifi- Pdcd1lg2 gene through direct binding to the EICE sequence in cation in the Pdcd1lg2 gene (A). The following data were obtained from the CNS3. Gene Expression Omnibus Accession Viewer (https://www.ncbi.nlm.nih.gov/ by guest on October 3, 2021 geo/query/acc.cgi?acc=GSE36099): PU.1, GCM881099; IRF4, GCM881147; Involvement of the acetylation of the H3K27 histone in the CNS H3K27Ac, GSM881080. The EICE sequence, which is the binding site of region in PD-L2 expression in BMDCs PU.1 and IRF4, is found in CNS1, 3, and 4 (B). The amount of genomic DNA in whole BMDCs immunoprecipitated by ChIP assays using anti-PU. As shown in Fig. 4A, K27 residues of histone H3 in CNS regions 1Ab(C) and anti-IRF4 Ab (D) was determined by quantitative PCR tar- are highly acetylated in DCs compared with those in another geting the Pdcd1lg2 gene. The data represent the mean 6 SD of three hematopoietic cell, mast cells, suggesting a relationship between independent assays performed in triplicate. *p , 0.05. the degree of the acetylation of CNS regions in the Pdcd1lg2 gene and the expression level of PD-L2. Recently, we found that PU.1 modulates the histone acetylation of the Ciita gene in DCs (17). In mobility shift assay (EMSA) using double-stranded oligo DNAs the current study, to clarify the involvement of PU.1 in H3K27 with the Pdcd1lg2 gene sequence containing EICE motifs as acetylation of the Pdcd1lg2 gene, we performed ChIP assays with probes (Fig. 3A). When the probe DNA was mixed with PU.1 anti-acetyl histone H3 (K27) Ab in BMDCs treated with Spi1 protein, the specific shifted bands appeared for all three probes siRNA. It was confirmed that H3K27 residues in CNS1, CNS0, (lanes 2, 8, and 14), suggesting that PU.1 bound to all tested DNA CNS2, and CNS3 were significantly acetylated in control sequences. The addition of IRF4 to the mixture of PU.1 and probe siRNA-transfected DCs (siNegainFig.4B).WhenPU.1siRNA DNA decreased the band intensity of the PU.1/probe complex and was introduced into DCs, the H3K27 acetylation levels of the in turn caused the appearance of new bands, which likely repre- Pdcd1lg2 gene were drastically decreased (siSpi1 in Fig. 4B). In sented the PU.1/IRF4/probe complex, at higher molecular weights contrast, IRF4 knockdown did not affect the acetylation levels (lanes 3, 9, and 15). Each complex composition was confirmed by (data not shown). the disappearance of specific bands and/or the appearance of the It was previously reported that PU.1 physically interacts with the supershifted bands in the presence of anti-PU.1 Ab and anti-IRF4 coactivators p300 and GCN5, which possess a HAT activity in Ab (lanes 5, 6, 11, 12, 17, and 18). Similar band shifts were ob- B cells and Th9 cells, respectively (23, 24). To reveal the in- served in an EMSA using a well-characterized EICE sequence in the volvement of p300 and GCN5 in the histone acetylation of the l1B gene (30), as shown in Fig. 3B. When the IRF4 binding se- Pdcd1lg2 gene in DCs, we performed the knockdown of p300 and quence in the CNS3 was mutated, the band intensity of PU.1/IRF4/ GCN5 by introducing siRNAs into BMDCs. The Ep300 mRNA probe was dramatically reduced (Fig. 3C). In Fig. 3A, the band level was reduced to ∼50% of that in control cells by the trans- intensity ratio of the PU.1/IRF4/CNS3 probe versus PU.1/CNS3 in fection of Ep300 siRNA into DCs in which the Pdcd1lg2 mRNA lane 9 was highest compared with that of CNS4 (lane 15), indicating level was significantly decreased (Fig. 4C). Under this experi- that the PU.1/IRF4 heterodimer prefers to bind to the EICE in CNS3 mental condition, the H3K27 acetylation status in all CNS regions rather than the EICE in CNS4. was reduced to the background level (Fig. 4D). In contrast, the The Journal of Immunology 5

FIGURE 3. Transactivation of the Pdcd1lg2 gene by PU.1 and IRF4 via the EICE sequence. EMSA profiles obtained using fluorescein-labeled dsDNA corresponding to CNS1, CNS3, or CNS4 (A) using a probe corre- sponding to well-characterized EICE sequence in the l1B gene (B) and using a mutant probe lacking the IRF4 binding motif in the CNS3 sequence (C). Relative luciferase activity in cells transfected with the pGL-4.10 vector, pGL4.10_CNS3_WT vector, pGL-4.10_CNS3_mut vector, or pGL4.10_Aldh1a2_EICE (22) (D). Rel- Downloaded from ative luciferase activity is shown as the ratio of luciferase activity in the transfected cells to that observed in mock-transfected cells, and the data represent the mean 6 SD of three independent assays performed in http://www.jimmunol.org/ triplicate. *p , 0.05. The amount of PU.1 binding to the chromosomal DNA in siRNA-introduced BMDCs (E). The data represent the mean 6 SD of three independent assays per- formed in triplicate. *p , 0.05. by guest on October 3, 2021

knockdown of GCN5 did not affect the Pdcd1lg2 mRNA level suggested that GM-CSF stimulation increased the expression of (data not shown). These results suggest that PU.1 and p300 are PD-L2 in Flt3L-BMDCs, probably because of the acceleration of involved in acetylation of the H3K27 in CNS3 in the Pdcd1lg2 PU.1 binding to the Pdcd1lg2 gene and the increase in the histone gene in DCs. acetylation level in the Pdcd1lg2 gene. Pdcd1lg2 gene expression under GM-CSF stimulation Involvement of PU.1, IRF4, and p300 in the Pdcd1lg2 gene The above-mentioned results suggested that PU.1 is crucial for the expression in DCs ex vivo expression of PD-L2 in GM-CSF–induced BMDCs. In our recent The experiments using GM-CSF– or Flt3L-induced BMDCs in- study, it was revealed that GM-CSF stimulation upregulated the dicated the involvement of PU.1, IRF4, and p300 in Pdcd1lg2 expression of IRF4 and the recruitment of PU.1 to chromosomal gene expression in DCs. To further confirm the contribution of DNA in Flt3L-derived BMDCs (22). Therefore, we hypothesized these transcriptional regulators to the expression of the Pdcd1lg2 that GM-CSF stimulation induces or upregulates the PD-L2 ex- gene in natural DCs ex vivo, we used DCs purified from mouse pression in Flt3L-BMDCs. Quantitative PCR showed that the spleen. We introduced Spi1, Irf4,orEp300 siRNA into CD11c+ Pdcd1lg2 mRNA level in whole Flt3L-BMDCs was signifi- splenic DCs and quantified the mRNA levels in harvested cells cantly increased by GM-CSF stimulation (Fig. 5A). By using after 2-d cultivation in the presence of GM-CSF. As shown in flow cytometry analysis, we found that conventional DCs (cDCs; Fig. 6, the knockdown of PU.1, IRF4, and p300 significantly de- CD11c+/PDCA-12) expressed PD-L2 at a low level in steady-state creased Pdcd1lg2 mRNA levels. Therefore, the in vitro finding condition, and PD-L2 expression was increased following GM- that PU.1, IRF4, and p300 are involved in Pdcd1lg2 gene ex- CSF stimulation (Fig. 5B). PD-L2 expression was also detected pression in DCs was supported by the ex vivo results. in GM-CSF–stimulated plasmacytoid DCs (CD11c+/PDCA-1+) that did not express PD-L2 in a nonstimulated state (Fig. 5B). A Discussion ChIP assay in a CD11c+ population of Flt3L-BMDCs revealed that The molecule PD-1 and its ligand PD-L1 GM-CSF stimulation increased the amount of PU.1 protein binding have been paid increasing attention in the field of cancer im- to CNS regions in the Pdcd1lg2 gene (Fig. 5C). Furthermore, munity. PD-L2, a second ligand of PD-1, has recently shown to be GM-CSF stimulation tended to increase the H3K27 acetylation involved not only in cancer but also in allergy, infection, and level of CNS regions in Flt3L-BMDCs (Fig. 5D). These results humoral immunity (5–7), which raises the possibility that PD-L2 6 PU.1/IRF4 TRANSACTIVATES THE PDCD1LG2 GENE IN DCs Downloaded from

FIGURE 4. Involvement of H3K27 acetylation in Pdcd1lg2 gene expression in BMDCs. ChIP-seq profiles showing the histone modification of the

Pdcd1lg2 gene (A). DC represents GSM881080; mast cell represents GSM1329816. ChIP assay of Spi1 siRNA- (B)orEp300 siRNA-treated (D) BMDCs http://www.jimmunol.org/ using anti-acetyl H3K27 Ab (closed bars) or its control (open bars) in (B and D). The mRNA expression of Pdcd1lg2 and the target gene in Ep300 siRNA- treated BMDCs (closed bars) and control siRNA-treated BMDCs (open bars) in (C). The data represent the mean 6 SD of three independent assays performed in triplicate [(C and D), and CNS0–3 in (B)]. CNS4 in (B) is data of single experiment performed in triplicate. *p , 0.05. n.s., not significant. may be a therapeutic target for various immune-related diseases. level of PD-L2 in DC-like cells and macrophage-like cells is very The most definitive difference between PD-L1 and PD-L2 is different (Fig. 1D–F). The expression level of PU.1 is similar their expression profiles: whereas the expression of PD-L1 is between DC-like cells and macrophage-like cells, whereas the ubiquitous and IFN-g inducible, PD-L2 is mainly detected in IRF4 expression level in DC-like cells is higher than that in by guest on October 3, 2021 APCs, and the factors that induce PD-L2 expression are IL-4, macrophage-like cells [(32), not shown in our data]. We found that IL-13, and GM-CSF (8). Previously, STAT6 was identified in the Gm-csfr gene was highly expressed in DC-like cells (data not studies analyzing the mechanism by which Pdcd1lg2 gene ex- shown). Considering that GM-CSF stimulation increases the activity pression is induced by IL-4/IL-13 in activated macrophages and of PU.1 (33), including its transactivation activity and/or degree of DCs (9, 10). However, considering that STAT6 is ubiquitous binding to chromosomal DNA accompanied by a high amount of transcription factor, the transcriptional regulatory molecules IRF4, it may cause the difference in PD-L2 expression level between that determine the cell type-specific expression of the Pdcd1lg2 DC-like cells and macrophage-like cells. Actually, overexpression of gene remained unclear. In the current study, we indicated that IRF4 rather than PU.1 upregulated the PD-L2 expression (Fig. 1H). the hematopoietic cell–specific transcription factor PU.1 plays a The observation in the overexpression experiment may support our critical role in Pdcd1lg2 gene expression as a transactivator with hypothesis that a high amount of IRF4 in cells in which PU.1 ex- a partner molecule IRF4 and as an epigenetic regulator with hibits enough activity is required for the expression of PD-L2. p300, which can explain the mechanism by which GM-CSF PU.1 binds to the EICE sequence as a heterodimer with IRF4 stimulation upregulates the expression of PD-L2 in a cell type- (28). Among the EICE sequences located in CNS1, 3, and 4, the specific manner. EICE in CNS3 was the most preferred sequence for the binding of First, we demonstrated the involvement of PU.1 and IRF4 in PU.1/IRF4 in vitro (Fig. 3A). In contrast to the binding of PU.1, Pdcd1lg2 gene expression by using Spi1 or Irf4 siRNA-treated which was observed in all analyzed CNS regions in living cells BMDCs (Fig. 1A, 1B). As previously reported, we found that (Fig. 2C), IRF4 binding was detected in CNS1 and CNS3 GM-CSF–generated BMDCs are heterogeneous and comprise two (Fig. 2D). A luciferase assay showed that PU.1 and IRF4 exhibited populations: a CD11bint/MHCIIhigh population (characterized as transactivation activity via the EICE in CNS3 in a synergistic DC-like cells) and a CD11bhigh/MHCIIint population (macrophage- manner (Fig. 3D), whereas the cooperative transactivation by PU.1 like cells) (31, 32). Flow cytometry analysis showed that PD-L2 and IRF4 was not observed in luciferase assays using reporter is expressed in DC-like CD11bint/MHCIIhigh cells but not in plasmids carrying the EICE sequence from CNS1 or CNS4 (data macrophage-like cells and that PU.1 knockdown and IRF4 not shown). Based on these results, we conclude that PU.1 and knockdown reduced the cell surface expression level of PD-L2 IRF4 transactivated the Pdcd1lg2 gene via the EICE sequence in (Fig. 1). These results indicated that PU.1 and IRF4 are positive CNS3. During the binding of IRF4 to the CNS1 region (Fig. 2D), regulators of Pdcd1lg2 gene expression in BMDCs. Furthermore, IRF4 may form a complex with transcription factor(s) other than the knockdown of PU.1 and IRF4 decreased the number of PU.1, as IRF4 cannot bind to dsDNA by itself and requires other CD11bint/MHCIIhigh cells (Fig. 1D, 1E), suggesting that PU.1 and molecule(s) to do so. IRF4 are involved in the development and/or maintenance of a so- Because H3K27 in all analyzed CNS regions was highly acet- called DC-like population in BMDCs. The cell surface expression ylated in DCs but not in mast cells in silico (Fig. 4A), H3K27 The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. The effect of GM-CSF on Flt3L-BMDCs. Flt3L-BMDCs generated from bone marrow cells by culture with 100 ng/ml of Flt3L for 6 d were stimulated by 10 ng/ml GM-CSF for 24 h. CD11c+ cells purified by microbeads are used for ChIP assays shown in (C) and (D). The mRNA expression of Pdcd1lg2 in GM-CSF–stimulated cells or nonstimulated cells (whole cells) (A). Surface expression level of PD-L2 in cDCs and plasmacytoid DCs (B). Shadow represents isotype control; line represents anti–PD-L2 Ab. ChIP assay of GM-CSF–stimulated cells or nonstimulated cells using anti-PU.1 Ab or its by guest on October 3, 2021 control (C) and anti-acetyl H3K27 Ab or its control (D). The data represent the mean 6 SD of three independent assays performed in triplicate (A, C,andD). A representative result of three independent experiments is shown (B). *p , 0.05. acetylation in CNS regions was also expected to contribute H3K27 acetylation in CNS regions were increased by GM-CSF to Pdcd1lg2 gene expression in DCs. We confirmed that the stimulation in Flt3L-induced DCs, which was accompanied by the knockdown of p300 decreased Pdcd1lg2 gene expression (Fig. 4C). strong upregulation of the Pdcd1lg2 mRNA level and an apparent As expected, the knockdown experiments showed that both PU.1 increase in PD-L2 protein, especially in the cDC population and p300 are involved in the H3K27 acetylation of CNS regions (Fig. 5). Based on these observations, we conclude that GM-CSF (Fig. 4B, 4D). Based on these results, we speculate that p300, which stimulation induces Pdcd1lg2 gene expression through enhancing is recruited to chromatin through a physical interaction with PU.1 the expression and/or function of PU.1 and IRF4. Finally, via in CNSs, enhances H3K27 acetylation in CNS regions, resulting experiments using freshly isolated splenic DCs, we confirmed that in the transcription of the Pdcd1lg2 gene. Although there has been PU.1, IRF4, and p300 govern Pdcd1lg2 gene expression in DCs ex a report that GCN5 recruited by PU.1 contributes to IL-9 ex- vivo (Fig. 6). pression in Th9 cells via histone acetylation (23), the involvement In addition to DCs, macrophages also express PD-L2, especially of GCN5 in Pdcd1lg2 transcription was not observed in our under GM-CSF stimulation (8). The role of PU.1 in PD-L2 ex- experimental conditions. In a previous study, p300 enhanced the pression in macrophages was not evaluated in the current study transactivation activity of PU.1 through the acetylation of lysine because of the nondetectable level of PD-L2 in macrophage-like residues in the transactivation domain of PU.1 (24). Although cells (Fig. 1D, 1E). However, we propose the possibility that PU.1 we did not analyze the effect of p300 on PU.1 modification, we plays a key role in PD-L2 expression in macrophages, as PU.1 is showed that p300 and PU.1 are required for H3K27 acetylation critical for gene expression in macrophages as well. Further and the transcription of the Pdcd1lg2 gene, which, to our knowl- studies using PD-L2–expressing macrophages will be needed to edge, is a novel finding. confirm the involvement of PU.1 and IRF4 in PD-L2 expression in It is well known that GM-CSF stimulation induces the differ- macrophages. entiation and characteristic gene expression of DCs. We previously PD-L2 has an impact on the regulation of helper T cells. PD-L2 found that GM-CSF stimulation increases the expression of PU.1 in DCs or activated macrophages suppresses Th2 responses and and the subsequent recruitment of PU.1 to chromosomal DNA induces Th1 responses (9, 10). Compared with CD8a+ DCs, (20, 22) and the IRF4 expression level in DCs was markedly CD8a2DCs favors the Ag presentation to CD4+ T cells (34). upregulated by GM-CSF stimulation (22). In this study, we found CD8a2 DCs may express PD-L2 under the influence of PU.1 that the amount of PU.1 binding in CNS regions and the level of because IRF4 is critical for the development of CD8a2 cDCs 8 PU.1/IRF4 TRANSACTIVATES THE PDCD1LG2 GENE IN DCs

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