Reciprocal regulation of RORγt acetylation and function by p300 and HDAC1

T helper 17 (Th17) cells not only play critical roles in protecting against bacterial and fungal infections but are also involved in the pathogenesis of autoimmune diseases. The retinoic acid-related orphan receptor (RORγt) is a key transcription factor involved in Th17 cell differentiation through direct transcriptional activation of interleukin 17(A) (IL-17). How RORγt itself is regulated remains unclear. Here, we report that p300, which has histone acetyltransferase (HAT) activity, interacts with and acetylates RORγt at its K81 residue. Knockdown of p300 downregulates RORγt protein and RORγt-mediated gene expression in Th17 cells. In addition, p300 can promote RORγt-mediated transcriptional activation. Interestingly, the histone deacetylase (HDAC) HDAC1 can also interact with RORγt and reduce its acetylation level. In summary, our data reveal previously unappreciated posttranslational regulation of RORγt, uncovering the underlying mechanism by which the histone acetyltransferase p300 and the histone deacetylase HDAC1 reciprocally regulate the RORγt-mediated transcriptional activation of IL-17.

T helper 17 (Th17) cells are involved in both innate immunity and adaptive immune responses. These cells not only play critical roles in protecting against bacterial and fungal infections but are also involved in the pathogenesis of autoimmune diseases, including multiple sclerosis, arthritis, Crohn's disease, uveitis and psoriasis 1,2 . Th17 cells, which produce interleukin 17 (A) (IL-17A) and IL-17F, have been described as a separate T helper cell subset distinct from Th1, Th2 and regulatory T (Treg) cells. IL-17A and IL-17F are expressed in activated peripheral blood CD4 + T cells and induce production of proinflammatory cytokines and chemokines, including IL6 and CXCL8 3 . Transforming growth factor-β (TGF-β ), IL-23 and proinflammatory cytokines (e.g., IL-1β and IL-6) are all essential for human Th17 differentiation and the expression of IL-17A, IL-17F, IL-23 receptor (IL-23R) and the retinoic acid-related orphan receptor (RORγ t) 4 . The regulation of these genes is augmented by the induction of IL-21, which acts in an autocrine manner 5 . Th17 differentiation has been shown to require the transcription factors RORγ t and RORα in conjunction with other essential transcription factors such as the signal transducer and activator of transcription 3 (STAT3), the aryl hydrocarbon receptor (Ahr), interferon regulatory factor 4 (IRF4), the Runt-related transcription factor 1 (Runx1), B-cell-activating transcription factor (BATF), Sox5 and c-MAF [6][7][8][9] . In addition, RORγ t-deficient T cells inhibit Th17 cell differentiation, attenuate the expression of IL-17A and IL-17F and resist autoimmune disease. Conversely, overexpression of RORγ t induces IL-17 expression Scientific RepoRts | 5:16355 | DOI: 10.1038/srep16355 and leads to more severe experimental autoimmune encephalomyelitis (EAE) than naturally occurs in wild-type mice 7,10,11 . Together, these studies suggest that RORγ t is a lineage-specifying transcription factor that plays a focal deterministic role in the differentiation of Th17 cells and directs the transcriptional activation of Th17-specific genes, including IL-17A, IL-17F, IL-21 and IL-23R.
Our previous data have shown that the E3 deubiquitinase USP17 positively regulates RORγ t in Th17 cells 12 . A recent study found that CNS2-deficient T cells showed decreased RORγ t-driven IL-17A and IL-17F expression in vitro, and that CNS2-deficient mice were resistant to EAE, which may have been due to the CNS2-mediated recruitment of JmjC domain-containing protein 3 (JMJD3) and the histone acetyltransferase p300 13 . In addition, Fanpan showed that HIF1α and RORγ t induced the transcription of RORγ t target genes and Th17 cell differentiation through the recruitment of p300 to the IL-17A promoter 14 . However, whether p300 plays an important role in regulating RORγ t and the mechanism involved remain unknown. p300 (also known as Ep300 or KAT3B), an adenovirus E1A-associated 300-kDa protein, is a transcriptional cofactor and nuclear phosphoprotein with intrinsic acetyltransferase activity, and it regulates histones to modulate chromatin organization. In addition, p300 can regulate non-histone proteins, including nuclear transcription factors such as p53, NF-κ B and Foxp3 [15][16][17] . Acetylation of these transcription factors can modulate their transcriptional activity by altering their stability, subcellular localization and/or DNA-binding activity 18 .
Histone acetyltransferases and histone deacetylases reciprocally affect the steady-state levels of histone acetylation. Histone acetyltransferases typically act as transcriptional activators, and histone deacetylases, which catalyze the deacetylation of histones, generally regulate chromatin structure, modify histone and non-histone proteins, and suppress gene expression 19,20 . Class I histone deacetylase subfamily members include HDAC1, HDAC2, HDAC3 and HDAC8 21 ; HDAC1, as a transcriptional coactivator, exerts histone deacetylation activity and plays an important role in biological processes such as cell proliferation, differentiation and cell cycle progression 20 . Recently, many studies have associated HDAC activity with diseases such as cancer, pulmonary hypertrophy and cardiac hypertrophy 22,23 .
Here, we report that RORγ t is acetylated in Th17 cells. p300 interacts with, stabilizes and acetylates RORγ t and knockdown of p300 downregulates RORγ t at the protein level and decreases RORγ t-mediated gene expression. p300 also promotes the RORγ t-mediated transcriptional activation of IL-17. Furthermore, HDAC1 interacts with and deacetylates RORγ t, leading to inhibition of RORγ t-mediated IL-17 transcription. Our results reveal a previously unknown mechanism by which p300 and HDAC1 reciprocally regulate the RORγ t-mediated transcriptional activation of IL-17.

Results
RORγt is acetylated in human Th17 cells. RORγ t is a master transcription factor in Th17 cells, and RORγ t expression determines Th17 differentiation. First, we tested whether RORγ t is acetylated in transiently transfected cells. Flag-RORγ t was transfected into HEK293T cells in the presence of HDAC inhibitors, and we observed that RORγ t was acetylated (Fig. 1A). Furthermore, we found that RORγ t was also acetylated in Th17 cells. In addition, RORγ t acetylation was significantly enhanced in the presence of HDAC inhibitors (Fig. 1B). Taken together, these data indicate that RORγ t is acetylated in vivo. p300 interacts with and stabilizes RORγ t. To assess whether certain acetyltransferases can upregulate RORγ t-mediated transcription activation, we cotransfected 5 HATs and RORγ t into HEK293T cells along with the IL-17 promoter to screen for the effects of HATs on RORγ t-mediated transcriptional activation, p300 significantly upregulated RORγ t-mediated transcription activation among the 5 HATs (Supplementary Figure 1). In addition, western blotting analysis comparing human naïve CD4 + T cells and Th17 cells showed that p300 protein level is higher in Th17 cells ( Fig. 2A). Subsequently, to determine whether p300 associates with RORγ t, coimmunoprecipitation was performed. HA-p300 and Flag-RORγ t were transiently transfected into HEK293T cells, and cell lysates were analyzed using an antibody against the HA-tag. Coimmunoprecipitation between p300 and RORγ t demonstrated that p300 interacts with RORγ t (Fig. 2B). In addition, endogenous IP also showed that p300 interacts with RORγ t in human Th17 cells (Fig. 2C). p300 localization was predominantly nuclear, and RORγ t was also observed in the nucleus. These results suggest that p300 colocalizes with RORγ t in HeLa cells, consistent with an interaction between p300 and RORγ t (Supplementary Figure 2).
Previous studies have shown that acetylation can affect protein stabilization 17,28 , thus, we examined whether p300 can stabilize RORγ t. We observed that a dose-dependent increase in the RORγ t protein (E) Flag-tagged RORγ t was cotransfected with His-tagged p300 into HEK293T cells. Cells were treated with CHX for the indicated periods and analyzed with western blotting. (F) Naïve CD4 + T cells were cultured under Th17-polarizing conditions for 7 days. Th17 cells were transduced with a lentivirus containing either shCK or shp300. Cells were treated with puromycin for 3 days and the protein levels were assessed. Each figure is representative of > 3 independent experiments.
Scientific RepoRts | 5:16355 | DOI: 10.1038/srep16355 level positively correlated with the protein level of p300 (Fig. 2D). To confirm this result, we transfected Flag-RORγ t into HEK293T cells either with or without His-p300 and then treated the cells with the protein synthesis inhibitor cycloheximide (CHX) at the indicated time points. Thus, we confirmed that RORγ t stabilization could be positively regulated by p300 (Fig. 2E). To further test the p300-mediated stabilization of RORγ t under more physiological conditions, we generated a shRNA construct targeting p300 in human Th17 cells to reduce the endogenous p300 levels and observed that knockdown of p300 decreased the level of RORγ t protein (Fig. 2F). p300 acetylates RORγt at the K81 residue. p300 is an acetyltransferase with intrinsic acetyltransferase activity. Previous data have shown that p300 can acetylate transcription factors such as Foxp3 and p53 15,29 . To determine whether p300 can acetylate RORγ t, Flag-tagged p300 and Myc-tagged RORγ t plasmids were transfected into HEK293T cells then we treated the cells with HDAC inhibitors. Immunoprecipitation was performed using an anti-Myc antibody, and the results were analyzed by western blotting using the indicated antibodies. Our data indicate that p300 acetylates RORγ t (Fig. 3A) and that it does so in a dose-dependent manner (Fig. 3B). In addition, to determine whether RORγ t acetylation is associated with p300 in human Th17 cells, we used a shRNA construct targeting p300 in human Th17 cells to reduce the endogenous p300 level and observed that knockdown of p300 decreased the RORγ t acetylation level (Fig. 3C).
Full-length RORγ t contains an N-terminal domain, a hinge region domain and a ligand-binding domain. To map the region in RORγ t that is acetylated by p300, either RORγ t truncation mutants or wild-type RORγ t was cotransfected with HA-tagged p300 into HEK293T cells. Immunoprecipitation was performed, and the results indicated that p300 acetylates RORγ t on the N-terminal region (Fig. 3D). Next, we screened the N-terminal region of RORγ t associated with p300 by immunoprecipitation and found that a point mutation at lysine 81 into arginine significantly decreased p300-mediated acetylation (Fig. 3E).

HDAC inhibitors increase RORγt acetylation and RORγt-mediated IL-17 transcription.
To investigate the effects of HDAC inhibitors on RORγ t acetylation and RORγ t-mediated transcription, we transfected Flag-tagged RORγ t and HA-tagged p300 into HEK293T cells in the presence of HDAC inhibitors. We observed that HDAC inhibitors significantly increased RORγ t acetylation compared to untreated control (Fig. 4A). Moreover, an IL-17A luciferase reporter was cotransfected with either HA-p300 or Flag-RORγ t into HEK293T cells in either the presence or the absence of HDAC inhibitors. In this experiment, we observed that RORγ t-mediated IL-17 transcription was dramatically increased in the presence of HDAC inhibitors compared to the DMSO-only control (Fig. 4B). Subsequently, when Th17 cells were treated with DMSO or HDAC inhibitors, RT-PCR analysis showed that the expression of RORγ t-mediated genes were dramatically upregulated in the presence of HDAC inhibitors compared to the DMSO-only control (Fig. 4C).

HDAC1 interacts with and deacetylates RORγt.
To further investigate which HDAC is responsible for the observed effects, we screened the effects of several HDACs on p300-mediated RORγ t acetylation and found that HDAC1 decreased p300-mediated RORγ t acetylation (Supplementary Figure 3). Subsequently, to determine the protein level of HDAC1 in naïve and Th17-polarized T cells, we used western blotting analysis and found that the protein level of HDAC1 was higher in Th17 cells compared to naïve CD4 + T cells (Fig. 5A). To verify whether HDAC1 is associated with RORγ t, we transfected Myc-HDAC1 and Flag-RORγ t into HEK293T cells, and the coimmunoprecipitation results showed that HDAC1 interacts with RORγ t (Fig. 5B,C). In addition, endogenous IP also showed that HDAC1 interacts with RORγ t in human Th17 cells (Fig. 5D,E). HDAC1 has histone deacetylase activity, and we therefore sought to determine whether HDAC1 could decrease the acetylation level of RORγ t. We found that HDAC1 decreased RORγ t acetylation (Fig. 5F). Furthermore, HDAC1 decreased the RORγ t acetylation mediated by p300 (Fig. 5G). Collectively, these data suggest that HDAC1 interacts with and deacetylates RORγ t. p300 and HDAC1 reciprocally regulate RORγt function. To investigate the mechanism by which p300 regulates RORγ t, we generated a shRNA construct targeting p300 to reduce the endogenous p300

. HDAC inhibitors increase RORγt acetylation and RORγt-mediated IL-17 transcription.
(A) Flag-tagged RORγ t and HA-p300 were cotransfected into HEK293T cells treated either with or without HDAC inhibitors. Cell lysates were immunoprecipitated with an anti-Flag antibody and analyzed with the indicated antibodies. (B) Flag-tagged RORγ t and HA-tagged p300 were cotransfected with an IL-17 luciferase reporter into HEK293T cells in the presence of HDAC inhibitors. The cells were lysed, and luciferase activity was measured. (C) Th17 cells were treated with DMSO or HDAC inhibitors and analyzed using RT-PCR. Each figure is representative of > 3 independent experiments. The data are presented as the mean ± SEM; *p < 0.05, **p < 0.01. levels and the RT-PCR data show that silencing p300 downregulated Th17-related genes, including IL-17A, IL-17F and IL-23R (Fig. 6A). Thus, knockdown of p300 decreases RORγ t-mediated gene expression in Th17 cells. In addition, to further assess the protein levels of Th17 cytokines, we used ELISA to show that knockdown of p300 decreased the expression of IL-17A as well as RORγ t (Fig. 6B). RORγ t is a transcription factor that mediates IL-17 promoter activity. To determine whether p300 promotes RORγ t-mediated IL-17 transcription, an IL-17 luciferase reporter was cotransfected with either HA-p300 or Flag-RORγ t into HEK293T cells. Luciferase expression was analyzed and showed that p300 enhances RORγ t-mediated IL-17 transcription in a dose-dependent manner (Fig. 6C). Additionally, when the IL-17 luciferase reporter was cotransfected with either HA-p300 or Flag-RORγ t into HEK293T cells in the presence of Myc-HDAC1, the results indicated that HDAC1 represses p300-dependent, RORγ t-mediated IL-17 transcription (Fig. 6D). Together, these data suggest that p300 enhances RORγ t-mediated transcription, which is inhibited by HDAC1.

The functional differentiation of CD4 + T cells is determined by lineage-specific transcription factors.
Previous studies have shown that T-bet plays a deterministic role in Th1 differentiation, whereas GATA3 and Foxp3 are important for Th2 and Treg cell differentiation, respectively 30,31 . Recent data have shown that RORγ t is a key transcription factor that, along with other transcription factors, drives Th17 cell differentiation 9 . Many studies have shown that post-translational modifications including acetylation, phosphorylation, methylation, sumoylation and ubiquitination affect these critical transcription factors. For example, the ubiquitin ligase Stub1 promotes Foxp3 degradation and thus negatively modulates Treg cell suppressive activity 32 , and TIP60 positively regulates ThPOK-mediated repression of eomesodermin in human CD4 + T cells 28 . Reciprocal regulation of Foxp3 acetylation and transcriptional repression occurs through the actions of the histone acetyltransferase Tip60 and the histone deacetylases HDAC7 and HDAC9 24 . The deubiquitinase USP17 positively regulates RORγ t-mediated IL-17 transcription 12 . However, whether histone acetyltransferases and deacetylases regulate RORγ t has remained unclear.
Here, we demonstrated that RORγ t is acetylated in Th17 cells in vivo and that RORγ t acetylation is significantly enhanced in the presence of HDAC inhibitors (Trichostatin A (TSA), nicotinamide (NAM) and EX-527). Together these HDAC inhibitors can inhibit a majority of the histone deacetylases 17,28 . TSA is an inhibitor for class I and II histone deacetylases, NAM is an inhibitor for class III histone deacetylases and EX-527 is a widely used inhibitor of sirtuin enzymes 33,34 . In a future study, we will identify which HDAC inhibitor is responsible for the observed effects. p300 interacts with, stabilizes and acetylates RORγ t at its K81 residue. Knockdown of p300 downregulates RORγ t at the protein level and decreases transcription of IL-17. Previous studies have shown that many post-translational modifications have critical effects on p53 stability and function 35 . Furthermore, acetylation plays an important role in the functional regulation of p53 by p300 15,36 . Appropriate small-molecule inhibitors of p300 have been shown to impair Foxp3 + Treg cell function and promote antitumor immunity 29 . Therefore, it will be interesting to study the acetylation and functional regulation Figure 6. p300 upregulates RORγt-mediated IL-17 transcription, which is inhibited by HDAC1. (A) Naïve CD4 + T cells were sorted from PBMCs and cultured under Th17-polarizing conditions for 7 days. The Th17 cells were transduced with a lentivirus containing either shCK or shp300. The cells were then treated with puromycin for 3 days, and analysis was performed using RT-PCR. (B) Naïve CD4 + T cells were differentiated into Th17 cells. Th17 cells were transduced with a lentivirus containing either shCK or shp300. ELISA was performed to detect IL-17A level in Th17 cells. (C) Flag-tagged RORγ t and HA-tagged p300 were cotransfected with an IL-17 luciferase reporter into HEK293T cells. The cells were lysed, and luciferase activity was measured. (D) Flag-tagged RORγ t, HA-tagged p300 and Myc-tagged HDAC1 were cotransfected with an IL-17 luciferase reporter into HEK293T cells. The cells were lysed, and luciferase activity was measured. (E) A working model showing reciprocal regulation of RORγ t acetylation and RORγ t function by p300 and HDAC1. Each figure is representative of > 3 independent experiments. The data are presented as the mean ± SEM; **p < 0.01. of RORγ t by p300. Previous reports have shown that p300 polyubiquitinates p53 through a ubiquitin ligase activity independent of its lysine acetyltransferase activity 37,38 , Stabilization of Foxp3 by p300 is associated with hyperacetylation of Foxp3, which prevents polyubiquitination and proteasomal degradation 17 . In addition, a similar mechanism for Smad7 and p53 has been previously described 39,40 . Therefore, whether the ubiquitin ligase activity of p300 may also regulate RORγ t necessitates further investigation.
HDAC inhibitors have been shown to reduce protein levels and activity and increase the global acetylation level, resulting in altered cell proliferation, apoptosis and gene expression 41,42 . In this report, we provided evidence that HDAC inhibitors increase RORγ t acetylation and RORγ t-mediated IL-17 transcription. Recent data have shown that the histone deacetylase inhibitor ITF2357 decreases IL-6R production and RORγ t expression, suppresses polarization toward Th17 cells and enhances Treg cell polarization through the IL-6-STAT3-IL17 pathway in mice 43 . The deacetylase inhibitor TSA promotes the suppressive function of Treg cells 44 . However, Zhijian showed that TSA decreases Foxp3 expression and the number of Treg cells 45 . Our results conflict with those of previous studies because of differences in factors such as treatment time, the class of the HDAC inhibitor used and the source of the specimens. Therefore, our results demonstrate that HDAC inhibitors can enhance gene transcription via inhibition of HDACs.
Protein acetyltransferases and deacetylases regulate the balance between acetylation and deacetylation of transcriptional factors, thereby affecting the expression of the involved genes. HATs, which add an acetyl group to lysine residues, are associated with gene transcription activity. However, HDACs, which attenuate acetylation levels by removing acetyl groups from their substrates, are associated with transcriptional repression 46 . In this report, we observed that HDAC1 interacts with and deacetylates RORγ t and inhibits RORγ t-mediated transcriptional activation, therefore, HDAC1 may be responsible for the observed effects. A previous paper showed that HDAC1, commonly considered a transcriptional corepressor, has histone deacetylase activity and represses gene transcription 20 .
RORγ t is involved in autoimmune diseases. Here, we demonstrate that RORγ t is acetylated, and this acetylation is reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase HDAC1. Our work suggests that p300 and HDAC1 may be novel targets for the treatment of RORγ t-mediated autoimmune diseases.
Coimmunoprecipitation. At 48 h after transfection, cells were harvested, washed with ice-cold PBS, and subsequently lysed on ice for 30 minutes with protein lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1% NP40, 0.5% NaDOC, and 10% glycerol) containing protein inhibitors (cocktail, 1 mM Na 3 VO 4 , 10 mM NaF, and 1 mM PMSF). The cell lysates were centrifuged at 4 °C and the supernatants were immunoprecipitated by rotating for 1 h at 4 °C with antibodies and then for 1 h with proteinA/G-agarose beads. The beads were then washed with lysis buffer 4 times, and western blotting and immunoprecipitation were performed as previously described 47 . HAT assay. A p300-expression plasmid and a RORγ t-expression plasmid were cotransfected into HEK293T cells. The cells were treated with HDAC inhibitors prior to cell harvesting (overnight treatment with 50 μ M EX-527 and 4 h treatment with 1 mM NAM and 400 nM TSA). At 48 h after transfection, the cells were harvested, and immunoprecipitation was performed.
Scientific RepoRts | 5:16355 | DOI: 10.1038/srep16355 Luciferase assays. IL-17 Luciferase reporter vectors, β -gal and plasmids were transfected into HEK293T cells. After 48 h, the cells were harvested, washed with ice-cold PBS, lysed on ice for 30 minutes with luciferase lysis buffer, and then analyzed using a dual luciferase reporter kit (Promega).
ELISA. The Human IL-17 Platinum ELISA kit was used in accordance with the manufacturer's protocol (eBioscience).
Confocal assay. Myc-tagged RORγ t and Flag-tagged p300 were cotransfected into HeLa cells, which were fixed, permeabilized and then stained with anti-Myc or anti-p300. The cells were also stained with DAPI to visualize the nuclei. Cells were examined by confocal microscopy.
Statistical analysis. The data are presented as the means ± SEM. Comparisons between two groups were performed using Student's t-test. Differences were considered Statistically significant at *p < 0.05.