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The abundant expression of IFNγ in Th-inducing POK (ThPOK)-deficient CD4+ T cells requires the activation of Eomesodermin (Eomes); however, the underlying mechanism of this phenomenon remains unclear. Here we report that ThPOK binds directly to the promoter region of the Eomes gene to repress its expression in CD4+ T cells. We identified the histone acetyltransferase TIP60 as a co-repressor of ThPOK-target genes, where ectopically expressed TIP60 increased ThPOK protein stability by promoting its acetylation at its Lys360 residue to then augment the transcriptional repression of Eomes. Moreover, knockdown of endogenous TIP60 abolished the stabilization of ThPOK in CD4+ T cells, which led to the transcriptional activation of Eomes and increased production of IFNγ. Our results reveal a novel pathway by which TIP60 and ThPOK synergistically suppresses Eomes function and IFNγ production, which could contribute to the regulation of inflammation.
TIP602 (Tat-interactive protein, 60 kDa) was identified as an HIV-1 TAT-interacting protein that augments TAT-mediated transactivation of the HIV-1 promoter (1). Further studies characterized TIP60 as a histone acetyltransferase that acetylates H2A, H3, and H4, but not H2B of the core histones to regulate gene expression (2). However, TIP60 is not only a chromatin modifier that regulates transcription as it also plays a role in DNA repair, cell apoptosis, oncogenesis, and serum deprivation-induced autophagy through exerting its function as a transcriptional co-regulator or through its enzymatic activity on transcription factors such as FOXP3, STAT3, c-Myc, p53, and protein kinases including ATM and UKL1 (3–9).
TIP60 deficiency in mice is lethal, whereas heterozygous deletion of TIP60 leads to haplo-insufficient tumor suppression (10). However, the role of TIP60 in regulating immune responses remains largely uncharacterized. In CD4+ CD25+ regulatory T (Treg) cells, we have shown that TIP60 binds directly to the proline-rich domain of the forkhead family transcription factor FOXP3, which is essential for FOXP3-mediated repression of IL-2 expression (8). In CD4+ T helper 2 (Th2) and Th17 cells, the proinflammatory cytokine IL-9 activates STAT3 to modulate its downstream gene expression (11) and is regulated by TIP60 through its interaction with both IL9Rα and STAT3 (9, 12).
The TCR signal-induced zinc finger and BR-C, Ttk, and Bab (BTB) domain-containing protein family transcription factor Th-inducing POK (ThPOK) is dominantly expressed in CD4+ T cells (13) and is necessary for T helper cell differentiation (14, 15). ThPOK binds to the silencers within the CD4 and ThPOK gene loci and inhibits silencer activity (13, 16). Moreover, a number of studies have shown how ThPOK regulates CD4 versus CD8 T cell lineage commitment by suppressing classical CD8 lineage genes such as CD8, Perforin, Granzyme B, and RUNX3 (14, 17–19). Others have also shown how the function of Eomesodermin (Eomes), a T-box transcriptional activator of IFN-γ, negatively correlates with ThPOK expression (17, 18, 20).
Here we report a previously uncharacterized mechanism by which the gene transcription of Eomes is directly repressed by ThPOK and how TIP60 is a cofactor for ThPOK-mediated repression of Eomes expression. This pathway in turn mitigates the activation of Eomes target genes such as IFNγ in human CD4+ T cells. As ThPOK contains a proline-rich domain, we hypothesized that TIP60 might also bind to its proline-rich domain to mediate T cell lineage differentiation and function and modulate inflammation through regulating the transcriptional induction of Eomes; however, we found that the C-terminal region of ThPOK interacted with TIP60 and is acetylated at the Lys360 residue. Our results thus reveal a direct molecular link between TIP60 function and the modulation of CD4+ T cell-mediated inflammation through cytokine production.
HEK 293T cells were cultured in DMEM containing 10% FBS and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Jurkat cells were maintained in RPMI 1640 medium containing 10% FBS. Transfection of Jurkat cells with plasmid DNA was performed by electroporation on a Gene Pulser X cell apparatus (Bio-Rad Laboratories). Jurkat cells were activated using soluble antibodies against CD3 (1 μg/ml, Hit3a; Biolegend) and CD28 (2 μg/ml, CD28.2; Biolegend).
Cells were washed with ice-cold PBS and lysed on ice for 30 min in 1× RIPA buffer (50 mm Tris-HCl, pH 7.5, 135 mm NaCl; 1% Nonidet P-40; 0.5% sodium DOC; 1 mm EDTA, 10% glycerol) containing protease inhibitor (1:100, P8340; Sigma-Aldrich), 1 mm NaF, and 1 mm PMSF. Cell lysates were cleared by centrifugation, and supernatants were immunoprecipitated with the appropriate antibodies (Abs) using protein A/G-agarose beads at 4 °C. After washing, 2× sample loading buffer was added to the immunoprecipitates. Samples were then used for immunoblot analysis with the indicated antibodies.
The following antibodies were used for flow cytometry analysis: anti-CD4-FITC (RPA-T4; Biolegend), anti-CD8-APC (RPA-T8; BD Biosciences), anti-TCRαβ-PE (IP26; eBioscience), and anti-IFNγ-APC (4S.B3; eBioscience). Fixable viability dye eFluor 780 was purchased from eBioscience. Anti-HA (F-7), anti-ThPOK (A-4), anti-TIP60 (N-17), and goat IgG (sc-2028) were from Santa Cruz Biotechnology. Anti-FLAG (M2), anti-β-actin, and anti-α-tubulin were from Sigma-Aldrich and Tianjin Sungene Biotech (China), respectively. Mouse IgG was from Millipore. Anti-acetyllysine Ab was obtained from Immunechem Pharmaceuticals (Canada). Protein A/G-agarose beads (A10001) were purchased from Abmart (China). Cycloheximide (C7698-5G) and nicotinamide (72340-100G) were purchased from Sigma-Aldrich. EX-527 (S1541) was purchased from Selleck. Human ThPOK was cloned into the pIP-HA2 vector, and pCMV2-FLAG-TIP60 has been described previously (8). Mutagenesis was carried out according to the manufacturer's instructions using the Toyobo mutagenesis kit. ThPOK was cloned into the FUGW plasmid (kindly provided by Lan Ke, Institut Pasteur of Shanghai, Chinese Academy of Sciences).
The 1000-bp region upstream of the human Eomes transcriptional starting site (NCBI: human, chromosome 3 NC_000003.11; mouse, chromosome 9 NC_000075.6) was cloned into the pGL3-Basic vector to generate the pGL3-Eomes-Luc reporter construct. Jurkat cells were co-transfected with the reporter plasmid and a Renilla luciferase encoding plasmid as a control, and/or FLAG-TIP60 as indicated. 48 h later, cells were lysed, and luciferase assays were performed using the Dual-luciferase reporter kit (Promega).
Primary human CD4+ T cells were stimulated using anti-CD3/CD28 dynal beads (Invitrogen) at a cell to bead ratio of 1:1 for the indicated time periods. Cells were then cross-linked with formaldehyde, and the chromatin was sonicated into ~500-bp fragments. The sheared chromatin was immunoprecipitated with anti-ThPOK antibody, and mouse IgG was used as a negative control. The pulled down DNA fragments were subjected to qPCR analysis. Primers that were used are as follows: Probe N forward, 5′-ccagtctaaggagggtgctg-3′ and reverse, 5′-tgaaatgggctttccttttg-3′; Probe B forward, 5′-agtcacaggcgacttgatcc-3′ and reverse, 5′-agatctttgtccccatccac-3′.
Total RNA was extracted using TRIzol reagent (Invitrogen). cDNA was synthesized using a reverse transcriptase kit (TaKaRa, Japan), followed by qRT-PCR analysis (SYBR Green; TaKaRa). The primers that were used are as follows: CD8 forward, 5′-cctgagcaactccatcatgt-3′ and reverse, 5′-gatatcacaggcgaagtccag-3′; CD4 forward, 5′-gctggaatccaacatcaagg-3′ and reverse, 5′-ctgaaaccggtgaggacact-3′; ThPOK forward, 5′-gtctgccacaagatcatcca-3′ and reverse, 5′-tcgtagctgtgcaggaagc-3′; TIP60 forward, 5′-agatcctgagcgtgaaggac-3′ and reverse, 5′-tctctctggagagccaggac-3′; Eomes forward, 5′-tgcaaaaggcttcagagaca-3′; and reverse, 5′-ctctgttggggtgaaaggag-3′; Tbx21 forward, 5′-ccgtgactgcctaccagaat-3′ and reverse, 5′-atctcccccaaggaattgac-3′; IFNγ forward, 5′-aaacgagatgacttcgaaaagc-3′ and reverse, 5′-atattgcaggcaggacaacc-3′; β-actin forward, 5′-ctcttccagccttccttcct-3′ and reverse, 5′-cagggcagtgatctccttct-3′.
Primary human CD4+ and CD8+ T cells from healthy donors were isolated by FACS on a BD FACS ARIA II sorter (BD Biosciences). Primary T cells were expanded using anti-CD3/CD28 dynal beads (Invitrogen) at a cell to bead ratio of 1:1 in X-VIVO-15 medium (Lonza, Switzerland) supplemented with 10% human AB serum, 1% GlutaMax (GIBCO), 1% sodium pyruvate (GIBCO), and 1% Pen/Strep (GIBCO).
In brief, CD4+ T cells were restimulated with phorbol 12-myristate 13-acetate (50 ng/ml), ionomycin (1 μm), and Golgi Stop for 4 h. At the end of stimulation, cells were stained with fixable viability dye eFluor 780 and anti-CD4-FITC, then washed with PBS. Staining of IFNγ was carried out with the IC Fixation Buffer (eBioscience catalog number 00-8222) and Permeabilization Buffer (10×) (eBioscience catalog number 00-8333) according to the manufacturer's instructions.
The shRNA lentiviral vector pLKO.1 shTIP60, pLKO.1 shThPOK, or pLKO.1 shCK was transfected into HEK 293T cells via calcium phosphate transfection with the lentivirus packing vector Delta 8.9 and VSVG envelope glycoprotein. Viral supernatants were harvested after 48 h. Primary T cells were transduced with virus along with anti-CD3/28 stimuli (1 cell to 1 bead). The following shRNA sequences were used: shCK, 5′-caacaagatgaagagcaccaa-3′; shTIP60, 5′-cctcaatctcatcaactacta-3′; shThPOK, 5′-ccgcctctctctagctcgatt-3′.
CD4+ and CD8+ T cells were isolated from human peripheral blood mononuclear cells (Fig. 1A) and subsequently examined for TIP60 mRNA and protein expression levels alongside the CD4 T cell lineage-specific transcription factor ThPOK as a control. We found that TIP60 mRNA was highly expressed in CD4+ T cells compared with CD8+ T cells (Fig. 1B). Also, protein expression was found to be consistent with the mRNA levels (Fig. 1C). Therefore, both TIP60 and ThPOK are higher expressed in CD4+ T cells.
Previous studies indicated that long term TCR stimulation triggers ThPOK transcription in CD4+CD8lo thymocytes (13). However, it remains unclear how short term TCR stimulation regulates ThPOK expression in CD4 single positive (SP) cells. To reveal the role of TCR signaling, resting CD4 SP T cells were stimulated with soluble antibodies against CD3 and CD28 for a short period (within 8 h) prior to the analysis of ThPOK protein and mRNA levels. We observed an increasing level of ThPOK protein upon TCR stimulation, as well as TIP60 (Fig. 1D). However, the transcription of both genes was slightly repressed (Fig. 1E), which indicated that ThPOK and TIP60 were stabilized posttranslationally. Consistent with the previous findings, we identified that stabilized ThPOK correlates with the repression of Eomes gene transcription, but not T-bet (Fig. 1E).
As ThPOK contains a proline-rich domain that could be potentially recognized by TIP60, we tested whether TIP60 and ThPOK could interact with each other by reciprocal immunoprecipitation. HA-tagged ThPOK (HA-ThPOK) and FLAG-tagged TIP60 (FLAG-TIP60) were ectopically expressed in HEK 293T cells, followed by reciprocal immunoprecipitation. We found a positive interaction between TIP60 and ThPOK (Fig. 2A). We then further showed that endogenous TIP60 could co-immunoprecipitate ThPOK in human primary CD4+ T cells (Fig. 2B).
TIP60 is a lysine acetyltransferase, which acetylates not only histones but also nuclear transcription factors such as c-Myc, p53, and FOXP3. We were interested in investigating whether TIP60 could similarly modify ThPOK by lysine acetylation. We co-transfected the HA-ThPOK-expressing plasmid with increasing amounts of FLAG-TIP60-expressing plasmid into HEK 293T cells; cells were then treated with protein deacetylation inhibitors. We found that the overexpression of TIP60 promoted ThPOK acetylation (Fig. 2C). To elucidate the role of TIP60 in primary T cells, lentivirus-mediated shRNA knockdown of endogenous TIP60 was performed. Loss of TIP60 significantly reduced the acetylation level of ThPOK (Fig. 2D). Previously, we found that short term TCR stimulation could stabilize ThPOK. Others have also shown that the acetylation of ThPOK increases its stability (21); therefore, we decided to check the acetylation level of ThPOK after TCR stimulation. As expected, short term TCR stimulation promoted the interaction between TIP60 and ThPOK, which most likely increases the acetylation of ThPOK (Fig. 2E).
We then further tested the relationship between acetylation and protein stability. We observed that the increase in ThPOK protein level positively correlated with TIP60 level, which indicated that the overexpression of TIP60 facilitated the stabilization of ThPOK (Fig. 3A). To fully confirm this result, we treated the cells with protein synthesis inhibitor cycloheximide and examined ThPOK protein stability. HA-ThPOK was co-transfected with or without FLAG-TIP60, and cells were then treated with cycloheximide for the indicated time periods. Here, we confirmed that ThPOK stability could be positively regulated by TIP60 (Fig. 3B). To further examine TIP60-mediated stabilization of ThPOK under more physiological conditions, knockdown of endogenous TIP60 was performed in primary human CD4+ T cells. The protein level of ThPOK decreased noticeably in TIP60-silenced CD4+ T cells. Meanwhile, CD4+ T cells transduced with shRNA constructs targeting ThPOK were tested as a control (Fig. 3C). We then mapped by co-IP for the regions of ThPOK that associates with TIP60 to the C-terminal domain (residues 310–539), but not the N-terminal subdomain, which contains the proline-rich region (Fig. 3, D and E). These results indicated that the acetylated lysines were likely at the C-terminal region. Furthermore, TIP60 specifically stabilized the C-terminal region (C1) of ThPOK but not the N-terminal region (N1) (Fig. 3F). TIP60 preferentially acetylates the lysine of G(X)GK motif of histones (22). When we screened all of the residues of ThPOK, we identified that lysine 360 (GAGK motif) was contained in the C-terminal region, and thus a potential acetylation site (Fig. 3D). We found that a point mutation of lysine 360 into arginine abrogated TIP60-mediated stabilization, whereas combinatorial mutation of Lys203, Lys206, and Lys212 (lysines located in the proline-rich domain) could not (Fig. 3F). Further experiments showed that the ThPOK-K360R mutant abolished TIP60-mediated acetylation, which indicates that lysine 360 is the target of TIP60 (Fig. 3G).
Because CD4+ T helper cells become lineage-committed upon activation to mediate inflammatory responses, our data suggested that TIP60 could regulate inflammatory cytokine expression through modulating the activity of ThPOK. Previous studies have revealed how Eomes activity is up-regulated in the absence of ThPOK or in the presence of insufficient ThPOK expression (17, 18). Overexpression of ThPOK in CD8+ T cells specifically represses Eomes but not T-bet (20). Thus, we intended to clarify whether ThPOK could bind to the Eomes gene promoter to repress its transcription in CD4+ T cells.
Jurkat and ThPOK-overexpressed Jurkat (ThPOK-Jurkat) cells were stimulated with soluble α-CD3/CD28 antibodies for the indicated time periods (Fig. 4A). We then examined the mRNA levels of Eomes and IFNγ. Both genes were down-regulated in ThPOK-Jurkat cells (Fig. 4A). We aligned the 1-kb region (−1,000 to 0) of the Eomes promoter from mouse and human. The alignment result showed that there was a consensus ThPOK binding sequence CCCTCCC (19, 23, 24), which suggested that Eomes is potentially a direct transcriptional target of ThPOK (Fig. 4B). We next subcloned the 1-kb region of the human Eomes promoter into the pGL3-Basic Luciferase vector and transfected the Eomes luciferase (Eomes-Luc) reporter plasmid into Jurkat or ThPOK-Jurkat cells. We found that the Eomes-Luc reporter activity was significantly repressed in ThPOK-Jurkat cells (Fig. 4C). To further confirm the involvement of ThPOK in Eomes gene transcription we analyzed whether ThPOK could bind to the Eomes promoter in primary CD4+ T cells using a ChIP assay. Human CD4+ T cells were stimulated with anti-CD3/CD28 dynal beads for the indicated periods, and the binding of ThPOK to the promoter (Probe B region with a consensus ThPOK binding site) was notably higher after anti-CD3/CD28 stimulation (Fig. 4D). Furthermore, mutation of the ThPOK binding site could reverse ThPOK-mediated repression of Eomes-Luc reporter activity (Fig. 4E), but not completely, perhaps due to other less consensus binding sites that may be responsible for ThPOK binding and activity at the Eomes promoter.
As we found that TIP60 could stabilize ThPOK in CD4+ T cells we decided to test whether TIP60 could promote ThPOK-mediated repression of Eomes expression. Jurkat and ThPOK-Jurkat cells were transfected with the Eomes luciferase reporter and FLAG-TIP60-encoding plasmids as indicated. We found that TIP60 stabilized ThPOK and increased the repression of the Eomes promoter as indicated by the decrease in luciferase activity (Fig. 4F).
ThPOK was reported to bind to the silencers of the CD4 and ThPOK gene loci to abolish their activity (16). We hypothesized that TIP60 could positively regulate ThPOK-mediated transcriptional regulation of CD4, ThPOK, and Eomes. To test this, primary CD4+ T cells were transduced with shRNA constructs targeting CK (control), TIP60, or ThPOK and then subjected to analysis by qRT-PCR or flow cytometry (Fig. 5A). Total RNA was then extracted, and the mRNA levels of CD8, CD4, ThPOK, TIP60, Eomes, Tbx21, and IFNγ were determined by qRT-PCR. No CD8 transcripts were detected, which indicated that the knockdown of either TIP60 or ThPOK did not induce CD8 expression in CD4+ CD8− T cells (Fig. 5B). Both CD4 and ThPOK were down-regulated upon knockdown of TIP60 or ThPOK, whereas Eomes and IFNγ were both noticeably up-regulated (Fig. 5B). However, the expression of Tbx21, another T-box transcriptional activator of IFN-γ, was not dramatically affected (Fig. 5B). The protein levels of CD4 and IFNγ were further tested by flow cytometry. Knockdown of either TIP60 or ThPOK in CD4+ CD8− T cells slightly decreased CD4 protein level, which was consistent with the previous mRNA expression result (Fig. 5C). Meanwhile, we observed a higher percentage of CD4+ IFNγ+ T cells in cells depleted of TIP60 or ThPOK (Fig. 5D). To test whether the up-regulation of Eomes mRNA in TIP60-depleted cells was ThPOK-dependent, we overexpressed ThPOK in TIP60-depleted cells. The overexpression of ThPOK significantly promoted the repression of Eomes (Fig. 5E), which indicated that the loss of ThPOK activity in TIP60-depleted CD4+ T cells was responsible for the increase in transcription of Eomes. Our data suggest that TIP60 and ThPOK could significantly repress Eomes expression and play a potential role in modulating inflammation (Fig. 6).
Eomes was originally characterized as an inflammation-inducing gene in CD8+ T cells and Natural Killer cells, indispensible for IFNγ production during host defense responses against intracellular bacteria and virus infections (25–28). Recently, more attention has been paid to the function of Eomes in CD4+ T helper cells; Eomes is required for Th1 cell differentiation and is generally repressed in other T helper cell lineages. For example, Eomes directs naïve CD4+ T cell polarization toward the Th1 lineage versus Th17, where TGF-β (required for Th17 skewing) suppresses Eomes expression via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway (29–31). Eomes is also suppressed in effector Th2 cells; GATA3-deficient naïve CD4+ T cells produce a significant amount of IFNγ during Th2 differentiation, which is dependent on Eomes but not T-bet (32). GATA3 was reported to induce ThPOK expression (33) and may suppress Eomes in a ThPOK-dependent manner. Moreover, in memory Th2 cells, Eomes is highly expressed and interacts with GATA3 to prevent its binding to the IL-5 promoter (34). Here, we have identified another novel molecular pathway which could explain the molecular basis by which Eomes is repressed in CD4+ T helper cells in a TIP60- and ThPOK-dependent manner.
As we postulated, TIP60 was found to augment ThPOK-mediated repression of Eomes through stabilizing ThPOK; we speculate that TIP60 may also recruit histone deacetylases to ThPOK transcriptional complexes to epigenetically silence Eomes by the deacetylation of its promoter, such as HDAC7/9 and SIRT1, which have been reported to associate with TIP60 (8, 9, 35). In addition, ThPOK can recruit HDAC4/5 to the CD8 gene loci to impair CD8 transcription (19). However, it needs to be further substantiated as to whether these histone deacetylases could be recruited to the promoter of Eomes in CD4+ T cells.
The incontrollable overproduction of IFNγ by CD4+ T helper cells may coincide with the development of autoimmune disease (36, 37). CD4+ CD25+ CD127low FOXP3+ regulatory T cells are essential for peripheral tolerance and the prevention of autoimmune diseases (38). Strong evidence shows that autoreactive Treg cells can suppress adaptive immune responses and dampen inflammation (39). Therefore, TIP60, through positive regulation of its function, may be a potential anti-inflammation target because it inhibits IFNγ production in CD4+ T helper cells and also strengthens the suppressive function of Treg cells by promoting FOXP3-mediated transcriptional repression as we had previously identified (8). Although TIP60 has been shown to be involved in DNA repair, apoptosis, and Treg function, here we give a new insight into the molecular mechanism by which TIP60 suppresses Eomes and attenuates the production of the inflammatory cytokine IFNγ in CD4+ T cells. Thus, our work may provide a novel strategy for therapeutic studies toward the treatment of Th1-related diseases such as cancer, autoimmunity, and infectious diseases (36, 40).
*This work was supported, in whole or in part, by National Institutes of Health-National Science Foundation of China Collaborative Grant 81161120417. This work was also supported by National Science Foundation of China Grants 30972702, 31170825, 81270083, 31150110337, 31200646, 31200647, and 81271835; Shanghai Rising Star Program 10QA1407900; the Novo Nordisk-Chinese Academy of Sciences Foundation; the Sanofi-Aventis-Shanghai Institutes for Biological Sciences scholarship program, and the Knowledge Innovation Program of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Grants 2010KIP205 and 2012KIP204.
2The abbreviations used are: