Transcription Factor Directs a Silencing Programme for TH7 Cell Differentiation The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Citation Published Version Accessed Citable Link Terms of Use Ouyang, Xinshou, Ruihua Zhang, Jianjun Yang, Qingshan Li, Lihui Qin, Chen Zhu, Jianguo Liu, et al.. Transcription factor directs a silencing programme for TH7 cell differentiation. Nature Communications : 34. doi:.38/ncomms3 May 3, 8 :3:6 AM EDT http://nrs.harvard.edu/urn-3:hul.instrepos:597876 This article was downloaded from Harvard University's DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:hul.instrepos:dash.current.termsof-use#laa (Article begins on next page)
Received 7 Aug Accepted 3 Apr Published 7 May DOI:.38/ncomms3 Transcription factor directs a silencing programme for 7 cell differentiation Xinshou Ouyang, Ruihua Zhang, Jianjun Yang, Qingshan Li, Lihui Qin, Chen Zhu 3, Jianguo Liu 4, Huan Ning 4, Min Sun Shin 5, Monica Gupta 6, Chen-Feng Qi 5, John Cijiang He, Sergio A. Lira, Herbert C. Morse III 5, Keiko Ozato 6, Lloyd Mayer & Huabao Xiong 7 cells are recognized as a unique subset of T helper cells that have critical roles in the pathogenesis of autoimmunity and tissue inflammation. Although is necessary for the generation of 7 cells, the molecular mechanisms underlying the functional diversity of 7 cells are not fully understood. Here we show that a member of interferon regulatory factor (IRF) family of transcription factors,, has a critical role in silencing 7-cell differentiation. Mice with a conventional knockout, as well as a T cell-specific deletion, of the Irf8 gene exhibited more efficient 7 cells. Indeed, studies of an experimental model of colitis showed that deficiency resulted in more severe inflammation with an enhanced 7 phenotype. was induced steadily and inhibited 7-cell differentiation during 7 lineage commitment at least in part through its physical interaction with. These findings define as a novel intrinsic transcriptional inhibitor of 7-cell differentiation. Immunology Institute, Department of Medicine, Mount Sinai School of Medicine, Gustave L. Levy Place, New York, New York 9, USA. Department of Pathology, Mount Sinai School of Medicine, New York, New York 9, USA. 3 Center for Neurologic Diseases, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts 5, USA. 4 Division of Immunobiology, Saint Louis University School of Medicine, St Louis, Missouri 634, USA. 5 Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 89, USA. 6 Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 89, USA. Correspondence and requests for materials should be addressed to H.X. (email: huabao.xiong@mssm.edu). nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 CD4 T helper (TH) T cell subsets are characterized by the secretion of unique cytokine profiles and have critical roles in orchestrating adaptive immune responses. In addition to and cells, 7 cells have been identified more recently as a third subset mediating inflammatory and autoimmune responses through the production of interleukin (IL)-7A, F and IL- (refs 4). 7 lineage commitment is initially driven by transforming growth factor (TGF)-β in the presence of IL-6 or IL- (refs 58), whereas IL-3 serves to expand or maintain 7 populations,5,9,. The orphan nuclear receptor, RORC, also known as, has been identified as the master transcription factor for 7 development. The differentiation of 7 cells is also regulated by several recently described positive and negative feedback loops involving IL-, IL-3R, IL- and IL-7 (refs 6, 7, 5), indicating that intrinsic genetic programmes may contribute to the silencing of 7 lineage commitment. There is increasing evidence that 7 cells are involved in the pathogenesis of various autoimmune/inflammatory diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and asthma 6. Thus, a more complete understating of the molecular mechanisms involved in the regulation of 7 immune responses should provide insights into the pathogenesis and treatment of these and possibly other inflammatory diseases. Several transcription factors, including, RORα, STAT3 and interferon regulatory factor (IRF)4, have been reported to be important for 7-cell differentiation. However, the silencing programme for 7-cell differentiation has not been fully examined., a member of the IRF family, is expressed by B cells, dendritic cells (DCs), macrophages 79 and activated T cells,, and has been shown to have a diverse roles in the regulation of innate and adaptive immune responses. has a DNA-binding domain in the amino (N)-terminal half of the protein and an IRF association domain in the carboxy (C) terminus that is responsible for heterodimerization with other transcription factors. functions as a transcriptional repressor or activator depending on the formation of different heterodimeric DNA-binding complexes with partners that include members of the ETS family and the IRF family. It is known that has critical roles in the differentiation of myeloid cells, promoting monocyte over granulocyte differentiation 3. It is also a crucial regulator of many aspects of DC development, differentiation and function 4, thereby having an essential role in the establishment of innate immune responses. Although is critical for the regulation of immune cell growth, differentiation and survival 5, the direct effects of on T-cell activation and differentiation are incompletely understood. In the present study, we show that mice deficient in because of a conventional knockout (KO) or with a T cell-specific conditional deletion exhibited enhanced 7-cell differentiation while exhibiting no significant effects on or cells. In addition, transfer of naive T cells from -deficient mice induced more severe colitis in Rag / mice than T cell from normal controls. Furthermore, we report that physically interacts with, resulting in inhibition of transcription. These findings suggest that has a suppressive role in the control of 7 differentiation and highlight the importance of intrinsic genetic programmes for the silencing of 7-dependent immune responses. Results deficiency enhances 7-cell differentiation. To investigate the function of in T cells, we first examined the expression of in CD4 T cells from normal or OT-II transgenic mice activated by different stimuli. We found that T-cell antigen receptor (TCR) engagement with anti-cd3 and anti-cd8 antibodies as well as stimulation of OT-II cells resulted in significant induction of protein expression, as determined by western blotting (Supplementary Fig. Sa,b). Interestingly, protein was more stably expressed in naive CD4 T cells polarized for to 7 h under 7-inducing conditions compared with - or - inducing conditions (Supplementary Fig. Sa). To clarify how 7-polarizing conditions induce stable expression, CD4 cells were stimulated with in the absence of TCR activation and the results showed that clearly induced expression at both 48 and 7 h (Supplementary Fig. Sc). In addition, mitogenactivated protein kinase inhibitors significantly blocked protein expression induced by TCR activation (Supplementary Fig. Sd) and STAT3 mutant mice showed impaired mrna expression (Supplementary Fig. Se). These data indicate that is consistently elevated in activated CD4 T cells and TCR signalling cascade is involved in induction of expression. We then assessed the contributions of to 7 differentiation by studying CD4 T cells from mice deficient in due to a conventional KO of the gene (Irf8 / mice). Naive CD4 T cells from Irf8 / or wild-type () littermate mice were primed in vitro for 4 days under or 7 polarizing conditions. The cells were then re-stimulated with phorbol myristate acetate (PMA)/ionomycin and examined for the percentages of -producing cells by intracellular staining using flow cytometry. Notably, the frequency of -producing cells generated from Irf8 / T-cell cultures was about threefold greater than cells from cultures (Fig. a). These observations correlated with enhanced secretion by Irf8 / cells generated under 7 polarizing conditions as determined by enzyme-linked immunosorbent assay (ELISA; Fig. b). In addition, -producing CD4 T cells were significantly increased among lamina propria lymphocytes isolated from Irf8 / mice as compared with littermate controls following in vitro activation under 7 conditions or at the basal levels (Fig. c and Supplementary Fig. S). To determine whether the effects of deficiency on 7 potential were cell type-specific, we next compared CD4 T cells from mice expressing Lck-Cre that were (wt/wt) or homozygous (fl/fl) for a conditional allele of Irf8 gene resulting in selective depletion of in the T-cell compartment. Quantitative real-time reverse transcrption(rt)pcr (qpcr) analyses revealed dramatically lower levels of Irf8 transcripts in sorted thymocyte subpopulations (DP, CD4SP, CD8SP) and splenic CD3 CD4 cells from Lck-Cre Irf8 fl/fl compared with cells from Lck-Cre Irf8 wt/wt littermate control mice (Supplementary Fig. S3), confirming that the Irf8 gene was efficiently deleted from Lck-Cre Irf8 fl/fl T cells. Splenic and lymph node CD4 T-cell subsets from mice with - deficient T cells as well as from mice homozygous for a conventional Irf8 null allele were normal in number as well as in expression of the T-cell activation markers CD6L, CD44, CD5 and CD69. Expression of FOXP3 in thymic and peripheral lymph node T cells from mice of both genotypes was also similar (Supplementary Fig. S4), indicating that CD4 T cells develop normally in the absence of. Naive CD4 T cells from Lck-Cre Irf8 fl/fl and Lck-Cre Irf8 wt/wt littermate controls were subjected to 7 polarization. As expected, cells from mice with a T cell-specific deficiency in showed a remarkable increase in the generation of -producing cells (Fig. d) in association with significantly elevated levels of secretion (Fig. e). These results excluded the possibility that the effects of deficiency on T cells from mice with a conventional KO of the gene could be attributed to the altered activities of other subsets of -deficient cells, such as B cells, DC or macrophages. 7 cells generated from splenic T cells of Irf8 / mice comprised a major portion of the β-tcr CD4 lymphocyte subset (Supplementary Fig. S5). Indeed, high levels of protein expression were detected in CD4 T cells polarized under 7 conditions as determined by immunoblot analyses (Fig. f). Accordingly, transcript levels of the iconic 7 cytokines A, F and IL- were enhanced in Irf8 / 7 cells (Fig. g). In contrast, or differentiation was not noticeably affected in Irf8 / T-cell cultures (Fig. ac). In addition, [ 3 H]-Thymidine incorporation assay showed that the nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 ARTICLE a 7 b c 5 4 3.4.56.4.3 3 4 5. 36.3..9 CD4 T cells (%) 5 4 3 7 7 (ng ml ) 9 6 3 TH TH TH7 TH TH7 5 4 3.94 5.6 97. 84.4 3.8 33.6 96. 3 4 5 CD4 7 66.4 d 7 7 e f Lck-Cre lrf8 wt/wt Lck-Cre lrf8 fl/fl 4 3.6. 5.6.88 3.85.5.57.7 3 4 g F IL- IL-4 6 5 Relative mrna level 5 4 3 4 3 4 6 4 3 5 4 3 7 7 7 7 7 4 3. 5..37.89 3..83 3 4 FOXP3.5.74 (ng ml ) 6 4 Lck-Cre lrf8 wt/wt Lck-Cre lrf8 fl/fl β-actin 7 36 kda Figure Increased production in deficient T helper cells. (a) Naive CD4 T cells from wild-type () or Irf8 / mice were differentiated under and 7 polarizing conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h, stained for intracellular and, and analysed by flow cytometry. Representative fluorescence-activated cell sorting (FACS) dot plots gated on CD4 cells and the percentages of - producing CD4 cells are shown. Data are from one experiment representative of three independent experiments. Error bars, s.d. (b) The cells prepared in a, and polarizing conditions were re-stimulated with PMA/ionomycin for 4 h and the supernatants were analysed for by ELISA. Data are from one experiment representative of three independent experiments. Error bars, s.d. (c) Wild-type or Irf8 / lamina propria lymphocytes (LPL) were differentiated under 7 conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h, stained for intracellular and analysed by flow cytometry. (d) Naive CD4 T cells from Lck-Cre Irf8 wt/wt and Lck-Cre Irf8 fl/fl mice were differentiated under and 7 polarizing conditions for 4 days and the cells were re-stimulated with PMA/ionomycin for 5 h for staining of, and FOXP3. Representative FACS dot plots gated on CD4 cells are shown. (e) The cells prepared in d were re-stimulated with PMA/ionomycin for 4 h and the supernatants were analysed for by ELISA. Data are from one experiment representative of two independent experiments. Error bars, s.d. (f) Naive CD4 T cells from C57BL/6 mice were differentiated under and 7 conditions for 4 days. The cells were re-stimulated with PMA/ionomycin for h and expression was analysed by western blot. (g) The cells prepared in a were re-stimulated with PMA/ionomycin for 5 h and mrna expression of indicated genes was determined by qpcr. The data shown were normalized to levels of ubiquitin expression as analysed by qpcr. The results are representative of three independent experiments. Error bars, s.d. proliferation of CD4 T cells from Lck-Cre Irf8 fl/fl and Lck-Cre - Irf8 wt/wt mice cultured under 7 conditions was comparable (Fig. d). Taken together, these results indicate that is induced during T-cell activation, and that 7-cell differentiation is enhanced in cells deficient in. T reg cells and autocrine cytokines are not altered in Irf8 / mice. To understand whether alterations in T reg cells might contribute to enhanced 7 differentiation in -deficient mice, we analysed FOXP3 CD4 T cells in these mice. There were no significant differences between the FOXP3 CD4 T-cell populations of and Irf8 / mice under 7- or T reg -inducing conditions (Fig. 3a,b). Thus, the more efficient generation of Irf8 / 7 cells in response to the combined effects of plus IL-6 was not because of alterations in -derived T reg suppression. -producing cells generated from Irf8 / T-cell cultures were greatly increased following stimulation with IL-6 plus (Fig. 3c). To further investigate how affects 7 differentiation, naive and Irf8 / CD4 T cells were subjected to 7 differentiation in the presence of IL-6, IL-3 and, either alone or nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 a c b.85.3 3.44 4.97. 3.54 3 4.8.6.48 4.3.4 3.55 3 4 IL-4 36. 36.3 5.8 53. IL-4 CD4 T cells (%) CD4 T cells (%) 5 4 3 8 6 4 (ng ml ) d 6 4 3 H-thymide uptake (CPM), 5,, 5, LcK-Cre lrf8 wt/wt Medium T cells IL-4 (ng ml ). 7 7..9.6.3 7 LcK-Cre lrf8 fl/fl 7 7 Figure and differentiation in Irf8 / CD4 T cells. Naive CD4 T cells of or Irf8 / mice were differentiated under or polarizing conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h and stained for intracellular as a marker (a) and IL-4 as a marker (b) by flow cytometry. Data are from one experiment representative of three independent experiments. Error bars, s.d. The cells prepared in a and b were re-stimulated with PMA/ionomycin for h and the supernatants were analysed for and IL-4 by ELISA (c). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 T cells from spleens and lymph nodes of Lck-Cre Irf8 wt/wt and Lck-Cre Irf8 fl/fl mice were prepared and the cells were differentiated under and 7 polarizing conditions for 3 days. [ 3 H]-Thymidine was added during the last 8 h of culture. Then the cells were collected and were counted with a beta-counter (d). Data are from one experiment representative of two independent experiments. Error bars, s.d. in various combinations, and then examined for the expression of lineage-specific genes by qpcr. Neither IL-6 nor alone induced significant levels of or F transcripts (Fig. 3d). In contrast, dramatic increases in and F transcripts were induced at multiple time points by the combination of IL-3, IL-6 and in cultures of both Irf8 / and T cells (Fig. 3d). Increased expression of was confirmed at the protein level by ELISA (Fig. 3eg). IL-3 induced low levels of and F transcripts with no significant differences being seen between CD4 T cells of and Irf8 / mice (Fig. 3d). This suggests that may not target the IL-3 signalling cascade, but may exert a major influence on 7 differentiation instead of 7 expansion and maintenance. IL-, an autocrine cytokine produced by CD4 T follicular helper cells and 7 cells 6,7, induces 7 differentiation in the presence of. -deficient T cells displayed enhanced induction of 7-associated molecules following stimulation by combined with IL-6 or IL- (Figs 3h and 4a). However, production of IL- and IL- was comparable in and Irf8 / 7 cells (Fig. 4be). These results suggest that an autocrine loop involving either IL- or IL- is not involved in the functional control of 7 differentiation by. We next determined whether the induction of 7-associated genes may be affected by forced expression of in T cells. Retroviral transduction of -IRES-GFP into naive CD4 T cells significantly decreased the percentage of -producing cells under 7 polarizing conditions (Fig. 5a), and -positive cells were moderately reduced (Fig. 5a). Similarly, retroviral transduction of into the EL4T lymphoma cell line stimulated with PMA/ionomycin resulted in significantly reduced transcripts for, but had no effect on the expression of interferon (IFN)-γ, IL-4, IL- or FOXP3 (Fig. 5b). Taken together, these results demonstrate a direct role for in suppressing 7-specific gene expression. interacts with and suppresses transcription. The above findings prompted us to probe the molecular basis for control of 7-cell differentiation. As many studies have demonstrated a critical role for in 7-cell differentiation both in vitro and in vivo, we asked if might affect mediated induction. EL4 cells were transiently transfected followed by stimulation with PMA/ionomycin. Overexpression of resulted in significantly increased expression of and F, whereas co-transfection with greatly reduced the expression of these genes, suggesting that inhibits induced expression of transcripts (Fig. 6a). Using a 6-kbp promoter reporter plasmid, we confirmed that strongly induced promoter reporter activity in 93T cells (Fig. 6b), nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 a b c IL-6 Relative cell number IL-6.94.37. 4.59 5.7 4.36.73 9.8.6 9.6.38 34.68.49 3.8.58.67 3.7 3 4 FOXP3 FOXP3.49 8.3 3.8 RA /IL-6 /IL-6 RA..64.5 4.53.4 6.4.3 7.7 3.7 59. 7.4 3.6 58. 7.48 3.8 4.9.35.7 3.73 3.3 4 3 4.7 3. 3.6 9.4.4.3.9.9 3 4 d Relative mrna level e 7,5 6, 4,5 3,,5, 7,5 5,,5 IL-6 IL-3.95 5.78 4 4.96 3.43.98 4.3 3.8 4 3.7 3 7.7.55 3 4 48 7 (h).4.4 None IL-3 IL-6 IL-3 f g h Relative mrna level (ng ml ). IL-6 IL-3 (ng ml ) 4 CCR6 6 4. 7.5 5..5 5 5 48 7 (h) TH α-il-4/α-/th7 TH TH7 α-il-4/α-/th7 4, ARTICLE A IL- IL-3R 3, 4, 5 3,, 5,,, 5 3,,, Foxp3,4,5 7 35 None IL-6/ IL-/ Figure 3 inhibits the expression of 7-associated genes. Naive CD4 T cells from wild-type and Irf8 / mice were stimulated with plate-bound anti-cd3 and anti-cd8 antibodies in the presence of IL-6 ( ng ml ) plus differing concentrations of (.,, 5 ng ml ) (a), or (5 ng ml ), (5 ng ml )/IL-6 ( ng ml ) with or without retinoic acid (RA, nm) (b). After 4 days of stimulation, and FOXP3 intracellular staining was performed and analysed by flow cytometry. (c) The cells prepared in a and b except for the presence of (5 ng ml ) plus various concentrations of IL-6 (5,, 5, ng ml ) were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular and and analysed by flow cytometry. (d) Naive CD4 T cells from wild-type and Irf8 / mice were stimulated with IL-6, IL-3, or different combinations of these cytokines for various intervals. Total RNA was extracted and analysed by RTPCR for mrna expression of (top panel) and F (bottom panel). Data are from one experiment representative of three independent experiments. Error bars, s.d. (e) Naive CD4 T cells from and Irf8 / mice were stimulated with the indicated cytokines for 4 days. Cells were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular and and analysed by flow cytometry. The results are representative of three independent experiments. (f) Cells were prepared as in e and the culture supernatants were collected after 4 days of stimulation. protein secretion was analysed by ELISA. (g) Naive CD4 T cells from and Irf8 / mice were prepared and stimulated with and IL-6 in the presence of neutralizing anti- ( µg ml ) and anti-il-4 ( µg ml ) antibodies. At 7 h after the stimulation, protein secretion in culture supernatants was analysed by ELISA. Data are the mean ± s.d. of triplicate cultures. (h) Naive CD4 T cells from wild-type or Irf8 / mice were stimulated with the indicated cytokine combinations for 48 h. Total RNA was extracted and analysed by qpcr for mrna expression of the indicated genes. Data are from one experiment representative of three independent experiments. Error bars, s.d. which was shown to be due to a direct effect of on the promoter 6,7. Co-transfection of in these cells suppressed -mediated promoter activity in a dose-dependent manner (Fig. 6c). Similar results were observed in EL4 cells (Fig. 6d). CNS is a conserved non-coding sequence (CNS) element ~5 kbp upstream of the locus that functions as a -dependent enhancer element required for optimal transcription 7,8. Overexpression of significantly suppressed CNS-enhanced promoter activity (Fig. 6e), indicating that inhibits transcription. To investigate whether can directly bind the CNS region of the promoter, we co-transfected an promoter reporter (containing CNS) and plasmids into 93T cells and performed chromatin immunoprecipitation (ChIP) using an -specific antibody. The precipitated chromatin DNA was analysed by qpcr using primers covering the CNS region of the promoter. The results showed that antibody specifically pulled down the CNS region sequences (Fig. 6f). To confirm the results, naive and Irf8 / CD4 T cells were stimulated under 7 polarizing conditions for 6 h and ChIP assay was performed as above. The precipitated chromatin DNA was analysed by PCR using primers covering the CNS region of the promoter. Similarly, antibody specifically pulled down the CNS region sequences of 7 cells from mice but not from Irf8 / mice (Fig. 6g). These results demonstrate that bound directly to this region of the promoter. Further analyses of the CNS sequence revealed several IRF consensus binding sequence elements (Supplementary Fig. S6a). We showed that mutation of a typical IRF-binding site nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
a c d IL- (pg ml ) 4 3 45 45 3 5.8 3..98 IL- None.. 3 4 7 7 /IL-.3 4.. 35.79.4 e.8 Relative IL- mrna level /IL-6.3.48 4 6.5.4 8.4.88 3 /IL-.74.6 3 4.4 3 5 b IL- (pg ml ) 75 5 5 IL-6 IL- None /IL-6 None /IL-6 Figure 4 IL- signalling and IL- production during 7-cell differentiation in Irf8 / mice. Naive CD4 T cells from and Irf8 / mice were prepared, and the cells were stimulated with anti-cd3 and anti-cd8 antibodies in the presence or absence of plus IL- or IL-6 for 7 h. Intracellular staining for and expression was performed and analysed by flow cytometry (a). The secretion of IL- protein in culture supernatants was analysed by ELISA (b). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 T cells were stimulated with anti-cd3 and anti-cd8 antibodies in the presence of plus IL-6 or IL-. At 96 h after stimulation, intracellular staining for and IL- was performed and analysed after re-stimulation with PMA/ionomycin by flow cytometry (c). Naive CD4 T cells from spleens and lymph nodes of and Irf8 / mice were prepared and the cells were stimulated under,, and 7 polarizing conditions for 4 days. Then the cells were re-stimulated with PMA/ionomycin for h and IL- protein secretion was analysed by ELISA (d). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 T cells were stimulated with anti-cd3 and anti-cd8 antibodies in the presence of plus IL-6 or IL- for 7 h. IL- expression was analysed by RTPCR (e). Data are from one experiment representative of three independent experiments. Error bars, s.d. nature communications DOI:.38/ncomms3 in the CNS region of promoter nullified the inhibition of -mediated promoter activation by (Supplementary Fig. S6b). These results provided a molecular basis for understanding the inhibitory effects of on transcription. It is likely that cooperates with other known transcription factors, such as FOXP3 or RUNX, or unknown factors in the coordinate regulation of 7 differentiation 6,9,3. We then co-transfected HA-tagged and T7- plasmids into 93T cells for co-immunoprecipitation. Immunoprecipitation of resulted in co-precipitation of (Fig. 7a), even in the presence of ethidium bromide or DNase I, indicating that and interact with each other without the involvement of DNA. Using confocal microscopy, we also determined that and co-localized in the nucleus of NIH3T3 cells co-transfected with GFP- and T7- constructs (Fig. 7b). Flow cytometric analyses of naive CD4 T cells stimulated under 7 polarizing conditions clearly revealed a population of CD4 cells with the majority of these cells also staining for (Fig. 7c). Furthermore, anti- antibodies were found to co-immunoprecipitate from lysates of primary CD4 T cells from but not from Irf8 / mice cultured under 7 polarizing conditions (Fig. 7d), indicating that endogenous and interact with each other. As shown in Figure 7e, is comprised of an N-terminal DNA-binding domain, a flanking internal region, and a C-terminal IRF association domain 3. To map the binding sites between on, we co-transfected 93T cells with T7-tagged and Flag-tagged full-length or one of a series of C-terminal truncation mutants (39, 356, 35, 53, 3, 9 and 54) followed by co-immunoprecipitation experiments. The results showed that amino-acid residues between 3 and 9 were important for the physical interaction with (Supplementary Fig. S7a, Fig. 7f). mutant (4), which is not bound to, did not suppress -mediated promoter activation as and other binding mutants did (Fig. 7g and Supplementary Fig. S7b), demonstrating that the inhibitory activity of on transcription is related to its interaction with. In addition, IRF4, another IRF family member that is required for the generation of 7 cells 3, was also found in co-immunoprecipitation experiments to interact with, whereas there was no interaction between IRF and (Supplementary Fig. S8a). Although transcript levels for IRF4 in cells deficient in did not change under 7 polarizing conditions (Supplementary Fig. S8b), we still cannot exclude possibility that and IRF4 mutually influence their activities in 7 cells. Thus, our results suggest that protein protein interactions between and have at least a partial role in -mediated inhibitory effects on transcription. controls 7-cell differentiation in vivo. To further assess the effects of on 7-cell differentiation in vivo, we performed adoptive transfer experiments using CD4 CD6L CD45RB hi CD5 cells from and Irf8 / mice to induce colitis in RAG KO (Rag / ) mice. Irf8 / mice did not develop spontaneous colitis during an observation period of.5 years (Fig. 8a,b). However, Rag / mice reconstituted with Irf8 / naive CD4 T cells began losing weight earlier and lost more weight than mice in the control group. Parallel histological studies of colon sections from Rag / mice reconstituted with Irf8 / T cells revealed more severe inflammatory cell infiltrates and significantly higher pathological scores than those observed in sections from mice reconstituted with T cells from mice (Fig. 8ce). In addition, mice reconstituted with Irf8 / cells had a significantly higher percentage of -producing cells than control mice (Fig. 8f). To determine whether T reg cells from Irf8 / mice could suppress effector T cells, we examined the population of T reg cells in Rag / mice after the transfer of naive or Irf8 / CD4 T cells. Rag / recipients of naive CD4 T cells from mice of either genotype generated a small percentage of CD4 FOXP3 and CD4 cells in the mesenteric lymph nodes (Fig. 8f). Furthermore, co-transfers of naive CD4 T cells with CD4 CD5 cells purified from or Irf8 / mice resulted in similar effects on body weight (Supplementary Fig. S9). These results indicate that the effects of deficiency on T cell-mediated inflammation nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 ARTICLE a 5 4 3 7 RV-MIG RV-MIG RV-MIG-.97 5.3 4.4 98 84.7 95.6 3 4 5 GFP % of Max 8 6 4 RV-MIG RV-MIG- 7 RV-MIG 7 3 4 b 5.3 IL- 6.8 IL-4 5.5 8.4 Relative mrna level. 4.6 FOXP3 6 IL-. 4,.8 8,. 5 5 3 4 5 5 3 4 5 5 3 4 5 5 3 4 (h) Figure 5 Transduction of inhibits 7-associated genes. (a) Naive CD4 T cells from C57BL/6 mice were infected with retrovirus encoding or empty vector and were activated under 7-inducing conditions for 4 days. The cells were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular and and analysed by flow cytometry. (b) EL4 cells were transduced with retroviruses encoding (black column) or GFP (white column) for 48 h and the transduced cells were then stimulated with PMA/ionomycin for various times as indicated. Total RNA was extracted and the transcript levels of 7-associated genes were analysed by qpcr as indicated. The results are normalized to ubiquitin levels. Data are from one experiment representative of three independent experiments. Error bars, s.d. could not be explained by influences on the function of CD4 T reg cells. Thus, deficiency promotes intestinal inflammation in a T cell-mediated model of colitis, suggesting that may have an inhibitory role in the control of 7-mediated immune responses. To further understand the role of in 7-cell differentiation in vivo, we extended our observations to an infection model, as 7 cells have also been proposed to have a role in inflammation against both intracellular and extracellular bacteria 33,34. Staphylococcus aureus is a Gram-positive bacterium that can induce production from CD4 T cells mainly through Staphylococcus aureus enterotoxin A (SEA), and humans deficient in 7 cells are highly susceptible to infection with this agent 35,36. To better understand the regulatory effects of on 7-cell differentiation in a broader sense, we used superantigenic S. aureus to induce production in vivo. Spleen cells from Lck-Cre Irf8 fl/fl and Lck-Cre Irf8 wt/wt littermate controls immunized with SEA 4 days previously were re-stimulated in vitro with SEA for an additional days and then examined for -producing CD4 T cells by flow cytometry and for secretion by ELISA (Supplementary Fig. S). - deficient mice generated significantly more -producing CD4 T cells than mice, further confirming that negatively regulates 7-cell differentiation in vivo. Discussion 7 cells represent a recently defined member of a still growing family of T helper cells. The mechanisms involved in the silencing programme for this T helper subset remain unclear. Here we demonstrate that serves as an intrinsic silencer for 7-cell differentiation. -deficiency in both conventional and T cell-specific conditional KO mice led to more robust 7-cell differentiation without effects on either or cell lineages. Furthermore, transfer of / CD4 CD45Rb hi cells into Rag / mice induced more severe colitis than transfer of CD4 CD45Rb hi cells. In addition, mice reconstituted with / cells had a significantly higher percentage of -producing cells than mice reconstituted with cells. In addition, we showed that physically interacts with resulting in suppression of transcription. These results suggest that negatively regulates the development of 7 immune response resulting in the control of inflammation. Many studies have demonstrated that has important functions in myeloid cells 4. Macrophages from Irf8 / mice did not produce IL- in response to and LPS. regulates IL- expression by binding to the IL- p4 promoter region, acting in synergy with IRF to activate IL- p4 gene expression 37. In addition, also induces the expression of other inflammatory proteins expressed by myeloid cells, including inos, IL-8 and IL- (refs 384). protein levels are controlled in part by Cbl-mediated ubiquitylation and subsequent proteasomal degradation 4. More recent studies have shown that transcriptional activation of IL-p4 by is enhanced following ubiquitylation by the E3 ubiquitin ligase, TRIM (ref. 4). Here, we showed that CD4 T cells clearly expressed protein on TCR engagement and that nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 a F FOXP3 b f Relative mrna level c,,5, 5 /IL-6, ** 4, ** 3,,, -Luc Vector d 75 9, 6, 3, * Relative Iuciferase activities, 75 5 5 e 6 * Relative binding g 6 4 Input IgG α- Relative luciferase activities 75 5 5 Relative luciferase activities 5 5 * Relative luciferase activities 4 bp IP IP Input IgG Input IgG CNS FOXp3 /IL-6 Figure 6 IRF-8 represses transcription. (a) -expressing EL4 cells (black column) or control cells (white column) were transiently transfected with a plasmid for 4 h, and the cells were treated with plate-bound anti-cd3 and anti-cd8 antibodies in the presence or absence of the indicated cytokines for 8 h. qpcr analyses of transcripts of the indicated genes were performed and the results were normalized to the levels of ubiquitin transcripts. **P <. versus cells transfected with (Student s t-test). Data are representative of multiple experiments. (b) 93T cells were cotransfected with an promoter reporter construct containing a 6-kbp promoter and increasing doses of a plasmid for 3 h. (c) 93T cells were co-transfected with an promoter reporter construct containing a 6-kbp promoter, a plasmid, a FOXP3 plasmid, and increasing doses of an plasmid for 3 h. (d) -expressing EL4 cells (black column) or control cells (white column) were co-transfected with an promoter reporter construct containing a 6-kbp promoter plus plasmid, and treated with plate-bound anti-cd3 antibody in the presence or absence of the indicated cytokines for 4 h. Luciferase activities in (b, c, d) were measured and normalized to β-galactosidase activity. Data are mean ± s.d. of triplicate cultures and are representative of three independent experiments. *P <.5 versus cells transfected with (Student s t-test). (e) -expressing EL4 cells (black column) or control cells (white column) were co-transfected with an promoter reporter construct containing a minimal.-kb promoter plus CNS, and plasmids. The cells were treated with plate-bound anti-cd3 and anti-cd8 antibodies and IL-6/ for 4 h. A luciferase assay was performed as described in (b), (c), and (d). *P <.5 versus cells transfected with (Student s t-test). (f) 93T cells were co-transfected with an promoter reporter construct (containing CNS) with either an overexpression plasmid or a control vector for 36 h, followed by ChIP analysis. 3 µg of anti- antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. qpcr was used to quantify the amount of precipitated DNA with primers flanking the CNS region of the promoter. Data were normalized to input DNA in each respective sample. (g) Naive CD4 T cells from wild-type or Irf8 / mice were cultured under 7-polarizing conditions for 6 h, followed by ChIP assay as described above. 3 µg of anti- antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the CNS region of the promoter. 7 polarization conditions induced stable protein expression. In addition, mitogen-activated protein kinase inhibitors completely blocked protein expression induced by TCR activation and STAT3 mutant mice showed impaired mrna expression, indicating that the TCR signalling cascade is involved in induction of expression. The percentages of CD4 T cells in tissues of Irf8 / and mice were comparable. Following stimulation under 7 polarizing conditions, however, the percentages of - producing CD4 T cells were greatly increased and the expression of 7 signature genes was significantly enhanced for cells from Irf8 / as compared with mice. These results suggest that is an important transcription factor in controlling CD4 T cell plasticity by targeting 7-cell differentiation. The balance between pathogenic 7 cells and suppressive T reg cells in the immune system depends on the presence of inflammatory cytokines such as IL-6 and IL- (ref. 43). The antiinflammatory cytokine,, combined with IL-6 or IL- can drive the conversion of a cell phenotype from -induced FOXP3-expressing T reg cells to -expressing 7 cells. inhibits the expression of STAT4 and GATA3, thereby preventing the differentiation of and cells, respectively, and concurrently facilitating 7-cell development. CD4 T cells from and Irf8 / mice yielded similar populations of FOXP3 cells following stimulation under 7- or T reg -inducing conditions. In addition, there were no significant differences between and Irf8 / 7 cells in the production of the autocrine cytokines, IL- and IL-. These results rule out the possibility that enhanced generation of 7 cells by CD4 T cells from Irf8 / mice stimulated with plus IL-6 was due to alterations in -derived T reg suppression or to an autocrine loop involving IL- or IL-. acts as a transcriptional repressor or a transcriptional activator depending on the target DNA sequence and interactions with different partner proteins, including PU., E47 and other IRFs 5. We demonstrated that interacts directly with, resulting in suppression of transcription. The association of with was not competed by FOXP3, another -binding nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 a c e f Lysate IP:T7 -T7 -HA 5 4 3 WB:HA WB:T7 WB:HA WB:T7 h 3.8.4 8. 36.9.5 3 4 5.69 4 375 44 DBD IAD -T7 FL -3-9 -54 -Flag IP:Flag IB:T7 IP:Flag IB:T7 7 h 8 kda DNase I d WB: WB: g Relative luciferase activities protein 44, indicating that antagonizes the effect of without the involvement of FOXP3. In addition, co-immunoprecipitation studies showed that IRF4, another IRF family member required for 7-cell differentiation 3, also interacts with. It is likely that cooperates with other transcription factors, such as FOXP3 or RUNX, or unknown factors in the regulation of 7 Lysate b GFP IP: lgg cells,5, 5 Alexa 594 * Lysate lgg IP: cells Merged FL -4 Vector Figure 7 interacts physically with RORγ. (a) 93T cells were transfected with HA-tagged and T7-tagged overexpression plasmids for 4 h and cell lysates were prepared in the presence or absence of DNase I or ethidium bromide. 5 µg of cell lysates were immunoprecipitated with an anti-t7 antibody and immunoblotted with specific antibodies as indicated. Data are representative of three independent experiments. (b) NIH3T3 cells were transiently transfected with GFP- and T7- for 4 h, and cells were fixed and stained red for followed by confocal microscopic analysis. Scale bar, 5 µm. (c) Naive CD4 T cells from mice were cultured under 7-polarizing conditions for 7 h and the expression of and was analysed by flow cytometry. The cells were gated on CD4 T cells. Data are representative of three independent experiments. (d) Naive CD4 T cells from wild-type or Irf8 / mice were cultured under 7-polarizing conditions for 6 h and the cell lysates were then immunoprecipitated with an anti- antibody and western blotted (WB) with anti- and anti- antibodies. Data represent three independent experiments. (e) Diagrams of protein domains. (f) 93T cells were co-transfected with plasmids containing Flag-tagged full-length, fragments (3, 9, 54) and T7-tagged plasmid for 4 h, and co-immunoprecipitation using anti-flag antibody from the cell extracts was performed and immunoblotted with anti-t7 antibody. Data are representative of three independent experiments. (g) 93T cells were co-transfected with an promoter reporter construct containing the 6-kbp promoter, a plasmid and either a full-length or the truncation mutant (4) construct for 3 h. Luciferase assays were performed as described in b. Data indicate mean ± s.d. of triplicate cultures and are representative of three independent experiments. *P <.5 versus cells transfected with mutant (Student s t-test). EtBr ARTICLE differentiation 6,93. It remains to be determined how these factors might collaborate with, the master transcription factor for 7 cells to regulate differentiation of this subset. 7 cells are critical pathogenic effector T cells in inflammatory disorders such as inflammatory bowel disease 4547. In the present study, we demonstrated that targets, resulting in the silencing of 7-cell differentiation. In addition, transfer of / CD4 CD45Rb hi cells into Rag / mice induced more severe colitis than transfer of cells. These results suggest that functions as an important transcription factor in the control of inflammation by modulating activity. A recent genome-wide association study identifying as a susceptibility locus in patients with multiple sclerosis 48 is supportive of this model for function in inflammatory diseases. As a result, our data may provide a molecular basis for identifying specific single-nucleotide polymorphisms associated with susceptibility to clinical immune pathologies. Taken together, our results demonstrate that is stably expressed during 7-cell differentiation and has a critical role in directing the silencing programme for 7-cell development. On the basis of these studies, we propose a novel molecular mechanism for the inhibitory effects of on 7 differentiation and cytokine expression that involves the modulation of activity (Supplementary Fig. S). Our observations support a pathogenic role for 7 cells in exacerbating inflammation and indicate that may be a therapeutic target for controlling 7-mediated autoimmune and inflammatory diseases. Methods Mice. C57BL/6 (B6) and B6-Irf8 / mice were maintained in the barrier facility at the Mount Sinai School of Medicine. GFP-FOXP3 mice were crossbred with Irf8 / mice to obtain Irf8 / GFP-FOXP3 mice. conditional knockout mice (Irf8 fl/fl ) were generated at Ozgene under a contract with NIAID by flanking exon and an inserted PGK-neo cassette with loxp sites. Following homologous recombination of the targeting vector in C57BL/6 ES cells and establishment of germ line transmission, the PKG-neo cassette, which was flanked by flippase recognition target (FRT) sites, was excised by crossing with a FLP transgenic mouse. Selective breeding was used to eliminate the FLP gene. Conditional deletion of in T cells was performed by crossing with Lck-Cre mice to generate Lck-Cre Irf8 fl/fl mice. The animal study protocols were approved by the Institutional Animal Care and Use Committees of Mount Sinai, NICHD and NIAID (protocol LIP-4). Antibodies. The following antibodies were purchased from BD Biosciences, as conjugated to FITC, PE, PE-Cy5, percp-cy5.5 or APC: CD4 (L3T4), CD8 (53-6.7), CD3e (45-C), CD5 (PC6.5), CD44 (IM7), CD6L (MEL-4), CD45RB (C363-6A), (TC-8H), (XMG.), TCRβ chain (H57-597) and isotype controls. Antibodies for IL- (JES6-A), IL-4 (B), IL- (JES5-6E3) and Foxp3 (FJK-6S) were purchased from ebiosciences. CD4 T cell preparation and differentiation in vitro. Naive CD4 T cells (CD6L CD44 lo ) were prepared by fluorescence-activated cell sorting from spleens and lymph nodes of Irf8 / and littermates. The sorted cells were primed for 96 h with anti-cd3 ( µg ml ; 45-C; BD Biosciences) and soluble anti-cd8 ( µg ml ; 37.5; BD Biosciences). The cells were rested for 48 h, and were then re-stimulated for 5 h with PMA plus ionomycin in the presence of brefeldin A and intracellular cytokines were measured by flow cytometry. Cells stimulated under neutral conditions were defined as cells. Cells were stimulated to differentiate into cells by supplementation with IL- plus anti-il-4 ( µg ml ; B; BD Bioscences) or into cells by supplementation with IL-4 and anti- ( µg ml ; XMG., BD Biosciences). For 7-cell differentiation, cells were stimulated with transforming growth factor-β (5 ng ml ), IL-6 ( ng ml ) and IL-3 ( ng ml ; all from R&D Systems) in the presence of anti-il-4 antibody ( µg ml ; B, BD Bioscences) and anti- antibody ( µg ml ; XMG., BD Biosciences). Intracellular staining and flow cytometry. Cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A before intracellular staining. Cells were fixed with IC Fixation Buffer (BD Biosciences), incubated with permeabilization buffer, and stained with PE-anti-mouse, APC-anti- and PE-Cy 5.5 anti-mouse CD4 antibodies. Flow cytometry was performed on a FACSCalibur (BD Biosciences) and LSR Fortessa (BD Biosciences). RNA isolation and quantitative real-time RTPCR. Total RNA was extracted using an RNeasy plus kit (QIAGEN) and cdna was generated with an oligo (dt) primer and the Superscript II system (Invitrogen) followed by analysis using nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.
nature communications DOI:.38/ncomms3 a Histological score 5 9 6 3 Young Old Young Old b old age old age c Change of baseline (%) 5 * * * * 95 9 85 CD45RB hi CD45RB hi 8 7 4 8 35 Days post T cell transfer d e CD45RB hi CD45RB hi f 5 * **.6 Histological score 5 CD45RB hi CD45RB hi CD4 cells (%) 8 6 4 CD4 cells (%) 8 6 4 FOXP3 CD4 cells (%).4.. Figure 8 Lack of enhances the 7 immune response in experimental colitis. and Irf8 / mice were maintained under specific pathogen free (SPF). conditions for up to 8 months. Mice were killed and intestines were removed for histological analysis. Histology of colon tissues (a) and disease score (b) from age-matched young (5 weeks) and old (78 months) and Irf8 / mice (three to four mice in each group). Scale bars, µm. CD4 CD45RB hi T cells were purified from spleens and lymph nodes of wild-type or Irf8 / mice and 5 5 cells were injected (i.p.) into recipient Rag / mice. Body weight change was monitored every week and mice were killed 7 weeks later. (c) Changes in body weight of Rag / mice (n = 56 mice per group) after intraperitoneal transfer of or Irf8 / CD4 CD45RB hi T cells were recorded. Data are presented as the mean ± s.d. of the percentage of initial body weight and are representative of two similar experiments. *P <.5 versus recipients of cells (ANOVA test and Student s t-test). Disease scores (d) and sections of colons with colitis (e) from Rag / mice (n = 56 mice in each group) on day 35 after naive T cell transfer as described in c. *P <.5 versus recipients of cells (MannWhitney test). Scale bars, µm. (f) The percentage of, and FOXP3-producing cells from mesenteric lymph nodes of Rag / mice in c (white column, transfer with cells; black column, transfer with Irf8 / cells). **P <. versus wild-type cell transferred mice (Student s t-test). Data are presented as the mean ± s.d. from four mice in each group. Two independent experiments were performed with similar results. icycler PCR with SYBR Green PCR master Mix (Applied Biosystems). Results were normalized based on the expression of ubiquitin. The following primer sets were used: A, F, IL-, IL-3R,,,, IRF IRF4 T-bet FOXP3, CCR6, IL-, IL-4, IL- and ubiquitin (Supplementary Table S). Transfection and luciferase reporter assay. 93T cells were transiently transfected with an promoter luciferase reporter plasmid together with in the presence of plasmid at different concentrations. For each transfection,. µg of plasmid was mixed with µl of Opti-MEM I medium (without serum and antibiotics) and 4. µl of Lipofectamine reagent. The mixture was incubated at room temperature for min and added to -well plates containing cells and complete medium. The cells were incubated for 3 h and collected using reporter lysis buffer (Promega) for determination of luciferase activity. Cells were co-transfected with a β-galactosidase reporter plasmid to normalize experiments for transfection efficiency. Generation of the mutant promoters. A predicted IRF-binding site adjacent to the downstream -binding site in the CNS region of the mouse A promoter was mutated using QuickChange XL Site-Directed Mutagenesis Kit (Stratagene) according to manufacture s instruction. Two mutations were introduced on the promoter regions 54 to 5 (TGG to CCC) and 5 to 599 (AAA to CCC) from the transcriptional initiation site ( ) using the following primer sets: 5 -GGTTGGAAAAAAAAACCCAAAGTTTTCTGACCCA-3 and 5 -TGGGTCAGAAAACTTGGGTTTTTTTTTCCAACC-3 for TGG to CCC; 5 -TGGAAAAAAAAATGGCCCGTTTTCT-GACCCACT-3 and 5 -AGTGGGTC AGAAAACGGGCCATTTTTTTTTCCA-3 for AAA to CCC. Mutations were verified by sequencing. Retroviral transduction of in CD4 T cells. To prepare pseudotyped virus human 93 EbnaT cells were seeded at a density of 4 6 cells in a -cm dish. The next day, cells were transfected using calcium phosphate with a mixture of.5 µg of plasmid pmd.g encoding vesicular stomatitis virus G protein, 7.5 µg of plasmid encoding gag-pol, and µg of a retroviral expression constructs encoding GFP or. At 48 h after transfection, the viral supernatant was collected, centrifuged at 8g, and used to infect cells. CD4 T-cell transduction was performed as previously described, sorted naive CD4 T cells were plated as above and cultured for 4 h in the presence of anti-cd3 ( µg ml ; 45-C, BD Biosciences) and soluble anti-cd8 ( µg ml ; 37.5; BD Biosciences). Activated cells were then transduced with fresh retrovirus supernatant by centrifugation for.5 h at g in the presence of polybrene (6 µg ml ; Sigma). After 4 h, the cells were re-transduced using the same procedure and cultured for an additional 4 h and were then stimulated with and IL-6. The cells were collected on day 5 or 6 for intracellular cytokine staining. T-cell proliferation assay. Naive CD4 T cells were purified from spleens and lymph nodes of Lck-Cre Irf8 fl/fl and Lck-Cre Irf8 wt/wt littermate controls. Cells ( 5 per well) were cultured in the absence or presence of anti-cd3 ( µg ml ) and anti-cd8 ( µg ml ) antibodies for 3 days in 96-well microplates. [ 3 H]-Thymidine was added during the last 8 h of a 7-h culture. The cells were then collected and counted with a beta-counter. Co-immunoprecipitation and immunoblotting analysis. Cells were washed with cold phosphate-buffered saline and lysed for 5 min on ice in.5 ml of lysis buffer (5 mm Tris-HCl, ph 8., 8 mm NaCl,.5% Nonidet P-4,. mm EDTA, mm EGTA, % glycerol and mm dithiothreitol) containing protease inhibitors. Cell lysates were clarified by centrifugation (4 C, 5 min,, r.p.m.), aliquots (5 µg) were incubated with µg of normal rabbit IgG for 4 h and µl of protein G-Sepharose was added to the mixture for h. After centrifugation, the supernatant was collected and incubated with µg of anti-flag antibody overnight at 4 C with gentle rocking, after which immune complexes were collected as described nature communications :34 DOI:.38/ncomms3 www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved.