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. 2020 Apr 29;23(5):101106. doi: 10.1016/j.isci.2020.101106

RORγt-driven TH17 Cell Differentiation Requires Epigenetic Control by the Swi/Snf Chromatin Remodeling Complex

Sungkyu Lee 1, Jieun Kim 1, Hyungyu Min 1, Rho H Seong 1,2,∗
PMCID: PMC7235640  PMID: 32434140

Summary

Epigenetic regulation, including chromatin accessibility and posttranslational modifications of histones, is of importance for T cell lineage decision. TH17 cells play a critical role in protective mucosal immunity and pathogenic multiple autoimmune diseases. The differentiation of TH17 cells is dictated by a master transcription factor, RORγt. However, the epigenetic mechanism that controls TH17 cell differentiation remains poorly understood. Here we show that the Swi/Snf complex is required for TH17-mediated cytokine production both in vitro and in vivo. We demonstrate that RORγt recruits and forms a complex with the Swi/Snf complex to cooperate for the RORγt-mediated epigenetic modifications of target genes, including both permissive and repressive ones for TH17 cell differentiation. Our findings thus highlight the Swi/Snf complex as an essential epigenetic regulator of TH17 cell differentiation and provide a basis for the understanding of how a master transcription factor RORγt collaborates with the Swi/Snf complex to govern epigenetic regulation.

Subject Areas: Molecular Mechanism of Gene Regulation, Immunity, Transcriptomics

Graphical Abstract

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Highlights

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    The Swi/Snf complex plays essential roles for TH17 differentiation

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    SRG3/mBAF155 deficiency abrogates the expression of major target genes of RORγt

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    RORγt-dependent TH17 transcriptional program requires the Swi/Snf complex

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    The Swi/Snf complex is required for RORγt-driven histone modifications

Molecular Mechanism of Gene Regulation; Immunity; Transcriptomics

Introduction

The differentiation of naive CD4+ T cells into functionally distinct effector TH cell subsets, including TH1, TH2, TH9, and TH17, is an essential process for adaptive immunity (Zhu et al., 2010). The functional specialization is directed by induction of distinct master transcription factors, depending on the external cues from a diverse array of pathogenic agents. In addition, dynamic changes in chromatin structure and histone modifications are also considered a key underlying mechanism that directs lineage-specific gene expression (Lim et al., 2013). Gaining insight into the mechanisms by which master transcription factors and epigenetic changes establish and maintain lineage-specific programs of gene expression is an area of intense interest.

Interleukin-17 (IL-17)-producing TH cells (TH17 cells) play important roles in protective mucosal immunity against extracellular pathogens and also promote autoimmune and chronic inflammation (Korn et al., 2009, Muranski and Restifo, 2013, Stockinger and Omenetti, 2017, Weaver et al., 2013). TH17 cell can be differentiated in vitro either by transforming growth factor β (TGF-β) and IL-6 (Bettelli et al., 2006, Mangan et al., 2006, Veldhoen et al., 2006) (hereafter referred to as TH17(β)) or by IL-6, IL-1β, and IL-23 (Ghoreschi et al., 2010) (hereafter referred to as TH17(23)) to mimic distinct functional subsets. Identification of key transcription factors and a coherent regulatory network has contributed to the understanding of TH17 cell differentiation (Ciofani et al., 2012). Although additional transcription factors are required to promote full TH17 differentiation program (Ciofani et al., 2012, Oestreich and Weinmann, 2012), retinoic acid-related orphan receptor γt (RORγt) is considered as the master transcription factor for TH17 cell differentiation that is necessary and sufficient to induce IL-17A expression (Ivanov et al., 2006). Moreover, RORγt-driven TH17 transcriptional program is essential for the expression of a core subset of TH17 signature genes, including IL-17F and IL-23R as well as IL-17A (Ciofani et al., 2012, Wang et al., 2015).

Numerous studies have reported that epigenetic programming commanded by master transcription factors is key to cellular differentiation (Boller et al., 2016, Ghisletti et al., 2010, Heinz et al., 2010, Johnson et al., 2018, Natoli, 2010, Sartorelli and Puri, 2018). It is not clear yet whether the mechanism by which RORγt controls target genes is a simple transcriptional regulation or whether RORγt plays a more fundamental role for establishing permissive chromatin environments by actively remodeling chromatin structure. If the latter is the case, it also remains to be determined which enzymes capable of altering chromatin structure are required to mediate RORγt-driven epigenetic regulation.

The Swi/Snf chromatin remodeling complex is a group of epigenetic regulators that physically remodel DNA-nucleosomal architecture to regulate gene expression with the energy derived from ATP hydrolysis (de la Serna et al., 2006, Mathur and Roberts, 2018). The Swi/Snf complex is composed of multiple subunits including Brg1 with ATPase activity, Srg3/mBaf155, Baf170, and Baf47/Snf5 as the core subunits of the complex. SRG3, a murine homolog of human BAF155, serves as a scaffold protein that controls the stability of the Swi/Snf complex through direct interaction with the other subunits of the complex (Panamarova et al., 2016, Sohn et al., 2007). As no components of the Swi/Snf complex have intrinsic DNA sequence specificity, the Swi/Snf complex is recruited to its genomic targets by sequence-specific transcription factors and serves as a coactivator for transcriptional activation.

In this study, we uncover the Swi/Snf complex as a critical epigenetic regulator in TH17 cell differentiation. Specifically, unbiased transcriptomic analyses comparing wild-type (WT) and SRG3-deficient TH17-polarized cells reveal that loss of SRG3 expression results in the specific downregulation of RORγt target genes such as IL-17A, IL-17F, and IL-23R. We also reveal that RORγt augments the accumulation of the Swi/Snf complex in IL17a-IL17f and IL-23R gene loci and functions as a key epigenetic regulator of those TH17 signature genes. Indeed, the Swi/Snf complex serves an indispensable role for TH17 cell differentiation by coordinating multiple layers of RORγt-mediated epigenetic program to govern histone modifications.

Results

The Swi/Snf Complex Is Essential for In Vitro TH17 Differentiation

To investigate the role of the Swi/Snf complex in TH17 differentiation, we generated mice with conditional deficiency of SRG3 in CD4+ T cells (CD4CreSRG3fl/fl or SRG3 cKO) by crossing SRGfl/fl mice (Choi et al., 2012) to mice expressing Cre recombinase from the CD4 promoter (CD4Cre mice). As reported in our previous study (Choi et al., 2015), in comparison with their WT littermates (CD4CreSRG3wt/wt), SRG3-deficient mice exhibited comparable characteristics in terms of the frequencies and numbers of peripheral CD4/CD8 T cells, the ratio of naive to memory CD4+ T cells, and their rate of proliferation. These allowed for further analysis of effector differentiation. Naive CD4+ T cells were isolated from SRG3 cKO mice and their WT littermates and differentiated under TH17(β) conditions. We found that SRG3-deficient CD4+ T cells showed a marked reduction in IL-17A and IL-17F production compared with WT CD4+ T cells (Figures 1A and 1B). In addition, knockdown of BRG1 by retroviral transduction greatly reduced IL-17A production, indicating that BRG1 is also required for TH17 differentiation (Figure S1). Contrary to the remarkable decrease in the expression of IL-17A and IL-17F, SRG3-deficient CD4+ T cells displayed a normal level of Foxp3 expression both in iTreg and TH17(β) conditions (Figures 1A and 1C). These results, given the possible reciprocal regulation of differentiation between iTreg and TH17 cells (Bettelli et al., 2006, Zhou et al., 2008), indicate that the defect of TH17 cell differentiation in SRG3-deficient CD4+ T cells was not attributed to dysregulation of iTreg cell differentiation. In addition, we found that SRG3-deficient CD4+ T cells were indistinguishable from WT CD4+ T cells in proliferative response, as monitored by CSFE staining, which indicates that the impaired production of IL-17A in SRG3-deficient CD4+ T cells was not due to a defect in culture proliferation (Figure 1D). To assess whether the Swi/Snf complex plays a cell-intrinsic role in TH17 differentiation, we mixed WT or SRG3-deficient CD4+ T cells (CD45.1-) with congenic naive CD4+ T cells (CD45.1+) and cultured them in TH17(β) conditions (Figure 1E). We found a cell-intrinsic defect in the production of IL-17A in SRG3-deficient CD4+ T cells, indicative again of the Swi/Snf complex as an essential cell-intrinsic factor for TH17 differentiation. To further confirm the importance of the Swi/Snf complex in TH17 cell differentiation, we cultured WT and SRG3-deficient CD4+ T cells under pathogenic TH17(23) conditions with individual cytokines or several cytokine combinations (Figure 1F). Robust IL-17A and IL-17F production were detected in WT CD4+ T cells under three combinations of cytokines (IL-6/IL-1β, IL-6/IL-23, and IL-6/IL-1β/IL-23), whereas SRG3 deficiency severely diminished IL-17A and IL-17F production in all conditions. These results indicate that the Swi/Snf complex is essential for TH17 cell differentiation in vitro.

Figure 1.

Figure 1

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The Swi/Snf Complex Is Essential for the In Vitro TH17 Differentiation

(A–C and F) Flow cytometry of intracellular staining for IL-17A/Foxp3 (A and C) or IL-17A/IL-17F (B and F) in WT or SRG3 cKO naive CD4+ T cells cultured under TH17(β) conditions (A and B), iTreg conditions (C), or in the presence of cytokines as indicated (F) for 60 h and restimulated with PMA/ionomycin.

(D and E) (D) Flow cytometry of intracellular staining for IL-17A in CFSE-labeled WT or SRG3 cKO naive CD4+ T cells cultured under TH17(β) conditions for 60 h followed by restimulation. (E) Flow cytometry of intracellular staining for IL-17A in CD45.1+ WT naive CD4+ T cells plus CD45.1- WT or SRG3 cKO naive CD4+ T cells cultured under TH17(β) conditions for 60 h followed by restimulation. Data are representative of four (A and C), six (B), or two (D–F) independent experiments with consistent results. (B, C, and F) Representative FACS data are shown in the left or top panel, and pooled data with mean ± SD from six (B), three (C), or four (F) independent experiments are shown on the right or bottom. Statistical analysis was performed using unpaired two-tailed t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

The Swi/Snf Complex Is Required for the In Vivo Generation of TH17 Cells

We next examined gut-resident homeostatic TH17 cells in WT and SRG3-deficient mice at steady state (Figure 2A). A substantial proportion of WT CD4+ T cells in the small intestinal lamina propria (SI LP) expressed IL-17A alone or both IL-17A and IL-17F. However, SRG3-deficient SI LP CD4+ T cells showed a marked reduction of IL-17A and IL-17F production. Thus, the Swi/Snf complex is indispensable for the generation of a subset of TH17 cells in SI LP.

Figure 2.

Figure 2

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The Swi/Snf Complex Is Required for the In Vivo Generation of TH17 Cells

(A) Flow cytometry of intracellular staining for IL-17A/IL-17F in CD4+ T cells isolated from lamina propria in WT or SRG3 cKO mice and stimulated with PMA/ionomycin for 4 h. (B) EAE disease scores in WT and SRG3 cKO mice. (C and D) Flow cytometry of intracellular staining for IL-17A/IFN-γ or the absolute number of IL-17A+ or IFN-γ cells in WT or SRG3 cKO CD4+ T cells isolated from spinal cords 21 days after EAE induction and stimulated with PMA/ionomycin for 4 h. (A and C) Representative FACS data are shown in the left panel and pooled data with mean ± SD from four (A) or three (C and D) independent experiments are shown on the right. Statistical analysis was performed using unpaired two-tailed t test (∗p < 0.05, ∗∗p < 0.01).

Next, we immunized SRG3-deficient and WT littermate control mice with myelin oligodendrocyte glycoprotein (MOG) peptide to induce experimental autoimmune encephalomyelitis (EAE) in which TH17 cells are the major pathogenic population. SRG3-deficient mice exhibited remarkably reduced EAE incidence and severity compared with WT mice (Figure 2B). Analysis of spinal cord infiltrates revealed that IL-17A production was also greatly reduced in immunized SRG3-deficient mice, whereas IFN-γ levels were largely unaltered (Figures 2C and 2D). Given that commensal microbiota has been implicated in the generation of IL-17-producing T cells in the intestine and spinal cord (Lee et al., 2011), we cannot rule out that altered microbiota may contribute to an unfavorable environment for IL-17 production in SRG3-deficient mice. However, these data collectively show a central role for the Swi/Snf complex in both in vivo and in vitro differentiation of TH17 cells.

The Loss of SRG3 Impairs the Expression of RORγt Target Genes

To identify the basis for the defects of TH17 differentiation in SRG3-deficient CD4+ T cells, we performed three biological replicate microarray analysis comparing WT and SRG3-deficient CD4+ T cells cultured under TH17(β) conditions (Figure 3A). We identified a total of only 39 genes with more than 1.5-fold differential expression in SRG3-deficient TH17(β)-polarized cells compared with WT TH17(β)-polarized cells. Of note, IL17a was topping the list of downregulated genes in SRG3-deficient TH17(β)-polarized cells. Moreover, other TH17 signature genes, IL17f and IL23r, were prominently featured in the downregulated gene category. These results also indicate that the failure to produce IL-17A and IL-17F in SRG3-deficient TH17-polarized cells lies at the level of cellular transcription.

Figure 3.

Figure 3

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The Loss of SRG3 Impairs the Expression of RORγt Target Genes

(A) Heatmap of relative expression (Z score) (left) and gene expression fold change (right) displaying microarray analysis of WT and SRG3 cKO naive CD4+ T cells cultured under TH17(β) conditions for 60 h. Genes with at least 1.5-fold change are shown.

(B and C) (B) qRT-PCR analysis of WT or SRG3 cKO naive CD4+ T cells cultured under THN or TH17(β) conditions. Error bars, SD. Statistical analysis was performed using one-way ANOVA. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant) (C) Heatmap displaying microarray data, log2(fold change) of TH17-related gene subsets in the two experimental settings: RORγt−/− versus WT and SRG3 cKO versus WT TH17(β)-polarized cells. (A and C) Data represent the combined analysis of three biologically independent samples.

Verification of the expression of a subset of TH17 signature genes by quantitative PCR with reverse transcription (qRT-PCR) confirmed that mRNA expression of IL17a, IL17f, and IL23r is consistently much lower in SRG3-deficient TH17(β)-polarized cells than in WT counterparts (Figure 3B). However, the expression of RORγt mRNA was comparable between WT and SRG3-deficient cells. In addition, the mRNA expression of other transcription factors that facilitate TH17 differentiation, including RORα, Nfkbiz, Batf, and Ahr, was also largely unchanged by the loss of SRG3. We also validated that RORγt protein expression and nuclear localization are similar between WT and SRG3-deficient TH17-polarized cells (Figure S2).

Considering reduced expression of RORγt target genes in SRG3-deficient TH17-polarized cells despite the normal induction of RORγt expression, we then decided to further clarify the relationship between RORγt and the Swi/Snf complex by performing the comparative analysis of the transcription profiles on a restricted panel of genes involved in the TH17 cell biology (Hasan et al., 2017, Wu et al., 2013) from WT, SRG3-deficient, and RORγt-deficient TH17(β)-polarized cells. Surprisingly, microarray analysis of SRG3-deficient versus WT TH17(β)-polarized cells exhibited a marked overlap with RORγt-deficient versus WT TH17(β)-polarized cells in differentially regulated genes (Figure 3C), implying the possibility that the activation of RORγt-dependent core TH17 transcriptional program might rely on the Swi/Snf complex.

The Activation of RORγt-Dependent TH17 Transcriptional Program Requires the Swi/Snf Complex

To address this issue, we directly investigated whether SRG3 deficiency compromises the functional activity of RORγt in TH17 differentiation. We first employed retroviral transduction to deliver RORγt to naive CD4+ T cells to compare the ability of RORγt to induce IL-17A expression in WT and SRG3-deficient CD4+ T cells. In the absence of exogenous TH17-polarizing cytokines (THN conditions), ectopic expression of RORγt induced 39.5% IL-17A production, compared with only 0.12% IL-17A production by a control retrovirus (Figure 4A). Strikingly, SRG3-deficient CD4+ T cells showed a marked reduction in IL-17A production (6.05%) upon ectopic expression of RORγt. To further validate impaired RORγt-directed IL-17 production in SRG3-deficient CD4+ T cells in the presence of TH17-polarizing cytokines, we used RORγ-deficient or RORγ/SRG3 double-deficient (DKO) CD4+ T cells. Again, re-expression of RORγt rescued IL-17A/IL-17F production in RORγt-deficient cultures, whereas it failed to potentiate IL-17A and IL-17F production in DKO cultures under both TH17(β) and TH17(23) conditions (Figure 4B). Thus, the Swi/Snf complex is required for RORγt to activate IL17a and IL17f gene expression.

Figure 4.

Figure 4

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The Activation of RORγt-dependent TH17 Transcriptional Program Requires the Swi/Snf Complex

(A) Flow cytometry of intracellular staining for IL-17A in WT or SRG3 cKO naive CD4+ T cells transduced with control retrovirus expressing GFP only (GFP) or retrovirus encoding GFP and RORγt (GFP-RORγt) and cultured under THN conditions followed by restimulation. (B) Flow cytometry of intracellular staining for IL-17A/IL-17F in RORγt−/− or RORγt−/− SRG3 cKO (DKO) naive CD4+ T cells transduced with control retrovirus (GFP) or retrovirus encoding GFP and RORγt (GFP-RORγt) and cultured under TH17(β) (left) or TH17(23) (right) conditions followed by restimulation. (A and B) Data are representative of three independent experiments with consistent results. Dot plots are gated on CD4+GFP+. (C) Proximity ligation assay (PLA) in WT or SRG3 cKO naive CD4+ T cells cultured under TH17(β) conditions using anti-RORγt, anti-SRG3, or anti-BRG1 antibodies (blue, DAPI; red, PLA signal), scale bar (20 μm). (D) ChIP-qPCR analysis of WT naive and WT, SRG3 cKO or RORγt−/− TH17(β)-polarized CD4+ T cells (upper) or TH17(23)-polarized CD4+ T cells (bottom) using anti-SRG3 antibody. (C and D) Data are representative of at least two independent experiments. Error bars, SD. Statistical analysis was performed using one-way ANOVA. (∗∗p < 0.01, ∗∗∗p < 0.001).

The Swi/Snf Complex Physically Interacts with RORγt

We next examined the possibility that the Swi/Snf complex might be in close proximity to RORγt and form a functional complex during TH17 differentiation. To validate this, we performed the proximity ligation assay (PLA), a sensitive technique to visualize protein-protein interactions via a fluorescent signal when proteins neighbor each other. We found that SRG3 and BRG1 are in close proximity with RORγt in situ in WT TH17(β)-polarized cells, indicating that the Swi/Snf complex interacts with RORγt (Figure 4C). We detected no PLA signal between RORγt and SRG3 in SRG3-deficient TH17(β)-polarized cells as a negative control. In addition, the PLA signals between BRG1 and RORγt were substantially reduced in SRG3-deficient TH17(β)-polarized cells compared with WT TH17(β)-polarized cells, which could result from the decreased expression of BRG1 in the absence of SRG3 (Figure S2A). In addition, we confirmed the interaction between RORγt and the Swi/Snf complex through conventional immunoprecipitation assay followed by immunoblot analysis (Figure S3). Taken together, our results demonstrate the physical association of the Swi/Snf complex and RORγt in TH17 cells.

The Swi/Snf Complex Directly Targets IL17a-IL17f and IL-23r Loci in an RORγt-Dependent Manner

To investigate whether the Swi/Snf complex directly targets IL17a-IL17f and IL-23r loci, we performed the chromatin immunoprecipitation assay followed by quantitative PCR (ChIP-qPCR). These loci were chosen because they were among the most differentially expressed TH17-specific genes in SRG3-deficient TH17-polarized cells compared with WT TH17-polarized cells. We observed that SRG3 bound to IL17 conserved noncoding region 2 (IL17 CNS2, essential for IL17a-IL17f transcription [Wang et al., 2012]), the IL17a promoter (IL-17AP), IL17f promoter (IL-17FP), and IL23r promoter (IL-23RP) in both WT TH17(β)-polarized and WT TH17(23)-polarized cells (Figure 4D). Of note, we observed a substantial reduction of the occupancy of SRG3 in the absence of RORγt, indicating that RORγt increases the recruitment of the Swi/Snf complex to these sites.

The Pattern of Alterations of Histone Modifications by the Loss of SRG3 Closely Parallels that by the Loss of RORγt

If RORγt and the Swi/Snf complex act through collaborative interactions, we would expect that epigenetic regulations mediated by either of them would be similar. Based on this hypothesis, we first examined the role of the Swi/Snf complex and RORγt in modulating chromatin accessibility. We used restriction enzyme accessibility assay coupled with ligation-mediated quantitative PCR (LM-qPCR) to assess the accessible regions in IL-17AP. The restriction enzymes XmnI, PstI, PvuII, and HaeIII were used to measure the accessibility (Figure S4). WT TH17(β)-polarized cells showed a great increase in chromatin accessibility in PstI-282 and PstI+758 sites compared with naive CD4+ T cells. However, SRG3-deficient TH17(β)-polarized cells exhibited reduced accessibility compared with WT TH17(β)-polarized cells, indicating that the Swi/Snf complex is required to facilitate the accessible chromatin structure in IL-17AP. Interestingly, the two PstI sites in RORγt-deficient TH17(β)-polarized cells were as less accessible as were those in SRG3-deficient TH17(β)-polarized cells, which reveals the role of RORγt as an epigenetic regulator.

Chromatin accessibility is typically associated with permissive histone modifications. To characterize chromatin features accompanying the loss of SRG3 or RORγt, we focused on the deposition of six marks and factors: H3K4me1, H3K4me2, H3K4me3, H3K27ac, H3K27me3, and RNA polymerase II. H3K4me3 is a key feature of active promoters, whereas H3K4me1, H3K4me2, and H3K27ac are present in both promoters and enhancers (Calo and Wysocka, 2013, Heintzman et al., 2009). H3K27me3 is associated with gene repression (Cao et al., 2002). Naive CD4+ T cells exhibited little constitutive enrichment for all the histone modifications tested, with the exception of considerable amounts of H3K4me1 (Figures 5A and 5B). Remarkably, WT TH17-polarized cells showed high levels of H3K4me2, H3K4me3, H3K27ac, and RNAPII at IL-17 CNS2, IL-17AP, IL-17FP, and IL-23RP in both TH17(β) and TH17(23) conditions, reflective of robust expression of those genes. Interestingly, the levels of these enrichments were severely diminished in RORγt-deficient TH17-polarized cells with the only exception of H3K4me2 at IL-17FP in TH17(β) conditions and IL-23RP in TH17(23) conditions. Furthermore, as was the case in the absence of RORγt, SRG3-deficient TH17-polarized cells exhibited similar, albeit to a lesser extent, reduction of enrichment. On the other hand, the absence of SRG3 or RORγt significantly increased the deposition of repressive H3K27me3 compared with WT TH17-polarized cells. The levels of H3K4me1 were largely independent of SRG3 or RORγt. Taken together, SRG3 and RORγt seem to make a substantial contribution to shaping the active epigenetic landscape. In addition, these data, along with results of restriction enzyme accessibility assay, reveal that the SRG3-deficient and RORγt-deficient TH17-polarized cells closely resemble each other in the altered patterns of epigenetic structure and modifications, strongly suggesting a high degree of correlation between RORγt and the Swi/Snf complex for epigenetic regulation.

Figure 5.

Figure 5

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The Pattern of Alterations of Histone Modifications by the Loss of SRG3 Closely Parallels That by the Loss of RORγt

(A and B) ChIP-qPCR analysis of WT naive and WT, SRG3 cKO or RORγt−/− TH17(β)-polarized CD4+ T cells or TH17(23)-polarized CD4+ T cells using anti-H3K4me1, anti-H3K4me2, anti-H3K4me3, anti-H3K27ac, anti-H3K27me3, and anti-RNA PolII-S2P antibodies. Data are representative of at least two independent experiments. Error bars, SD. Statistical analysis was performed using one-way ANOVA. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant).

The Swi/Snf Complex Is Required for RORγt-Driven Histone Modifications

To directly investigate the ability of RORγt and a functional link between RORγt and the Swi/Snf complex in the histone modifications, we re-expressed RORγt in RORγt-deficient and RORγt/SRG3 double-deficient (DKO) CD4+ T cells and cultured them under TH17(β) conditions followed by ChIP-qPCR (Figure 6). Although RORγt re-expression had little if any effect on the levels of H3K4me1, it led to a dramatic increase in the accumulation of H3K4me3 and H3K27ac and a significant decrease in the deposition of H3K27me3, which further confirm the active role of RORγt for establishing epigenetic landscape. However, in the absence of SRG3, RORγt re-expression was not able to rescue the induction of H3K4me3 and H3K27ac and the reduction of H3K27me3 in DKO TH17(β)-polarized cells, supporting a model wherein the Swi/Snf complex is required for RORγt-mediated histone modifications and complete activation of TH17 signature genes.

Figure 6.

Figure 6

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The Swi/Snf Complex Is Required for RORγt-Driven Histone Modifications

ChIP-qPCR analysis of WT naive or, RORγt−/− or RORγt−/− SRG3 cKO (DKO) naive CD4+ T cells transduced with control retrovirus (GFP) or retrovirus encoding GFP and RORγt (GFP-RORγt) and cultured under TH17(β) conditions using anti-H3K4me1, anti-H3K4me3, anti-H3K27ac, and anti-H3K27me3 antibodies. Data are representative of at least two independent experiments. Error bars, SD. Statistical analysis was performed using one-way ANOVA. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant).

Discussion

This study reveals a critical requirement for the Swi/Snf complex in TH17 cell differentiation. Although the Swi/Snf complex does not regulate the expression of TH17-related transcription factors including RORγt, a master transcription factor of TH17 cells, we showed that defective TH17 differentiation of SRG3-deficient CD4+ T cells is primarily due to impaired RORγt activity to drive TH17 differentiation. Mechanistically, SRG3 loss results in the impaired expression of RORγt target genes by preventing RORγt-mediated histone modifications, which is a possible mechanism by which RORγt contributes to transcriptional control of a core subset of TH17 signature genes such as IL-17A, IL-17F, and IL-23R. Our results thus uncover that the Swi/Snf complex partners with RORγt and coordinates the epigenetic regulation of target genes of RORγt and ultimately TH17 cell differentiation.

It has been recognized that RORγt is a master transcription factor in TH17 cell differentiation (Ivanov et al., 2006). However, less is appreciated about how RORγt integrates with and regulates epigenetic framework to control lineage-specific gene expression in TH17 cells in spite of its pivotal role in defining the TH17 transcriptional program. In general, a set of pioneer factors are needed to actively open the otherwise inherent repressive chromatin architecture and shape the epigenetic landscape during CD4+ T cell differentiation, in turn allowing the binding of other transcriptional regulators in differentiating T cells. In TH17 cells, BATF and IRF4 were recently shown to cooperatively serve as pioneer factors to regulate initial chromatin accessibility without help from the specialized chromatin remodeling enzymes, thereby setting up the genomic landscape for TH17 cell differentiation (Ciofani et al., 2012). Although chromatin remodeling appears to be initiated by the cooperation between BATF and IRF4, our present study reveals a number of intriguing aspects regarding the epigenetic role of RORγt in the regulation of IL-17 gene expression. First, we found unexpected critical contributions of RORγt to not only establishing the permissive epigenetic landscape but also preventing the repressive one, as evidenced by comparing WT and RORγt-deficient TH17-polarized cells, indicating that RORγt has key roles for setting up the epigenetic landscape to govern a diverse array of histone modifications. Second, RORγt is involved in the regulation of chromatin accessibility to a comparable degree as SRG3, as shown by restriction enzyme accessibility assay. Of particular note, the epigenetic alterations such as chromatin accessibility and histone modifications in the absence of RORγt are dramatically paralleled by those in the absence of SRG3, which calls attention to the notion that the ability of transcription factors to remodel chromatin is dependent on the recruitment of dedicated chromatin remodeling complex. Re-expression of RORγt in RORγt-deficient and DKO cells led us to find that the Swi/Snf complex orchestrates the RORγt-mediated epigenetic regulations, including both histone methylation and acetylation. Thus, our results identify the Swi/Snf complex as a central link between the master transcription factor and its epigenetic control of target regions. Third, H3K4me1 deposition is largely unaffected by loss of SRG3 or RORγt, which indicates that H3K4me1 deposition is not regulated by SRG3 and RORγt. In addition, it also implies that the levels of H3K4me1 do not reflect reduced gene expression in the absence of SRG3 or RORγt. Given that H3K4me1 marks genomic regulatory elements irrespective of their activation status (Calo and Wysocka, 2013), it is likely that other transcription factors including pioneer factors are responsible for the deposition of H3K4me1 prior to binding or even expression of RORγt to mark specific genomic regulatory sites to recruit additional transcriptional activators. In this context, it is reasonable to assume that pre-conditioning of genomic target sites of RORγt accompanied by H3K4me1 deposition is a prerequisite for RORγt binding and that their activation states are subsequently potentiated by RORγt in conjunction with the Swi/Snf complex to culminate in robust gene expression.

Obviously, RORγt increased the accumulation of the Swi/Snf complex at IL-17 CNS2, IL-17AP, IL-17FP, and IL-23RP. However, it is worth noting that substantial levels of residual SRG3 and BRG1 were observed in the absence of RORγt. Thus, we cannot rule out the possibility that a significant amount of the Swi/Snf complex is recruited in an RORγt-independent manner and plays RORγt-independent roles for TH17 differentiation. However, the environmental cues or intracellular transcriptional factors to prompt the targeting of the Swi/Snf complex to IL17 locus in the absence of RORγt remain to be determined. Furthermore, it is also unclear whether the residual Swi/Snf complex is involved in subsequent RORγt binding and transcriptional activity. The answers to these questions are required to better understand the role of the Swi/Snf complex for TH17 differentiation.

Our data also suggest a potential role for chromatin remodeling by the Swi/Snf complex in the histone modifications. In line with this, recent studies have revealed the link between the Swi/Snf complex and a number of histone modifications (Alver et al., 2017, Hodges et al., 2018, Kia et al., 2008, Stanton et al., 2017, Wang et al., 2017, Wilson et al., 2010). Although histone modifying enzymes and ATP-dependent chromatin remodeling complex serve totally distinct biochemical functions, they act in a cooperative and closely integrated manner, thereby forming a reciprocal regulatory network to determine the chromatin states and the levels of gene expression. It is likely that most of these chromatin regulators exert their function as a large multiprotein complex that contains both histone modifying and nucleosome remodeling activities to regulate gene expression in a cooperative manner. We speculate that the Swi/Snf complex may regulate histone modifications by recruitment or expulsion of relevant enzymes in the target regions.

In this study, we demonstrate that RORγt requires the Swi/Snf complex to establish chromatin landscape to fully activate TH17 cell phenotype. However, in addition to its role in TH17 cell differentiation, RORγt plays essential roles in the development of thymocytes, lymphoid tissue inducer (LTi) cells, type 3 innate lymphoid cells, and IL-17 production in Tc17, TCRγδ, and natural killer T cells. Therefore, it will be interesting to examine the correlation between RORγt and the Swi/Snf complex in other cell types to determine whether it is a cell-type-specific mechanism.

Delineating the precise molecular mechanisms that dictate specification to the TH17 cell lineage is critical to the development of potential therapeutic applications that target TH17 cells. Collectively, our data may provide a new perspective on the epigenetic approach for therapeutic intervention in TH17-related diseases.

Limitations of the Study

The aim of this study is to investigate the role of the Swi/Snf complex in the TH17 generation. Even though we performed extensive in vitro molecular analyses to determine the mechanism by which the Swi/Snf complex regulates RORγt-mediated transcriptional program, further work is needed to validate the RORγt-independent role of the Swi/Snf complex during TH17 differentiation. Also, detailed study is necessary to reveal which histone-modifying enzymes require and cooperate with the Swi/Snf complex to understand their comprehensive epigenetic networks for the regulation of TH17 differentiation.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rho. H. Seong (rhseong@snu.ac.kr).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

GEO, GSE129132 (Microarray analysis).

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (NRF- 2016R1A2B3013865) and by Korea Mouse Phenotyping Project (NRF-2014M3A9D5A01073789) of the Ministry of Science, ICT and Future Planning through the National Research Foundations.

Author Contributions

S.L. performed most of the experiments with help from J.K. and H.M. J.K. performed restriction enzyme accessibility experiments. S.L. and R.H.S. designed the research, analyzed results, and wrote the manuscript.

Declaration of Interests

The authors declare no competing interests.

Published: May 22, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101106.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S4, and Table S1
mmc1.pdf (700.4KB, pdf)

References

  1. Alver B.H., Kim K.H., Lu P., Wang X., Manchester H.E., Wang W., Haswell J.R., Park P.J., Roberts C.W. The SWI/SNF chromatin remodelling complex is required for maintenance of lineage specific enhancers. Nat. Commun. 2017;8:14648. doi: 10.1038/ncomms14648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bettelli E., Carrier Y., Gao W., Korn T., Strom T.B., Oukka M., Weiner H.L., Kuchroo V.K. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. [DOI] [PubMed] [Google Scholar]
  3. Boller S., Ramamoorthy S., Akbas D., Nechanitzky R., Burger L., Murr R., Schubeler D., Grosschedl R. Pioneering activity of the C-terminal domain of EBF1 shapes the chromatin landscape for B cell programming. Immunity. 2016;44:527–541. doi: 10.1016/j.immuni.2016.02.021. [DOI] [PubMed] [Google Scholar]
  4. Calo E., Wysocka J. Modification of enhancer chromatin: what, how, and why? Mol. Cell. 2013;49:825–837. doi: 10.1016/j.molcel.2013.01.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cao R., Wang L., Wang H., Xia L., Erdjument-Bromage H., Tempst P., Jones R.S., Zhang Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science. 2002;298:1039–1043. doi: 10.1126/science.1076997. [DOI] [PubMed] [Google Scholar]
  6. Choi J., Jeon S., Choi S., Park K., Seong R.H. The SWI/SNF chromatin remodeling complex regulates germinal center formation by repressing Blimp-1 expression. Proc. Natl. Acad. Sci. U S A. 2015;112:E718–E727. doi: 10.1073/pnas.1418592112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choi J., Ko M., Jeon S., Jeon Y., Park K., Lee C., Lee H., Seong R.H. The SWI/SNF-like BAF complex is essential for early B cell development. J. Immunol. 2012;188:3791–3803. doi: 10.4049/jimmunol.1103390. [DOI] [PubMed] [Google Scholar]
  8. Ciofani M., Madar A., Galan C., Sellars M., Mace K., Pauli F., Agarwal A., Huang W., Parkhurst C.N., Muratet M. A validated regulatory network for Th17 cell specification. Cell. 2012;151:289–303. doi: 10.1016/j.cell.2012.09.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. de la Serna I.L., Ohkawa Y., Imbalzano A.N. Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers. Nat. Rev. Genet. 2006;7:461–473. doi: 10.1038/nrg1882. [DOI] [PubMed] [Google Scholar]
  10. Ghisletti S., Barozzi I., Mietton F., Polletti S., De Santa F., Venturini E., Gregory L., Lonie L., Chew A., Wei C.L. Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages. Immunity. 2010;32:317–328. doi: 10.1016/j.immuni.2010.02.008. [DOI] [PubMed] [Google Scholar]
  11. Ghoreschi K., Laurence A., Yang X.P., Tato C.M., McGeachy M.J., Konkel J.E., Ramos H.L., Wei L., Davidson T.S., Bouladoux N. Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling. Nature. 2010;467:967–971. doi: 10.1038/nature09447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hasan Z., Koizumi S., Sasaki D., Yamada H., Arakaki N., Fujihara Y., Okitsu S., Shirahata H., Ishikawa H. JunB is essential for IL-23-dependent pathogenicity of Th17 cells. Nat. Commun. 2017;8:15628. doi: 10.1038/ncomms15628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heintzman N.D., Hon G.C., Hawkins R.D., Kheradpour P., Stark A., Harp L.F., Ye Z., Lee L.K., Stuart R.K., Ching C.W. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature. 2009;459:108–112. doi: 10.1038/nature07829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heinz S., Benner C., Spann N., Bertolino E., Lin Y.C., Laslo P., Cheng J.X., Murre C., Singh H., Glass C.K. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell. 2010;38:576–589. doi: 10.1016/j.molcel.2010.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hodges H.C., Stanton B.Z., Cermakova K., Chang C.Y., Miller E.L., Kirkland J.G., Ku W.L., Veverka V., Zhao K., Crabtree G.R. Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers. Nat. Struct. Mol. Biol. 2018;25:61–72. doi: 10.1038/s41594-017-0007-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ivanov I.I., McKenzie B.S., Zhou L., Tadokoro C.E., Lepelley A., Lafaille J.J., Cua D.J., Littman D.R. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006;126:1121–1133. doi: 10.1016/j.cell.2006.07.035. [DOI] [PubMed] [Google Scholar]
  17. Johnson J.L., Georgakilas G., Petrovic J., Kurachi M., Cai S., Harly C., Pear W.S., Bhandoola A., Wherry E.J., Vahedi G. Lineage-determining transcription factor TCF-1 initiates the epigenetic identity of T cells. Immunity. 2018;48:243–257.e10. doi: 10.1016/j.immuni.2018.01.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kia S.K., Gorski M.M., Giannakopoulos S., Verrijzer C.P. SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus. Mol. Cell Biol. 2008;28:3457–3464. doi: 10.1128/MCB.02019-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Korn T., Bettelli E., Oukka M., Kuchroo V.K. IL-17 and Th17 cells. Annu. Rev. Immunol. 2009;27:485–517. doi: 10.1146/annurev.immunol.021908.132710. [DOI] [PubMed] [Google Scholar]
  20. Lee Y.K., Menezes J.S., Umesaki Y., Mazmanian S.K. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. U S A. 2011;108:4615–4622. doi: 10.1073/pnas.1000082107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lim P.S., Li J., Holloway A.F., Rao S. Epigenetic regulation of inducible gene expression in the immune system. Immunology. 2013;139:285–293. doi: 10.1111/imm.12100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mangan P.R., Harrington L.E., O'Quinn D.B., Helms W.S., Bullard D.C., Elson C.O., Hatton R.D., Wahl S.M., Schoeb T.R., Weaver C.T. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006;441:231–234. doi: 10.1038/nature04754. [DOI] [PubMed] [Google Scholar]
  23. Mathur R., Roberts C.W.M. SWI/SNF (BAF) complexes: guardians of the epigenome. Annu. Rev. Cancer Biol. 2018;2:413–427. [Google Scholar]
  24. Muranski P., Restifo N.P. Essentials of Th17 cell commitment and plasticity. Blood. 2013;121:2402–2414. doi: 10.1182/blood-2012-09-378653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Natoli G. Maintaining cell identity through global control of genomic organization. Immunity. 2010;33:12–24. doi: 10.1016/j.immuni.2010.07.006. [DOI] [PubMed] [Google Scholar]
  26. Oestreich K.J., Weinmann A.S. Master regulators or lineage-specifying? Changing views on CD4+ T cell transcription factors. Nat. Rev. Immunol. 2012;12:799–804. doi: 10.1038/nri3321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Panamarova M., Cox A., Wicher K.B., Butler R., Bulgakova N., Jeon S., Rosen B., Seong R.H., Skarnes W., Crabtree G. The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo. Development. 2016;143:1271–1283. doi: 10.1242/dev.131961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sartorelli V., Puri P.L. Shaping gene expression by landscaping chromatin architecture: lessons from a master. Mol. Cell. 2018;71:375–388. doi: 10.1016/j.molcel.2018.04.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sohn D.H., Lee K.Y., Lee C., Oh J., Chung H., Jeon S.H., Seong R.H. SRG3 interacts directly with the major components of the SWI/SNF chromatin remodeling complex and protects them from proteasomal degradation. J. Biol. Chem. 2007;282:10614–10624. doi: 10.1074/jbc.M610563200. [DOI] [PubMed] [Google Scholar]
  30. Stanton B.Z., Hodges C., Calarco J.P., Braun S.M., Ku W.L., Kadoch C., Zhao K., Crabtree G.R. Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin. Nat. Genet. 2017;49:282–288. doi: 10.1038/ng.3735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stockinger B., Omenetti S. The dichotomous nature of T helper 17 cells. Nat. Rev. Immunol. 2017;17:535–544. doi: 10.1038/nri.2017.50. [DOI] [PubMed] [Google Scholar]
  32. Veldhoen M., Hocking R.J., Atkins C.J., Locksley R.M., Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24:179–189. doi: 10.1016/j.immuni.2006.01.001. [DOI] [PubMed] [Google Scholar]
  33. Wang C., Collins M., Kuchroo V.K. Effector T cell differentiation: are master regulators of effector T cells still the masters? Curr. Opin. Immunol. 2015;37:6–10. doi: 10.1016/j.coi.2015.08.001. [DOI] [PubMed] [Google Scholar]
  34. Wang X., Lee R.S., Alver B.H., Haswell J.R., Wang S., Mieczkowski J., Drier Y., Gillespie S.M., Archer T.C., Wu J.N. SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation. Nat. Genet. 2017;49:289–295. doi: 10.1038/ng.3746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang X.H., Zhang Y.B., Yang X.X.O., Nurieva R.I., Chang S.H., Ojeda S.S., Kang H.S., Schluns K.S., Gui J.F., Jetten A.M. Transcription of Il17 and Il17f is controlled by conserved noncoding sequence 2. Immunity. 2012;36:23–31. doi: 10.1016/j.immuni.2011.10.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weaver C.T., Elson C.O., Fouser L.A., Kolls J.K. The Th17 pathway and inflammatory diseases of the intestines, lungs, and skin. Annu. Rev. Pathol. 2013;8:477–512. doi: 10.1146/annurev-pathol-011110-130318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wilson B.G., Wang X., Shen X., McKenna E.S., Lemieux M.E., Cho Y.J., Koellhoffer E.C., Pomeroy S.L., Orkin S.H., Roberts C.W. Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell. 2010;18:316–328. doi: 10.1016/j.ccr.2010.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wu C., Yosef N., Thalhamer T., Zhu C., Xiao S., Kishi Y., Regev A., Kuchroo V. Induction of pathogenic Th17 cells by inducible salt sensing kinase SGK1. J. Immunol. 2013;190:513–517. doi: 10.1038/nature11984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhou L., Lopes J.E., Chong M.M., Ivanov I.I., Min R., Victora G.D., Shen Y., Du J., Rubtsov Y.P., Rudensky A.Y. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature. 2008;453:236–240. doi: 10.1038/nature06878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu J., Yamane H., Paul W.E. Differentiation of effector CD4 T cell populations (∗) Annu. Rev. Immunol. 2010;28:445–489. doi: 10.1146/annurev-immunol-030409-101212. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods, Figures S1–S4, and Table S1
mmc1.pdf (700.4KB, pdf)

Data Availability Statement

GEO, GSE129132 (Microarray analysis).

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