IL-4 Inhibits the Biogenesis of an Epigenetically Suppressive PIWI-Interacting RNA To Upregulate CD1a Molecules on Monocytes/Dendritic Cells This information is current as of October 2, 2021. Xue Zhang, Xin He, Chao Liu, Jun Liu, Qifei Hu, Ting Pan, Xiaobing Duan, Bingfeng Liu, Yiwen Zhang, Jingliang Chen, Xingru Ma, Xu Zhang, Haihua Luo and Hui Zhang J Immunol published online 11 January 2016

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 11, 2016, doi:10.4049/jimmunol.1500805 The Journal of Immunology

IL-4 Inhibits the Biogenesis of an Epigenetically Suppressive PIWI-Interacting RNA To Upregulate CD1a Molecules on Monocytes/Dendritic Cells

Xue Zhang, Xin He, Chao Liu, Jun Liu, Qifei Hu, Ting Pan, Xiaobing Duan, Bingfeng Liu, Yiwen Zhang, Jingliang Chen, Xingru Ma, Xu Zhang, Haihua Luo, and Hui Zhang

The discovery of PIWI-interacting RNAs (piRNAs) revealed the complexity of the RNA world. Although piRNAs were first deemed to be germline specific, substantial evidence shows their various roles in somatic cells; however, their function in highly differentiated immune cells remains elusive. In this study, by initially screening with a small RNA deep-sequencing analysis, we found that a piRNA, tRNA-Glu–derived piRNA [td-piR(Glu)], was expressed much more abundantly in human Downloaded from monocytes than in dendritic cells. By regulating the polymerase III activity, IL-4 potently decreased the biogenesis of tRNA-Glu and, subsequently, td-piR(Glu). Further, we revealed that the td-piR(Glu)/PIWIL4 complex recruited SETDB1, SUV39H1, and heterochromatin 1b to the CD1A promoter region and facilitated H3K9 methylation. As a result, the transcription of CD1A was significantly inhibited. Collectively, we demonstrated that a piRNA acted as the signal molecule for a cytokine to regulate the expression of an important for lipid Ag presentation. The Journal of Immunology, 2016, 196: 000–000. http://www.jimmunol.org/

onocytes derived from bone marrow progenitors are Monocytes can be induced to DCs in vitro with several mononuclear phagocytes with the capacity to differ- cytokines, including GM-CSF and IL-4 (4). DCs bridge the in- M entiate into macrophages and, subsequently, dendritic nate- and adaptive-immune responses by a series of steps to cells (DCs), and they play a critical role in the immune response capture, process, and present Ags to Ag-specific T cells (5). They (1). Two distinct populations of “inflammatory” and “patrol- are divided into four subsets: conventional DCs that are derived ling” monocytes were defined in mice (2). In humans, based on directly from bone marrow precursors and have a short half-life, different functional properties, CD14+CD162 and CD14+CD16+ plasmacytoid DCs that can secrete a large amount of type I IFN by guest on October 2, 2021 monocytes are classified into inflammatory subsets. In contrast, after viral challenge, Langerhans cells, and monocyte-derived CD14dimCD16+ monocytes are classified into patrolling subsets (3). DCs (6). Many functional markers are manipulated by cytokines during the differentiation into DCs (7). PIWI-interacting RNAs (piRNAs) are small noncoding RNAs Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen that are predominantly expressed in germline and specifically University, Guangzhou, Guangdong 510080, China; Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun interact with PIWI (8). piRNAs are widely known for Yat-sen University, Guangzhou, Guangdong 510080, China; and Guangdong Engi- their function to silence the transposon elements and maintain neering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong the stability of the entire genome (9). However, additional evi- 510080, China dence indicates that piRNAs not only existed in germline but also Received for publication April 10, 2015. Accepted for publication November 9, 2015. appeared in somatic cells, such as ovarian somatic cells of This work was supported by the National Special Research Program for Important Drosophila, neuron cells of Aplysia, and human cancer cells (10– Infectious Diseases (Grant 2013ZX10001004), the Guangdong Innovative Research 12). In the somatic cells of Drosophila, the piRNA/PIWI complex Team Program (Grant 2009010058), the National Basic Research Program of recruits epigenetic factors to regulate histone modification of China (973 Program) (Grant 2010CB912202), and the National Natural Science Foundation of China (Grant 30972620) (to H.Z.). target sites (13). In the neuron cells of Aplysia, the piRNA/PIWI The sequences presented in this article have been submitted to the National Center complex mediates the DNA methylation of CpG islands in the for Biotechnology Information Expression Omnibus under accession number CREB2 promoter to facilitate long-term memory (11). In the early GSE73945. embryo of Drosophila, the piRNA pathway is involved in the Address correspondence and reprint requests to Prof. Hui Zhang, Sun Yat-sen Uni- degradation of nos mRNA through CCR4-mediated deadenylation versity, Room 1308, New Technology Building, No. 74, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. E-mail address: [email protected] (14). Although some piRNAs play roles in cell proliferation and via- The online version of this article contains supplemental material. bility, the mechanism remains to be clarified in human cancer cells (12). There is little information on how piRNA functions in the Abbreviations used in this article: ChIP, chromatin immunoprecipitation; DC, den- dritic cell; GEO, Omnibus; HA, hemagglutinin; HP1, heterochro- human immune system. In this study, we demonstrate that the matin protein 1; miRNA, microRNA; NCBI, National Center for Biotechnology highly expressed tRNA-derived piRNA (td-piR) in monocytes Information; piNC, piRNA negative control; piRNA, PIWI-interacting RNA; qPCR, quantitative PCR; RIP, RNA-binding protein immunoprecipitation; RT-qPCR, significantly repressed CD1A transcription by inducing H3K9 reverse transcription quantitative PCR; siRNA, small interfering RNA; td-piR, methylation at its promoter region. We also discovered that its tRNA-derived piRNA; tiRNA, tRNA-derived stress-induced RNA; tRF, tRNA- biogenesis was controlled by IL-4. Our work suggests that derived small RNA. piRNA could function as a mediator for signal transduction in Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 highly differentiated immune cells.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500805 2 A piRNA IS A TRANSDUCER OF IL-4 TO REGULATE CD1a EXPRESSION

Materials and Methods RNA-binding protein immunoprecipitation Cell culture and transfection RNA-binding protein immunoprecipitation (RIP) was performed with the PBMCs were isolated from healthy human donors through Ficoll gradient EZ-Magna RIP Kit (Millipore), according to the manufacturer’s instruc- 3 7 centrifugation. CD14+ monocytes were isolated with BD IMag anti- tions, with modified procedures. Approximate 1 10 human monocytes human CD14 magnetic particles, according to the magnetic labeling pro- were collected and fixed in 1% formaldehyde for 10 min at room tem- tocol supplied by the manufacturer. The cells were cultured in RPMI 1640 perature, and the unreacted formaldehyde was quenched with glycine for medium (Invitrogen) supplemented with 10% FBS (Life Technologies), 5 min at room temperature. After washing the fixed cells three times with 100 U/ml penicillin, and 100 U/ml streptomycin (HyClone). GM-CSF cold PBS, 500 ml RIP lysis buffer containing protease inhibitor mixture was used to resuspend the cell pellet. The lysate was incubated on ice for (1000 U/ml) and IL-4 (500 U/ml) were used to induce the differentiation 2 of monocytes into DCs for ∼7 d, and the medium was changed every 3 d. 5 min and stored at 80˚C to complete the lysis process. The magnetic Lipofectamine RNAiMAX was used to transfect various small RNAs beads were washed with RIP washing buffer twice and incubated with into monocytes/DCs following the manufacturer’s instructions (Invi- different Abs (mouse IgG, rabbit IgG, anti-human PIWIL1, and anti- hu- trogen). HEK293T cells were obtained from American Type Culture man PIWIL4) for 30 min at room temperature separately. Then the mag- Collection and grown at 37˚C in DMEM supplemented with 10% FCS netic beads were washed twice with RIP washing buffer. RIP buffer (Life Technologies), 100 U/ml penicillin, and 100 U/ml streptomycin containing RNase inhibitor and 20 mM EDTA was add to the magnetic (HyClone). Lipofectamine 2000 (Invitrogen) was used to transfect plas- beads. RIP lysate was thawed and centrifuged at 14,000 rpm for 10 min mids and small RNAs into HEK293T cells. at 4˚C. Then, 100 ml supernatant was added to the beads-Ab complex in RIP buffer. All of the tubes were incubated with rotation for 5–6 h at 4˚C. Plasmid construction The immunoprecipitation tubes were centrifuged briefly and placed on the magnetic separator, and the supernatant was discarded. The beads- The coding sequence regions of PIWIL4, SUV39H1, SETDB1, and Ab-protein-RNA complex was washed with 500 ml cold RIP washing heterochromatin protein 1 (HP1)b, tagged with hemagglutinin (HA) or buffer three times. RNA in the immunoprecipitation complex was extracted

FLAG, were obtained by RT-PCR, with the mRNA of human PBMCs with TRIzol reagent after the protein digestion by proteinase K. Finally, Downloaded from as the template, and then inserted separately into the pcDNA3.1 vector. the amount of RNA in the beads-Ab-protein-RNA immunoprecipitation The accuracy of all clones was confirmed by DNA sequencing. complex was determined by qPCR.

Cell line generation Chromatin immunoprecipitation The T cell line CD8-2 was established as described previously, with Chromatin immunoprecipitation (ChIP) was performed with the EZ- minor modifications (15). In brief, CD8+ T cells were isolated from a Magna ChIP A/G Kit (Millipore), according to the manufacturer’s in- random healthy donor with a BD Human CD8 T Lymphocytes En- structions. For each ChIP, ∼1.5 3 107 cells were collected and fixed in http://www.jimmunol.org/ richment Set, according to the protocol supplied by the manufacturer, 1% formaldehyde for 10 min at room temperature, and glycine was and cultured with an organic extract of Mycobacterium tuberculosis in added to quench the unreacted formaldehyde for 5 min at room tem- the presence of an equal number of monocytes that had been treated perature. After washing the fixed cells three times with cold PBS, cell with 500 U/ml GM-CSF and 300 U/ml IL-4 for 3 d to induce CD1 lysis buffer containing protease inhibitor mixture was used to resuspend expression. Cultures were restimulated every 12 d with autologous the cell pellet, and the suspension was incubated on ice for 15 min, CD1a+ monocytes (three times) and thereafter with allogeneic CD1a+ followed by centrifugation at 800 3 g at 4˚C for 5 min. The cell pellet monocytes (once). was resuspended in nucleic lysis buffer and sonicated to shear the DNA into 500–1000-bp fragments. After centrifugation at 10,000 3 g at 4˚C Reverse transcription quantitative PCR for 10 min to remove insoluble materials, 5 mlthesupernatantwastaken

out as “input,” and 50 ml was used for immunoprecipitation with dif- by guest on October 2, 2021 To measure mRNA levels, total RNA was reverse transcribed with ferent Abs and protein A/G magnetic beads for ∼8hat4˚C.Theprotein oligonucleotide primer, and a reverse transcription quantitative PCR A/G bead–Ab-chromatin complex was washed with a low-salt/high-salt/ (RT-qPCR) assay was performed with primers specific to different LiCl washing buffer and TE buffer. Subsequently, the protein-DNA , according to the supplier’s recommendations (SYBR Premix complex was eluted, and the free DNA was obtained through reverse ExTaq; Takara), on a CFX96 Real-Time System (Bio-Rad). The ex- cross-linking. Finally, the free DNA was purified using spin columns and pression of these genes was normalized to GAPDH mRNA. To measure subjected to qPCR. the level of tRNA-Glu–derived piRNA [td-piR(Glu)], total RNA was reverse transcribed with a reverse transcription primer for U6 and a Preparation of lipid Ags specific stem-loop reverse transcription primer to td-piR(Glu), and following quantitative PCR was performed with primers specific to The organic extract was purified from whole, lyophilized Mycobacterium td-piR(Glu) and U6. The expression of td-piR(Glu) was normalized to tuberculosis strain H37Ra. To obtain the lipid Ag, we followed the U6. Quantitative PCR data were processed using Bio-Rad CFX Man- protocol described previously, with minor modifications (15). For the ager (Version1.5). extraction of lipid Ags from M. tuberculosis strain H37Ra, bacteria were grown in Middlebrook 7H9 medium. After 3–4 wk, the bacteria were Northern blot collected and extracted with 2:1 (v/v) chloroform/methanol at 20˚C for Total RNA was extracted with TRIzol reagent (Invitrogen), and the con- 2 h to yield solutions of mycobacterial lipids, which were dried in ni- centration was determined using a NanoDrop 2000C (Thermo).The same trogen. The residue was resuspended in acetone at 4˚C with 2% (v/v) of amount of RNA from different cells was separated on 10% Urea-PAGE gel. 10% (m/v) MgCl2 in methanol for 1.5 h to precipitate anionic phos- After transferring onto a nitrocellulose membrane, a g-32P–labeled oli- pholipids. The supernatants were dried, and the residue was redissolved gonucleotide probe completely complementary to td-piR(Glu) was hy- in 100:25 hexanes/chloroform and loaded onto silica solid-phase ex- bridized overnight at 50˚C. The membrane was washed three times for 10 traction columns (Sigma). These were eluted with hexane/chloroform/ min with washing buffer (stringent and nonstringent buffer) at 50˚C. The isopropanol/acetic acid solutions at ratios from 100:25:0:0 to 50:75:15:1, phosphor imaging system was used to detect the signal. followed by pure methanol. The antigenic activity was found in the 50:75:15:1 solutions. Coimmunoprecipitation and Western blot Abs HEK293T cells were collected and lysed with immunoprecipitation lysis buffer after they were transfected with pcDNA3.1-based constructs Several Abs were used for flow cytometry analysis: anti-CD40 (5C3), anti- containing interested genes using Lipofectamine 2000 (Invitrogen) for CD80 (2D10.4), anti-CD86 (IT2.2), anti-CD209 (eBh209), anti–HLA-DR 48 h. The lyses were incubated with 40 ml of anti-HA beads (Sigma) (LN3), anti-CD1a (HI149), anti-CD4 (OKT4), anti-CD8a (RPA-T8), anti- from4hto6hat4˚C,thenthebead–protein mixtures were washed three CD8b (SIDI8BEE), anti-CD28 (CD28.2), anti-TCRab (IP26). All of these times with cold lysis buffer. The immunoprecipitated samples were an- Abs were purchased from eBioscience, with the exception of anti-CD8a, alyzed by Western blotting with primary Abs (anti-HA, anti-FLAG, or which was purchased from BD. The following Abs were used for CHIP, anti-PIWIL4 Abs) followed by secondary Abs (goat anti–mouse IRDye RIP, and Western blotting: PIWIL4 Ab (Abcam; 87939), PIWIL1 Ab 680, goat anti–rabbit IRDye 800, or HRP-conjugated anti–mouse IgG). (Sigma; SAB4200365), HA Ab (MBL; M180-3), FLAG Ab (MBL; The blotting was analyzed with the Odyssey infrared imaging system or PM020), b-actin Ab (CST; 4967), JNK1 Ab (Abcam; ab10664), p53 Ab electrochemiluminescent detection system. (Santa Cruz; sc-126), and BRF1 Ab (Abcam; 74221). The Journal of Immunology 3

Flow cytometry Array containing 47,000 transcripts. Hybridization was performed at 45˚C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 640). Cells were collected and washed in PBS with 0.5% BSA, and the single- The GeneChip arrays were washed and stained (streptavidin-PE) on an cell suspensions were labeled on ice for 30 min with various Abs. Flow Affymetrix Fluidics Station 450, followed by scanning on a GeneChip cytometry was performed on a BD Fortessa and analyzed with FlowJo Scanner 3000. The hybridization data were analyzed using GeneChip software. Operating software (GCOS 1.4). In a comparison analysis, we applied a two-class unpaired method, using Significant Analysis of Microarray Deep-sequencing experiment software, to identify genes that had statistically significant differential expression between piRNA negative control (piNC) and td-piR(Glu), us- RNAs extracted from monocytes and DCs were deep sequenced to analyze , . the small RNA fraction, at lengths of 18–40 nt, by the Beijing Genomics ing a false discovery rate 5% and a fold change 1.8 as selection Institute (Shenzhen, China). Before the construction of the small RNA thresholds. The sequences presented in this article have been submitted library, an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) was to the NCBI GEO database under accession number GSE73945 (http:// used to examine sample integrity and concentration, and a NanoDrop was www.ncbi.nlm.nih.gov/geo/). used to examine inorganic ions or polycarbonate contamination. This step was intended to provide a reference for library construction and later Statistical analysis analysis. The small RNA library was constructed using a TruSeq Small Statistical significance between two groups was determined by the Student RNA Sample Pre Kit (Illumina). In brief, the following steps were t test using GraphPad Prism 6. The p values , 0.05 were considered performed: 1) separating 18–40 nt small RNA fragment from 500 ng statistically significant. RNA sample using PAGE gel (ssRNA Ladder Marker; TAKARA); striped, and recycled; 2) adaptor ligation (TruSeq Small RNA Sample Kit; Illumina); 3) RT-PCR: after reverse transcription (Invitrogen), several Results rounds of PCR amplification, using PCR Primer Cocktail and PCR Identification of a class of small RNAs with piRNA features in

Master Mix, were performed to enrich the cDNA fragments (reaction human monocytes/DCs Downloaded from conditions: 98˚C for 30 s; 11 cycles of 98˚C for 10 s, 60˚C for 30 s, and 72˚C for 15 s; 72˚C for 10 min; 4˚C hold); 4) purification of PCR products To investigate epigenetic regulation during DC differentiation, with PAGE gel and dissolution of the recycled products in EB solution; total small RNAs (20–40 nt) in human monocytes or DCs were and 5) quantitation of the final library in two ways: an Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents) was used to determine the deep sequenced. In addition to the major distribution peak of average molecule length, and RT-qPCR (TaqMan Probe) was used to microRNAs (miRNAs), a class of small RNAs with lengths of 26– quantify the library. Then, the qualified libraries were amplified on cBot to 33 nt, distinguishable from miRNAs, was differentially expressed generate the cluster on the flow cell. The amplified flow cell was se- between monocytes and DCs. These small RNAs were much more http://www.jimmunol.org/ quenced with single end using the HiSEquation 2000 system. Before abundant in monocytes than in DCs (Fig. 1A, Supplemental Fig. analysis of this sequencing result, some contaminant reads, including low- quality reads, reads with 59 primer contaminants, reads without 39 primer, 1A). In addition to the existence of the annotated piRNAs, further reads without the insert tag, reads with poly A, and reads shorter than analysis of these small RNAs sequences revealed that they could 18 nt, were removed from the fq file. Then, the length distribution of be mainly derived from small nucleolar RNAs or tRNAs (Fig. 1B), these clean reads was summarized. To avoid sequencing errors, the small which were reported to be processed into small RNAs by different RNA sequences of ultra-low reads (reads per million , 1) were not in- cluded in the bioinformatic analyses. Classification of the small RNA se- pathways (16–20). Although some of these small RNAs were quences was based on GenBank (http://www.ncbi.nlm.nih.gov/genbank) derived from mitochondrial tRNAs in monocytes, most were de- and Rfam (http://rfam.xfam.org) databases using the local Blastn rived from nuclear tRNAs (Supplemental Fig. 1A). The abundance by guest on October 2, 2021 program with an E value threshold of 1E-2. At this step, the sequences of the tRNA-derived small RNA prompted us to explore the origin with more than two mismatches (including gaps) were filtered out after of these small RNAs. According to their sequences, these small alignment. RNAs were derived primarily from tRNA-Glu, although some PIWIL4 expression analysis using the Gene Expression of them were from tRNA-Gly, tRNA-Pro, and so on (Fig. 1C). We Omnibus database initially called these small RNAs “td-piR(X),” because their lengths were similar to those of classical piRNAs. td-piR(Glu) was All of the gene-expression profiles for PIWIL4 in mouse/human mono- cytes and DCs in the National Center for Biotechnology Informa- one of the most abundant small RNAs in monocytes, but it was tion (NCBI) Gene Expression Omnibus (GEO) Profiles database (http:// markedly reduced in DCs. The deep-sequencing of monocytes and www.ncbi.nlm.nih.gov/geoprofiles) were collected by searching for key DCs showed that the expression of td-piR(Glu) in monocytes was words. In GEO Profiles, the values representing the expression level of all significantly higher than several miRNAs that were reported to genes within one GeneChip hybridization were sorted, divided into 10 groups, and placed into percentile bins (1 to 100: lowest level to highest play important roles in monocytes or DCs (Fig. 1D). The existence level; http://www.ncbi.nlm.nih.gov/geo/info/profiles.html). The key words and differential expression of td-piR(Glu) were confirmed by RT- “PIWIL4” and “dendritic” were used to retrieve the gene-expression qPCR (Fig. 1E) and Northern blot (Fig. 1F). The expression of profile data for PIWIL4 in mouse/human DCs from the NCBI GEO Pro- td-piR(Glu) was also much higher in monocytes than in B and files database, whereas the key words “PIWL4” and “monocytes” were T lymphocytes (Supplemental Fig. 1C). We further determined used to retrieve the gene-expression profile data for PIWIL4 in human monocytes. Next, the GEO ID numbers of all related profiles were shown whether td-piR(Glu) had the characteristics of classical piRNAs. and classified according to the expression levels of the samples in each The results of reverse transcription at low concentrations of gene-expression profile research. The percentile bin into which the ex- deoxynucleotide triphosphates, followed by PCR (21), proved that pression value of PIWIL4 was placed indicated the expression level of 29-O-methylation was modified at the 39 end of td-piR(Glu) (22) PIWIL4. For more details, please see http://www.ncbi.nlm.nih.gov/geo/ (Fig. 1G). RIP was conducted to test the interaction between info/profiles.html. PIWIL proteins and the small RNAs. To examine the expression GeneChip array of PIWILs in human monocytes and DCs, we assessed the mRNA levels of PIWILs by RT-qPCR; all of them were detectable, with Total RNA was extracted using TRIzol reagent and purified further using a QIAGEN RNeasy Mini Kit, according to the manufacturer’s instructions. the exception of PIWIL3 in monocytes and DCs (Supplemental RNA quality was assessed by formaldehyde agarose gel electrophoresis Fig. 1D). The proteins levels of PIWILs in monocytes and and quantitated spectrophotometrically. An aliquot of 0.1 mg total RNA DCs were also measured (Supplemental Fig. 1E). Although it is was used to synthesize double-stranded cDNA and produce biotin-tagged reported that the expression of PIWIL proteins is low in mouse cRNA using the MessageAmp Premier RNA Amplification Kit. The resulting biotin-tagged cRNAs were fragmented into strands of 35–200 bp, immune cells, the expression of PIWIL4 is moderate to high in according to the protocols from Affymetrix. The fragmented cRNA was human monocytes and DCs based on the data that we systemat- hybridized to an Affymetrix GeneChip U133 Plus 2.0 ically collected and analyzed from the NCBI GEO Profiles database 4 A piRNA IS A TRANSDUCER OF IL-4 TO REGULATE CD1a EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. A class of small RNAs derived from tRNAs was identified with piRNA features in human monocytes/DCs. (A) Deep-sequencing data revealed a fraction of small RNAs with a length of 26–35 nt (shown in gray) that are different from miRNAs (shown in dark gray) in human monocytes and DCs. The y-axis for reads per million (RPM) of miRNAs is at left side, whereas that for piRNAs is on the right side. (B) The source of small RNAs with lengths of 26–33 nt was classified by searching the Rfam database, and their abundance was compared between monocytes and DCs. (C) tRNA-derived small RNAs with the length of 26–33 nt were categorized. (D) Abundance of td-piR(Glu) and miRNAs in monocytes and DCs according to the deep- sequencing data. The differential expression of td-piR(Glu) in monocytes and DCs was confirmed by stem-loop RT-qPCR (E) and Northern blot (F). (G) The detection of 29-O-methylation modification at the 39 end of td-piR(Glu) via reverse transcription at low concentrations of dNTPs followed by PCR assay. Ctrl piR and Ctrl sR, 29-nt ssRNAs synthesized chemically in vitro, with or without a 29-O-methylation modification at the 39 end, were used as a positive and negative control. (H) td-piR(Glu) enriched by PIWIL1 or PIWIL4 in monocytes was isolated from the RIP complex and detected by RT-qPCR. td- piR(Glu) from IgG immunoprecipitation was used as a negative control. (I) Sequences of tRNA-Glu and td-piR(Glu) (upper panel). Structure of tRNA-Glu; arrows indicate the cleavage sites for td-piR(Glu) (lower panel). Normalized levels of tRNA-Glu (J) and td-piR(Glu) (K) in monocytes were determined by RT-qPCR after knockdown of RNase Z and POP4 with siRNAs. U6 was used as a reference control. The Journal of Immunology 5

(www.ncbi.nlm.nih.gov/geoprofiles) (Supplemental Fig. 1F, 1G). or without the Ag that was the organic extract of M. tuberculosis. The enrichment of td-piR(Glu) by PIWIL1 or PIWIL4 proteins in The result showed that the ability of CD1a to present lipid Ag was monocytes was also confirmed (Fig. 1H). Collectively, td-piR(Glu) significantly inhibited by td-piR(Glu) (Fig. 2G). However, other that is highly and specifically expressed in human monocytes and functions of DCs, such as the ability to secrete IL-12 and stimulate significantly decreased in DCs had the characteristics of a piRNA. the proliferation of total CD4+ T cells, were not affected by td- In addition to the well-known functions of tRNAs in translation, piR(Glu) (Supplemental Fig. 2E, 2F). some small RNA fragments derived from tRNAs expand a new aspect of tRNA biological function (16, 18, 20). Because the td-piR(Glu) is downregulated by IL-4 and acts as a signaling sequences of td-piR(Glu) and the 59 end of mature tRNA-Glu molecule of the IL-4–JNK1 and IL-4–p53 pathways to regulate were identical, it seemed that td-piR(Glu) was cleaved from the CD1a expression 59 endofmaturetRNA-Glubyanendonucleolyticenzymeor Although we demonstrated that td-piR(Glu) played an important 39–59 exonuclease (Fig. 1I). Because some stress-induced tRNA role in regulating CD1a expression, the signal transduction for fragments, known as tRNA-derived stress-induced RNAs (tiR- regulating the expression of td-piR(Glu) needs to be unraveled. NAs), are also 30–40 nt in length and cleaved from the 59 end of Because of the significant decrease in td-piR(Glu) during the mature tRNAs, it was important to investigate whether td-piR differentiation of monocytes into DCs, it is possible that certain was actually identical to tiRNA, which is generated from tRNA cytokine(s) could directly affect its expression. To this end, we through an RNase, angiogenin (19). With small interfering RNA treated monocytes with various cytokines, including GM-CSF, (siRNA)-mediated effective knockdown of angiogenin, the bio- IL-4, IL-1b, IL-6, or TNF-a, which were reported to affect the genesis of ti-piR(Glu) was not affected (Supplemental Fig. 1H). differentiation of monocytes (28). td-piR(Glu) was significantly Further, to confirm that tRNA-Glu is the direct precursor of td- decreased by IL-4 but not by othercytokines(Fig.3A).Impor- Downloaded from piR(Glu), the RNase Z and POP4, both of which are involved in tantly, we also found that IL-4 significantly increased CD1a tRNA biogenesis (23, 24), were knocked down by siRNAs in mo- expression (Fig. 3B). In an IL-4 dose-dependent experiment, we nocytes (Supplemental Fig. 4A). We noticed that, consistent with found a clear inverse correlation between the expression of td- the reduction in mature tRNA-Glu (Fig. 1J), td-piR(Glu) was also piR(Glu) and CD1a (Fig. 3C). Therefore, it is highly likely that downregulated (Fig. 1K). IL-4 is the major regulator that determines the downregulation of td-piR(Glu) during the differentiation of monocytes into http://www.jimmunol.org/ td-piR(Glu) specifically downregulates the expression of CD1a DCs. molecules on monocytes/DCs Because it is known that IL-4 plays a role in the differentiation Because the expression level of td-piR(Glu) in monocytes is much of monocytes into DCs, and polymerase III transcription activity higher than in DCs, it could be involved in the differentiation of is decreased during the differentiation of mammalian cells (29, monocytes into DCs. We cultured CD14+ human monocytes with 30), we assessed the level of tRNA-Glu at different time points GM-CSF and IL-4 for 72 h and then transfected the chemically after IL-4 treatment. IL-4 treatment significantly decreased the synthesized td-piR(Glu) into these DC-like cells at a high efficiency levels of td-piR(Glu) and tRNA-Glu (Fig. 3D), indicating that (Supplemental Fig. 2A). After 48 h, the change in gene expression the reduction in td-piR(Glu) could be caused by IL-4–mediated by guest on October 2, 2021 caused by td-piR(Glu) was examined by mRNA profiling analysis. downregulation of tRNA-Glu. It was also shown that tRNA Approximately 300 genes that were differentially expressed with a transcription is regulated by various pathways (29). To inves- fold change $ 1.8 or # 0.56 were identified (Fig. 2A). Based tigate the pathways that were involved in the regulation of on the analysis, some of them are related to DC tRNA-Glu by IL-4, the mRNA levels of various genes that functions, including cell migration, Ag binding, and Ag process- participate in the regulation of polymerase III transcription ing (Supplemental Fig. 2B). Interestingly, we noticed that CD1a, were examined in monocytes. IL-4 treatment significantly in- a specific and functional marker of DCs that can present self creased the mRNA and protein levels of p53, whereas it sig- and nonself lipid Ag to T lymphocytes (25, 26), was significantly nificantly decreased the mRNA and protein levels of JNK1, downregulated by the overexpression of piRNA. However, the BRF1, and TBP (Fig. 3E, 3F). To determine the roles of these mRNA levels of other CD1 genes were not affected (Fig. 2A, 2B). proteins in the IL-4 signaling pathway, siRNAs specific for JNK1, To confirm the alteration in CD1a expression and investigate p53, or BRF1 were transfected into monocytes (Supplemental Fig. whether the expression of other DC markers was also altered, 4A). The siRNA-mediated knockdown of JNK1 or BRF1 de- we performed RT-qPCR and flow cytometry analysis. By ana- creased the level of td-piR(Glu) but increased the mRNA level lyzing specific DC markers, including CD1a, CD40, CD80, CD86, of CD1a, which is consistent with IL-4 treatment (Fig. 3G, 3H), HLA-DR, and DC-sign, which indicate the complete differentia- whereas the knockdown of p53 counteracted the effects of IL-4 tion of DCs from monocytes (27), we found that only CD1a on the treatment on CD1a and td-piR(Glu) (Fig. 3I). Further, the ex- cellular surface was significantly decreased (Fig. 2C, 2D), which pression of BRF1 was reduced after the knockdown of JNK1 in was consistent with the mRNA profiling data. Further, RT-qPCR monocytes (Fig. 3G). These results were substantiated by ChIP- showed that the reduction in CD1a mRNA by td-piR(Glu) was qPCR of TBP in monocytes (Supplemental Fig. 3A). Together, dose dependent (Supplemental Fig. 2C). Alternatively, we trans- our data indicated that IL-4 repressed TFIIIB activity by inhib- fected the chemically synthesized antisense RNA, which is com- iting the expression of JNK1 to cause the reduction in BRF1, as pletely complementary to td-piR(Glu), into monocytes and found well as by increasing the expression of p53, which can interact with a significant increase in CD1a expression. The effect of td- TBP proteins and, therefore, inhibit the promoter occupancy by piR(Glu) antisense was also dose dependent (Fig. 2E, 2F). To TFIIIB (31). examine whether the function performed by CD1a on DCs was also affected by td-piR(Glu), importantly, to investigate the effect Transcription of CD1A is downregulated by td-piR(Glu) of td-piR(Glu) on the lipid Ag presentation activity of CD1a, we through H3K9me3 methylation in the promoter region established a CD8-2 T cell line expressing the TCRab hetero- The reduction in CD1A mRNA levels caused by td-piR(Glu) could dimer restricted by CD1a (15) (Supplemental Fig. 2D). The cell be due to the decreased transcription or facilitated posttranscrip- line was cocultured with td-piR(Glu)– or piNC-treated DCs, with tional decay. The latter could be regulated by the small RNA- 6 A piRNA IS A TRANSDUCER OF IL-4 TO REGULATE CD1a EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. td-piR(Glu) specifically represses CD1a expression on the surface of monocytes or DCs. (A) mRNA profiling microarray analysis of human DCs. The gene-expression scatterplot of td-piR(Glu) versus control is shown. Significantly upregulated genes are shown in red, and down- regulated genes are shown in green. (B) td-piR(Glu) or piNC was transfected into DC-like cells induced from monocytes with GM-CSF and IL-4 on the third day. After 48 h, mRNA levels of CD1A, CD1B, CD1C,andCD1D in these cells were determined by RT-qPCR. (C and D) Flow cytometry analysis of DCs treated as in (B); mean fluorescence intensity (MFI) of six markers is shown. (E) Isolated monocytes were transfected with different amounts of antisense td-piR(Glu) or piNC, and the relative level of CD1A mRNA was detected by RT-qPCR at 48 h. Relative level of CD1A mRNA under different concentrations of anti-piR(Glu) was normalized to the one under the corresponding concentrations of anti-piNC. (F) The protein level of CD1a was detected by flow cytometry after the transfection of monocytes with antisense td-piR(Glu) or piNC for 72 h. The concentration of 100 nM anti-piNC was used to be a representative for the different concentrations of anti-piNC. (G) Monocyte-derived DCs (1 3 105/well) were transfected with piNC or td-piR(Glu) and cocultured with 1 3 105 CD8-2 T cells. The organic extract from M. tuberculosis (200 ng/ml), used as Ag, was added or not to the culture. After coculture for 72 h, IL-2 in the supernatant of cells was examined by ELISA. Data are mean 6 SD and are representative of three in- dependent experiments. *p , 0.05, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. IL-4 increases CD1a expression by downregulating the biogenesis of td-piR(Glu) through the JNK1 and p53 signaling pathways. Freshly isolated monocytes were treated with different cytokines for 48 h, and the levels of td-piR(Glu) (A) and CD1A mRNA (B) were determined by RT-qPCR. (C) The levels of CD1A mRNA and td-piR(Glu) were determined by RT-qPCR in monocytes treated by increasing concentrations of IL-4. (D) Expression of td-piR(Glu) and tRNA-Glu in monocytes treated with IL-4 at the time points indicated. RNA was extracted from the cells and subjected to Northern blot analysis. A total of 40 g of RNA (left panel)or5gofRNA(right panel) was used. (E) Freshly isolated monocytes were treated with IL-4 (50 ng/ml) for 48 h. The mRNA levels of p53, CK2, Rb, p107, p130, c-Myc, JNK1, JNK2, BDP1, BRF1, and TBP were detected by RT-qPCR. (F) Protein levels of JNK1, p53, and BRF1 were examined by Western blot in monocytes treated or not with IL-4 for 48 h. b-actin was used as a loading control. (G) CD1A, td- piR(Glu), and BRF1 RNA levels in monocytes were examined by RT-qPCR after JNK1 was knocked down by specific siRNAs for 48 h. (H and I) CD1A and td-piR(Glu) RNA levels were examined by RT-qPCR after the indicated treatments. siP53+IL-4, p53 was knocked down in monocytes for 24 h, followed by IL-4 treatment for 48 h. Data are mean 6 SD and are representative of three independent experiments. *p , 0.05, **p , 0.01, *** p , 0.001. mediated mRNA degradation. To investigate the mechanism by possibility of CD1A mRNA nuclear degradation, Mtr4 and Trf4 which td-piR(Glu) repressed CD1a expression, proteins involved (Supplemental Fig. 4C), which are the major components of the in miRNA- or siRNA-mediated mRNA decay, including DCP1A, TRAMP complex and act as major cofactors for the exosome DCP1B, DCP2, CNOT1, EXOSC10, RRP44, XRN1, and XRN2 complex to control RNA quality in the nucleus, were also knocked (32), were knocked down by specific siRNAs (Supplemental Fig. down (33). The result indicated that the repression of CD1a 4C); the results showed no recovery in the repression of CD1a caused by td-piR(Glu) was not affected by the TRAMP complex caused by td-piR(Glu) (Supplemental Fig. 3B). We then examined in the nucleus (Supplemental Fig. 3C). Alternatively, it was shown CD1A mRNA levels in nuclear and cytoplasmic fractions from that piRNAs and PIWI proteins induce transcriptional silencing DCs transfected with td-piR(Glu) and found that the reduction in of genome-wide transposon elements by recruiting HP1a and his- CD1A mRNA began in the nucleus (Fig. 4A). To investigate the tone methyltransferase Su(Var)3–9 to facilitate H3K9 methylation 8 A piRNA IS A TRANSDUCER OF IL-4 TO REGULATE CD1a EXPRESSION

(H3K9me2/3) (13, 34, 35). We then hypothesized that the de- RNA hybrid analysis without any seed sequence setting (http:// crease in CD1A mRNA mediated by td-piR(Glu) could be due to bibiserv.techfak.uni-bielefeld.de/rnahybrid/), we found two pu- histone modification. To this end, the Abs specific for H3K4me3, tative binding sites at positions +138 and +330 bp of transcrip- H3K9me3, and H3K27me3 were used to examine the chromatic tion start site on the CD1A promoter, with minimum free energy state of the CD1A gene. Eight pairs of primers located in different of 225.7 and 225.3 kcal/mol, respectively (Fig. 4G). positions of CD1A gene loci were designed for ChIP-qPCR (Fig. b 4B). After the overexpression of td-piR(Glu) in DCs, H3K9me3 PIWIL4, SUV39H1, SETDB1, and HP1 participate in the at the CD1A 59 region was significantly increased, and the level regulation of CD1A transcription of polymerase II on the CD1A gene was also significantly de- To screen the possible proteins involved in CD1A transcriptional creased, indicating that CD1A transcriptionwasrepressedintd- regulation via H3K9 methylation, we knocked down Argonaute piR(Glu)–treated DCs (Fig. 4C–F). Using the online program subfamily and PIWI subfamily proteins (Supplemental Fig. 4D), Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. CD1a is downregulated by td-piR(Glu) via mediating the H3K9 modification at its promoter region. (A) After treatment of DCs with piNC or td-piR(Glu) for 48 h, CD1A mRNA level in the whole cell, as well as in the cytoplasmic and nuclear RNA fractions, was detected by RT-qPCR. GAPDH or U6 was used as a reference control for the cytoplasmic or nuclear fraction, respectively. (B) Diagram of the CD1A genomic locus. The full length of the CD1A gene is 4234 bp. It was divided into eight parts; the numbers 1 through 8 represent the different positions of the ChIP-qPCR primers. ChIP of H3K9me3 (C), H3K4me3 (D), H3K27me3 (E), and polymerase II (F) was performed to identify the different histone modifications and chromatin state on the CD1A gene after treatment of piNC or td-piR(Glu) in DCs. Sites 1–8 correspond to the diagram in (B). The relative enrichment of these modifications was calculated by normalizing the quantity of CD1A DNA against the quantity of input. (G) Schematic representation of the CD1A gene. piRNA and its potential targeting sites in the CD1A promoter region are shown. Data are mean 6 SD and are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. ATG, translation start site; P, promoter region. The Journal of Immunology 9 which were closely related to small noncoding RNA functions similar to MHC class I and can present Ag to T cells to activate (36). Only the knockdown of PIWIL4 protein significantly rescued immune responses to pathogens. It presents a range of Ags, in- the piRNA-induced repression of CD1a (Fig. 5A), indicating that cluding lipid, glycolipid, and lipopeptide, as well as other small PIWIL4, but not other proteins, was involved in the regulation of molecules (25, 41). An in-depth study showed that lipid Ag- CD1a expression. Further, G9a, GLP, SUV39H1, SUV39H2, lipopeptide didehydroxymycobatin related to mycobactin meta- and SETDB1, all of which are H3K9 methyltransferases and bolism can be specifically presented by the CD1a molecule to involved in heterochromatin and gene repression (37–39), were ab T cells (15, 26). It was shown that CD1a expression can be knocked down by gene-specific siRNAs in td-piR(Glu)–treated regulated by various cytokines. In addition to GM-CSF and IL-4, DCs (Supplemental Fig. 4E). The td-piR(Glu)–mediated decrease IL-6, IL-1b, or TNF-a can upregulate CD1a expression on human in CD1A mRNA was affected by the knockdown of SUV39H1 or monocytes, whereas IL-10 can downregulate it (42, 43). The ac- SETDB1 and not by other H3K9 methyltransferases (Fig. 5B). tivating transcription factors ATF-2 and CREB-1 are able to bind Furthermore, in td-piR(Glu)–treated DCs, we also knocked down to the minimal proximal promoter region of CD1A and play a role heterochromatin proteins, including HP1a,HP1b,andHP1g in regulating CD1a expression in human monocytes (28). CD1a (Supplemental Fig. 4F), which can recognize and bind to the H3K9 expression varies in different autoimmune disease models, in- methylation region in euchromatin and heterochromatin (40). Our cluding allergic bronchial asthma and psoriatic arthritis (44, 45), data indicated that only HP1b was involved in the regulation of which raises the possibility that its expression level could be re- CD1a expression (Fig. 5C). Meanwhile, when siRNAs specific for lated to the intensity of the T cell immune response. However, PIWIL4, SUV39H1, SETDB1, and HP1b were transfected into the mechanism of CD1a regulation during the differentiation of monocytes (Supplemental Fig. 4B), CD1a expression was signifi- monocytes remains to be elucidated. cantly increased, indicating that the epigenetically inhibitory mode By analyzing the deep-sequencing data, we found that a group Downloaded from for the CD1A gene occurred in monocytes (Fig. 5D, 5E). In addi- of small RNAs (25–33 nt) was differentially expressed between tion, after blocking endogenous td-piR(Glu) in monocytes with its monocytes and DCs and was primarily produced from tRNAs. antisense RNA, the level of H3K9 methylation and the enrichment Among them, abundant td-piR(Glu) could interact with PIWIL4 of PIWIL4 and HP1b proteins at the CD1A 59 region were sig- protein to form a complex and then recruit H3K9 methyl- nificantly reduced, suggesting that td-piR(Glu) specifically medi- transferases (SETDB1 and SUV39H1) to the CD1A promoter ated this epigenetically suppressive mode at the CD1A gene (Fig. region. Subsequently, H3K9 methylation at this region interacted http://www.jimmunol.org/ 5F–H). Therefore, we concluded that H3K9me3 methyltransferases with HP1b. Although PIWI proteins can interact directly with (SUV39H1, SETDB1), PIWIL4, and HP1b played an important HP1a in Drosophila (46), we showed that the interaction be- role in regulating CD1A transcription in monocytes and DCs. tween PIWIL4 and HP1b proteins was dependent on SETDB1 or SUV39H in human monocytes. The results of ChIP-qPCR after SUV39H1 and SETDB1 interact with PIWIL4 directly and then the knockdown of SETDB1 and HP1b confirmed this phenome- mediate the interaction of PIWIL4 and HP1b non. The discovery that the td-piR(Glu)/PIWIL4 complex induces The data from siRNA knockdown and ChIP-qPCR prompted us to H3K9 methylation at the promoter region provides a novel epi- further explore how piRNA/PIWIL4 complexes recruited these genetic mechanism of CD1a regulation. In monocytes, highly by guest on October 2, 2021 proteins to specifically control CD1a expression. To examine the expressed td-piR(Glu) inhibits CD1A transcription as a result of interaction between these proteins that are involved in the regu- the facultative heterochromatic state. During the differentiation of lation of CD1A transcription, including PIWIL4, SUV39H1, monocytes into DCs, td-piR(Glu) was downregulated, in line with SETDB1, and HP1b, the coimmunoprecipitation results showed the decrease in H3K9 modification at the CD1A 59 end region. As that PIWIL4 stably interacted with SUV39H1 or SETDB1 a result, the CD1A promoter region is converted into a euchro- (Fig. 6A, 6B), and these three proteins interacted with HP1b matic state and could be recognized by different transcription (Fig. 6C, 6D). Notably, siRNA-mediated SETDB1 or SUV39H1 factors to enhance its transcription. Although it was reported that knockdown impaired the interaction between PIWIL4 and HP1b the depletion of all three piwi genes (Miwi, Mili, Miwi2) does not (Fig. 6E), indicating that PIWIL4 interacted with SETDB1 or affect normal hematopoiesis and, subsequently, the numbers of SUV39H1 and then interacted indirectly with HP1b. To further progenitors or committed cells in mice, it is unclear whether the verify the function and interaction of these proteins in monocytes, functions of subsets of lymphoid and myeloid cells are affected ChIP-qPCR was performed after the knockdown of PIWIL4, (47). In addition, we and other investigators demonstrated that SETDB1, and HP1b. The knockdown of PIWIL4 decreased the PIWIL4 is expressed in human monocytes/DCs at a significantly amounts of SETDB1 and HP1b at the CD1A promoter region higher level than in mouse monocytes/DCs. Importantly, specific (Fig. 6F). The knockdown of SETDB1 affected the level of HP1b, siRNA knockdown of PIWIL4 expression significantly decreased but not PIWIL4, at the CD1A promoter region (Fig. 6G), whereas td-piR(Glu)–mediated CD1a repression (Fig. 5A). Collectively, the amounts of PIWIL4 and SETDB1 at the CD1A promoter re- therefore, our data clearly indicated that PIWIL4 specifically in- gion were not affected by the knockdown of HP1b (Fig. 6H). teracts with td-piR(Glu) and participates in the td-piR(Glu)–me- Taken together, our data indicated that SETDB1 and SUV39H1 diated function. A previous report indicated that a PIWI-like RNA were first recruited by the td-piR(Glu)/PIWIL4 complex to the derived from the antisense transcripts of KLD3L1 existed in NK specific site at the CD1A promoter to methylate the lysine residue cells to repress KLD3L1 expression by inducing DNA methyl- at the ninth position of histone 3 in this region. HP1b was then ation at the promoter region (48). However, it was not verified recruited by SETDB1 or SUV39H1 to this complex to stabilize whether this small RNA is of the features of piRNA or not, be- histone modification and sustain the facultative heterochromatic cause no evidence was shown for the direct interaction with PIWI state (Fig. 7). protein(s). Therefore, we showed the abundance and important function of certain piRNAs in human immune system. Discussion The study of small noncoding RNAs revealed that tRNAs are The human CD1 family consists of five CD1 proteins: CD1a, a source of small RNAs that are derived from their 59 or 39 end CD1b, CD1c, CD1d, and CD1e. All of them are cell surface (16). It was proposed that these tRNA-derived small RNA frag- glycoproteins, with the exception of CD1e. The CD1 molecule is ments (tRFs) are not generated randomly as by-products of tRNA 10 A piRNA IS A TRANSDUCER OF IL-4 TO REGULATE CD1a EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. H3K9 methyltransferases and heterochromatin proteins are involved in the regulation of CD1a expression in monocytes and DCs. DC-like cells, which were induced using GM-CSF and IL-4 for 3 d from monocytes, were transfected with different specific siRNAs for AGO proteins, PIWIL proteins (A), H3K9 methyltransferases (B), or heterochromatin proteins (C) for 24 h. After these cells were treated with piNC or td-piR(Glu) for 48 h, CD1A mRNA levels were determined by RT-qPCR. The fold change was calculated by normalizing the CD1A mRNA level with td-piR(Glu) treatment to that of piNC treatment. CD1a expression was detected by RT-qPCR (D) and flow cytometry (E) after monocytes were treated with different siRNAs for 48 h. The levels of H3K9me3 (F), PIWIL4 (G), and HP1b (H) on the CD1A gene were determined in monocytes after treatment with antisense piNC or td-piR(Glu) for 48 h. Data are mean 6 SD and are representative of three independent experiments. *p , 0.05, **p , 0.01. degradation or decay but rather by some precise mechanisms that proteins (16, 18). Alternatively, it was reported that tRNAs can are involved in dicer or RNase Z (18). The length of these tRFs be cleaved within the anticodon loop by a secreted RNase, usually ranges from 18 to 23 nt. Further studies suggested that angiogenin, under the conditions of arsenite, heat shock, or UV these tRFs have important biological functions in cell proliferation radiation (19). These stress-induced and tRNA-derived small and global gene silencing via association with different Argonaute RNAs are 30–40 nt in length and are known as tiRNAs. Some The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. PIWIL4 interacts directly with SUV39H1 and SETDB1 and recruits HP1b to form a repression complex to decrease CD1a expression. (A) HA-tagged PIWIL4 and FLAG-tagged SUV39H1 plasmids were cotransfected into 293T cells. After 48 h, coimmunoprecipitation was performed with anti-HA Ab-conjugated beads, and immunoblots were assayed by HA and FLAG Abs. GFP-HA was used as a negative control. (B) The interaction between PIWIL4 and SETDB1 was determined. After cotransfecting plasmids of PIWIL4 tagged with FLAG and SETDB1 tagged with HA into 293T cells, the coimmunoprecipitation was performed as in (A), except that the immunoblots were assayed by anti-HA or anti-PIWIL4 Abs. (C)The interaction of PIWIL4 and HP1b, as well as SETDB1 and HP1b, was determined by coimmunoprecipitation, as described above. (D)FLAG-tagged SUV39H1 and HA-tagged HP1b plasmids were used in the coimmunoprecipitation to determine the interaction of these two proteins, as described above. (E) The effects of various siRNAs on the interaction of PIWIL4 and HP1b. The numbers indicate the quantification of HP1b precipitated with PIWIL4 protein under different conditions. (F) The levels of SETDB1 and HP1b i989 in different regions of the CD1A gene were assayed by ChIP- qPCR after siRNA-mediated knockdown of PIWIL4 in monocytes. (G)TheassociationofPIWIL4andHP1b in the CD1A gene was assayed by ChIP- qPCR after siRNA-mediated knockdown of SETDB1 in monocytes. (H) The association of PIWIL4 and SETDB1 in the CD1A gene was assayed by ChIP-qPCR after siRNA-mediated knockdown of HP1b in monocytes. Sites 1, 2, and 4 correspond to the sites in Fig. 4B; site 4 was used as a negative control to confirm the viability of the experiment. The relative enrichment was calculated by normalizing the quantity of CD1A DNA enriched by a specific Ab to the quantity of input (F–H). Data are mean 6 SD and are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. N, not detectable. 12 A piRNA IS A TRANSDUCER OF IL-4 TO REGULATE CD1a EXPRESSION Downloaded from

FIGURE 7. Signaling pathway used by IL-4 to regulate the biogenesis of td-piR(Glu) and, subsequently, the expression of CD1a in human monocytes. http://www.jimmunol.org/ IL-4 downregulated the expression of JNK1 and then repressed the expression of BRF1, which is a downstream molecule of JNK1. In addition, TBP was significantly downregulated by IL-4. Furthermore, the expression of p53 was increased by IL-4. Taken together, the occupancy of TFIIIB on the promoterof tRNA-Glu was reduced. In line with the reduction in TFIIIB transcription activity on tRNA-Glu, the RNA level of tRNA-Glu was downregulated, and the level of td-piR(Glu) was also reduced. The association of H3K9 methyltransferases on the CD1A promoter region was subsequently reduced. As a result, the transcription of CD1A increased. reports indicated that they directly inhibit protein synthesis downregulated tRNA-Glu expression through the JNK1 and p53 (19, 20). In this study, we identified a class of small RNAs in signaling pathways. Although it is well-known that IL-4 can by guest on October 2, 2021 human monocytes that was generated from the 59 end of tRNAs guide the differentiation of monocytes into DCs but not mac- and that had characteristics of classical piRNAs, such as 29-O- rophages (4), there is no evidence with regard to the underlying methylation at the 39 end and specific interaction with PIWIL mechanism. In this study, we found that IL-4 increases the proteins. The level of td-piR(Glu) was not affected by siRNA- transcription of CD1A by downregulating the level of its mediated knockdown of dicer or angiogenin, which suggested inhibitor, td-piR(Glu). Therefore, our work demonstrates that that the generation of td-piR(Glu) may be through a novel piRNAs can function as a transduction molecule to transfer the pathway. Furthermore, tRFs havearelativepreferencefor signal from IL-4 to the specific expression of CD1a. This dis- Argonaute3–4 proteins (18), tiRNA is only associated with YB-1 covery suggested that IL-4 regulates the levels of a variety of small (20), and td-piR(Glu) interacts with PIWIL1/4 proteins but not noncoding RNAs through different pathways to orchestrate the dif- Argonaute proteins. Moreover, tRFs are 59-phosphorylated and ferentiation of monocytes. 39-hydroxylated (18), tiRNAs are likely to be 29-, 39-cyclic In this study, we uncovered the role of td-piR(Glu)/PIWIL4 in phosphorylated at the 39 end (19), and td-piR(Glu) harbors a 29- regulating CD1a expression; we also revealed that td-piR(Glu) O-methylation at its 39 end. Taken together, td-piR(Glu) with can be regulated by IL-4 through the JNK1 and p53 signaling characteristics of piRNAs is different from tRFs or tiRNAs. pathways and acts as a functional signaling molecule (Fig. 7). Our data showed that the level of td-piR(Glu) was significantly These results should prompt further exploration of the function, reduced during differentiation, and this was attributed to IL-4 biogenesis, and regulation of piRNAs in the mammalian immune treatment that decreased TFIIIB activity to repress the tran- system. In addition, the novel mechanism that we propose in this scription of tRNA-Glu. Although tRNAs play roles in protein article provides a feasible and specific method to control CD1a translation and have many other functions, including the regu- expression to treat some autoimmune diseases, as well as to lation of apoptosis in mammalian cells, use in retroviral tran- avoid immune rejection of transplants and immune evasion of scription, and involvement in signal transduction pathways tumors caused by aberrant CD1a expression. responding to nutrient deprivation (49), our work demonstrates that tRNAs can act as a precursor of the signaling molecule td- piR(Glu) and participate in the regulation of gene expression Acknowledgments during the differentiation of immune cells. Nevertheless, it is We thank Dr. Chuan Bai (Sun Yat-sen University) for guiding the prep- aration of lipid Ag and Dr. Xiaomin Lai (Sun Yat-sen University) for possible that the level of tRNA-Glu is also controlled by other providing M. tuberculosis strain H37Ra. transduction pathways that could regulate exonuclease or endo- nuclease activity. As a multifunctional cytokine, IL-4 activates several signaling pathways and plays an important role in the Disclosures immune response. In this study, we showed that IL-4 potently The authors have no financial conflicts of interest. The Journal of Immunology 13

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Supplemental Figure 1. The existence of td-piR(Glu) and PIWIL proteins in human monocytes and DCs. (A) Deep sequencing data showed the differential expression of small RNAs in length of 25~37 nts between monocytes and DCs according to the deep sequence data. (B) The expression of tRNA-derived small RNAs in the whole cell and the different fractions was compared between monocytes and DCs. (C) The levels of td-piR(Glu) in monocytes, B cells, CD4+ T cells or CD8+ T cells were examined by stem loop RT-qPCR. GAPDH was used as a reference control. (D) The mRNA levels of PIWIL1, PIWIL2 and PIWIL4 in monocytes and DCs were examined by RT-qPCR. GAPDH was used as a reference control. (E) Protein levels of PIWIL4 in human DCs and monocytes that were knocked down of PIWIL4 were detected by western blotting. Hela cells were used as a positive control and β-actin was used as loading control. (F) Gene expression profiles of PIWIL4 in mouse/human dendritic cells. The relative expression levels of all genes within a mRNA chip sample were ordered, and then split into 10 groups (1-9: lowest; 90-100: highest) shown on the x-axis. The percentile rank gives an indication of the relative expression level of PIWIL4 among all genes. The values shown on the y-axis represent the percentages of the mRNA genechip sample numbers with various ranked PIWIL4 expression within the total mRNA genechip sample numbers of mouse (n= 98) or human (n= 113) dendritic cells. The orange bar represents the expression of GAPDH as a reference control. (G) Gene expression profiles of PIWIL4 in human peripheral blood monocytes. The relative expression levels of all genes within a mRNA chip sample were rank ordered, and then split into 10 groups (1-9: lowest; 90-100: highest) shown on the x-axis. The percentile rank gives an indication of the relative expression level of PIWIL4 among all genes. The values shown on the y-axis represent the percentages of the mRNA genechip sample numbers with various ranked PIWIL4 expression within the total mRNA genechip sample numbers of human peripheral blood monocytes (n=1440). For more details, please see Table S3. Since none of the gene expression profiles of PIWIL4 in mouse peripheral blood monocytes was found in NCBI GEO Profiles database, only the result of the expression profiles of PIWIL4 in human peripheral blood monocytes was shown. The orange bar represents the expression of GAPDH as a reference control. (H) Td-piR(Glu) level was determined by qRT-PCR in monocytes after the specific siRNA-mediated knockdown of Angiogenin.

Supplemental Figure 2. The function of td-piR(Glu) in human monocytes or DCs. (A) Td-piR(Glu) level in human DCs after transfection of synthesized td-piR(Glu) into DCs at the concentration of 50 nM for 24 h. The level of td-piR(Glu) in monocytes was used as a positive control. (B) GO analysis of the genes that were affected by the overexpression of td-piR(Glu) in DCs with the fold-change (≥ 1.8 or ≤ 0.56) according to the mRNA profiling data. (C) The dose-dependent effect of td-piR(Glu) on decreasing CD1a mRNA level in human DCs. CD1a mRNA level was determined by RT-qPCR, and GAPDH was used as a reference control. (D) The molecules on the surface of constructed CD8-2 T cell line was analyzed by flow cytometry. (E) The monocyte-derived DCs transfected with piNC or td-piR(Glu) were co-cultured with CD4+ T cells at different ratio with or without the organic extract from M. tuberculosis for 4 days. Then the ability of DCs to stimulate CD4+ T cells proliferation was assessed by measuring CD4+ T cell numbers. (F) The IL-12 mRNA level was detected by RT-qPCR in DCs treated as indicated for 48 h.

Supplemental Figure 3. (A) The analysis of TBP binding on tRNA-Glu gene in monocytes treated as indicated. IL-4 reduced the level of TBP proteins on tRNA-Glu gene region, but this reduction was relieved by siRNA-mediated knockdown of p53. (B-C) The screening experiments to identify the proteins that participate in the td-piR(Glu)-mediated regulation of CD1a expression on DCs. The proteins that are involved in the mRNA decay in cytoplasm were knocked down by specific siRNAs in DC-like cells for 24 h. After the cells were treated with piNC or td-piR(Glu) for 48 h, CD1a mRNA level was examined by RT-qPCR. GAPDH was used as a reference control and the fold change was calculated by normalizing CD1a mRNA level of td-piR(Glu) treatment to that of piNC treatment (B). DC-like cells were treated with specific siRNAs that are involved in the mRNA degradation in nuclei for 24 h, and then the effect of td-piR(Glu) on CD1a mRNA level was assessed by RT-qPCR (C).

Supplemental Figure 4 The efficiency of siRNA-mediated knockdown of specific target genes in monocytes or DCs. (A-B) After monocytes were transfected with specific siRNAs for 48 h, the mRNA level of target gene was assessed by RT-qPCR respectively. (C-F) The mRNA level of different target gene was detected by RT-qPCR after DCs were transfected with specific siRNAs for 48 h. GAPDH was used as a reference control.