Thymocytes undergo apoptosis unless a functional TCR is assembled. Steroid receptor coactivators (SRCs) regulate nuclear receptor-mediated transcription by associated histone acetyltransferase activity. However, it has been a challenge to demonstrate the in vivo function of SRCs due to the overlapping functions among different members of SRCs. In this study, we show that recruitment of SRCs is required for thymic-specific retinoic acid-related orphan receptor γ (RORγ)t-regulated thymocyte survival in vivo. An activation function 2 domain, identified at the carboxyl terminus of RORγt, is responsible for recruiting SRCs. A mutation in the activation function domain (Y479F) of RORγt disrupted the interaction with SRCs and abolished RORγt-mediated trans-activation but not its ability to inhibit transcription. Transgenes encoding the wild-type RORγt, but not the mutant, restored thymocyte survival in RORγ null mice. Our results thus clearly demonstrate that RORγt recruits SRCs to impose a gene expression pattern required to expand the life span of thymocytes in vivo, which increases the opportunities for assembling a functional TCR.

Thymocytes are subject to a selection process critical for the development of fully functional mature T cells. The life span of thymocytes limits the progression of T cell development (1). Antiapoptotic molecules such as Bcl-xL extend the life span of thymocytes to ensure completion of the developmental process. Thymocytes lacking Bcl-xL undergo premature apoptosis (2, 3), whereas overexpression of Bcl-xL increases thymocyte survival and the chance for completing the selection process (1). Previously we, and subsequently others, have shown that retinoic acid-related orphan receptor γ (RORγ),3 a member of the steroid nuclear receptor family, regulates thymocyte survival via up-regulation of Bcl-xL (4, 5). RORγ was initially cloned by its sequence homology to retinoid hormone receptor (6). Another isoform, thymus-specific RORγ (RORγt), was subsequently cloned while screening for the molecules that inhibit activation-induced cell death in a T cell hybridoma (7). RORγt is a truncated form of RORγ lacking the first 24 aa due to use of a downstream translation start site. Although RORγ is expressed in multiple tissues, RORγt is expressed exclusively in thymocytes and a population of lymphoid tissue inducers at the embryonic stage (8, 9). Knockout of the RORγ gene, disrupting the expression of both RORγ and RORγt, resulted in massive apoptosis of thymocytes and defective development of lymph nodes (4, 5). However, less is known about the molecular mechanisms responsible for RORγt-regulated thymocyte survival.

A specific and heritable pattern of gene expression is imposed upon T cell lineage during the development in the thymus (10). Modification of histone by acetylation is a critical step in determining the pattern of gene expression by controlling the magnitude of gene activity (11, 12). Steroid receptor coactivators (SRCs) have the intrinsic or associated histone acetyltransferase activity necessary to remodel the chromatin structure by acetylation of histone. Acetylation is believed to relieve the chromatin-mediated transcriptional repression, resulting in active transcription (13). SRCs consist of three proteins with 50–55% homology, SRC1 (NcoA1), glucocorticoid receptor-interacting protein (GRIP)1 (TIF2), and SRC3 (p/CIP/RAC3/ACTR/AIB1/TRAM1) (14). In vitro transfection analysis demonstrated that the LXXLL motifs, present on the surface of SRCs, make contact with activation function 2 (AF2) domains located at the carboxyl terminus of ligand-binding domains of nuclear receptors (15, 16, 17, 18), resulting in the recruitment of SRCs to nuclear receptors (19, 20). However, it has been a challenge to demonstrate the in vivo function of recruitment of SRCs due to overlapping functions among various members (14). Knockout of an individual SRC member only results in a relatively minor phenotype (21, 22, 23), whereas double knockout of SRC1 and GRIP1 is lethal (24). Thus, the in vivo function of a given coactivator may not be revealed by a traditional knockout approach.

In this study, we show that RORγt recruits SRCs via its AF2 domain to stimulate transcription. In addition, by an AF2-domain-independent pathway, RORγt is able to inhibit the activity of NFAT. We created a mutation in the AF2 domain of RORγt that disrupted its interaction with SRCs, but not its ability to inhibit NFAT activity. RORγ null mice were reconstituted with a wild-type RORγt or an AF2 mutant incapable of binding to SRCs or a DNA-binding mutant that does not bind to its target DNA sequence. The massive thymocyte apoptosis was reversed by the transgene encoding the wild-type RORγt, but not the DNA-binding mutant, which acts as a negative control. Similar to DNA-binding mutant, AF2 failed to restore thymocyte survival, clearly demonstrating that recruitment of coactivators by RORγt is required to establish a gene expression pattern critical for thymocyte survival in vivo.

The plasmids pSG5-HA-GRIP1 and its LXXLL motif mutant were gifts from Dr. Michael Stallcup (Department of Pathology, University of Southern California, Los Angeles, CA). The third motif of GRIP1 was mutated to LXXAA by PCR-based mutagenesis. To generate expression plasmids encoding RORγt and its mutants, the DNA fragment containing RORγt cDNA was cloned in the pEF-Bos vector that contains a promoter of the elongation factor. RORγt reporter plasmid was a generous gift from Dr. Anthony Means (Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC). The following Abs for FACS analyses were purchased from BD Pharmingen: PE-anti-TCRβ (catalog no. 553172), biotin-anti-CD4 (catalog no. 553649), streptavidin-PE-Cy5 (catalog no. 554062), annexin V-PE (catalog no. 556421), PE-anti-CD8 (catalog no. RM2204), and anti-Bcl-xL. The hamster anti-RORγt Ab was previously described (4). Anti-GRIP1 (MS-1140-P1ABX) and anti-SRC1 (MS-1130-P1ABX) were purchased from Neomarkers; actin (SC8432) and p27kip1 (SC528G) Abs were obtained from Santa Cruz Biotechnology.

293T cells were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. 293T cells (2 × 105) were plated in 12-well dishes for 20 h. The cells were then transfected with 10 ng of a reporter construct, 100 ng of RORγt (wild type or mutants), 200 ng of SRC1 or GRIP1 expression plasmids, or with the indicated expression plasmids using the calcium phosphate method. The cells were fed with fresh medium 16 h after transfection. Then, after 24 h, cells were lysed in 200 μl of lysis buffer (137 mM NaCl, 50 mM Tris, and 0.5% Nonidet P-40), and 5 μl was used for luciferase assays as described in the protocol (Promega). A SV40-β-galactosidase expression plasmid was cotransfected for internal controls.

Thirty-six hours after transfection of the indicated expression plasmids, 293T cells were lysed in 1 ml of immunoprecipitation lysis buffer (150 mM NaCl, 50 mM Tris, 4 mM KCl, 1 mM MgCl2, 1 mM Na3VO4, 10% glycerol, 1% Nonidet P-40, and protease inhibitor). In the case of using thymocytes, 1 × 107 cells or sorted GFP+ thymocytes were lysed. Lysates were then precleared with 50 μl of 50% slurry protein A (Zymed Laboratories) for 3 h. The protein A beads were spun down, and the supernatant was incubated with 1 μg of monoclonal anti-SRC1 or GRIP1 Ab for 1 h. Twenty microliters of protein A beads was then added to the above mixtures for an additional 20 min. The protein A beads were spun down and washed four times with washing buffer (lysis buffer without protease inhibitor). The protein A pellets were then boiled in 50 μl of SDS loading buffer and resolved on a 10% SDS gel. Western blot analyses were performed with the indicated Abs and developed with ECL (Amersham Biosciences).

Three Tg constructs were cloned as shown in Fig. 4 a, encoding either wild-type or mutants RORγ driven by a CD4 promoter containing CD4 enhancer but not silencer. The NotI DNA fragment was microinjected into fertilized eggs by Transgenic Production Service in UIC. The founder mice were screened by Southern blot and FACS analyses of GFP expression. The Tg mice were then bred to RORγ null mice that were backcrossed to C57BL/6 for at least six generations. All of the mice used in this study were housed in a specific pathogen-free facility at the University of Illinois, Chicago.

FIGURE 4.

The wild-type RORγtTg but not the RORγt-DBDTg and RORγt-AF2Tg restored the survival of RORγ−/− thymocytes. a–d, Apoptosis assays. Thymocytes obtained from different genotypes of mice were cultured in medium for the indicated times, and then apoptotic cells were detected by annexin V and propidium iodide staining. Percentage of surviving cells was averaged from at least five mice with the error bar denoting SD. a, RORγ−/− thymocytes undergo rapid apoptosis. Apoptosis of the wild-type (ROR+) and RORγ−/− thymocytes (ROR) at various times after removal from mice and cultured in medium. b, Wild-type RORγtTg restored thymocyte survival. Apoptosis of the GFP+ and GFP thymocytes obtained from the RORγ null mice reconstituted with RORγtTg (RORγ/RORγtTg). c, RORγt-DBDTg failed to restore thymocyte survival. Apoptosis of the GFP+ and GFP thymocytes obtained from the RORγ null mice reconstituted with RORγt-DBDTg (RORγ/RORγt-DBDTg). d, RORγt-AF2Tg failed to restore thymocyte survival. Apoptosis of the GPF+ and GFP thymocytes obtained from the RORγ null mice reconstituted with RORγt-AF2Tg (RORγ/RORγt-AF2Tg). e, Wild-type RORγtTg but not RORγt-DBDTg and RORγt-AF2Tg restored thymic cellularity of the RORγt null mice. Total thymocyte number was averaged from five mice of each genotype. f, Wild-type RORγtTg but not RORγt-DBDTg and RORγt-AF2Tg restored the levels of Bcl-xL and p27kip1 in RORγ−/− thymocytes. Expression of RORγt, Bcl-xL, and p27kip1 was detected by Western blot analyses of the GFP+ cells sorted from different genotypes of mice. Actin was used as a control for equal loading. Data shown are representative of three independent experiments.

FIGURE 4.

The wild-type RORγtTg but not the RORγt-DBDTg and RORγt-AF2Tg restored the survival of RORγ−/− thymocytes. a–d, Apoptosis assays. Thymocytes obtained from different genotypes of mice were cultured in medium for the indicated times, and then apoptotic cells were detected by annexin V and propidium iodide staining. Percentage of surviving cells was averaged from at least five mice with the error bar denoting SD. a, RORγ−/− thymocytes undergo rapid apoptosis. Apoptosis of the wild-type (ROR+) and RORγ−/− thymocytes (ROR) at various times after removal from mice and cultured in medium. b, Wild-type RORγtTg restored thymocyte survival. Apoptosis of the GFP+ and GFP thymocytes obtained from the RORγ null mice reconstituted with RORγtTg (RORγ/RORγtTg). c, RORγt-DBDTg failed to restore thymocyte survival. Apoptosis of the GFP+ and GFP thymocytes obtained from the RORγ null mice reconstituted with RORγt-DBDTg (RORγ/RORγt-DBDTg). d, RORγt-AF2Tg failed to restore thymocyte survival. Apoptosis of the GPF+ and GFP thymocytes obtained from the RORγ null mice reconstituted with RORγt-AF2Tg (RORγ/RORγt-AF2Tg). e, Wild-type RORγtTg but not RORγt-DBDTg and RORγt-AF2Tg restored thymic cellularity of the RORγt null mice. Total thymocyte number was averaged from five mice of each genotype. f, Wild-type RORγtTg but not RORγt-DBDTg and RORγt-AF2Tg restored the levels of Bcl-xL and p27kip1 in RORγ−/− thymocytes. Expression of RORγt, Bcl-xL, and p27kip1 was detected by Western blot analyses of the GFP+ cells sorted from different genotypes of mice. Actin was used as a control for equal loading. Data shown are representative of three independent experiments.

Close modal

Thymocytes were stained in the dark with the indicated Abs on ice for 20 min, washed with FACS buffer (PBS with 0.02% sodium azide/1% FBS), and analyzed by a BD Biosciences FACSCalibur with CellQuest software. To analyze the PBLs, RBCs were lysed with ACK buffer (0.15 M NH4Cl, 1 mM KHCO3, and 1 mM EDTA) (Cambrex Bioscience) on ice for 5 min. PBLs were then stained with the indicated Abs. For isolation of GFP+CD4+CD8+ cells, thymocytes were stained with both CD8-PE and CD4-PE-Cy5 and sorted by a DakoCytomation MoFlo high-speed sorter. The GFP+CD4+CD8+ cells were sorted with >98% purity.

Thymocytes isolated from each genotype of mice were cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C for different times. The dead cells were then detected by annexin V-PE and propidium iodide staining for 20 min as described previously (4).

To investigate whether RORγt recruits other proteins, we first performed sequence analyses to identify the conserved domains known to be important for nuclear receptors. An AF2 domain (LYKELF) was identified at the carboxyl terminus of the RORγt ligand-binding domain, and it was conserved in all members of the ROR family (Fig. 1,a). RORγ, which shares the same DNA-and ligand-binding domains with RORγt, also contains this AF2 domain. Ligand-binding domains of nuclear receptors have a common structural feature with 12 α helices (H1–H12) (13). To determine whether the putative AF2 domain of RORγt is also conserved in a three-dimensional structure in addition to the primary sequence, we developed a computerized model for the RORγt ligand-binding domain (Fig. 1 b). Similar to other nuclear receptors, the ligand-binding domain of RORγt also contains 12 α helices, and the AF2 domain of RORγt is located at the H12 in the predicted model.

FIGURE 1.

Identification of an AF2 domain of RORγt responsible for recruiting SRCs. a, Identification of a conserved AF2 in RORγt ligand-binding domain. Conserved sequence (LYKELF) of the AF2 domain among members of the ROR family. b, A model of RORγt ligand-binding domain was predicted by a computer based on the homology to the ligand-binding domains of other nuclear receptors. The AF2 domain is located at helix 12 (H12). The critical tyrosine 479 (Y479) is indicated. c, RORγt and RORγt-DBD but not RORγt-AF2 were coimmunoprecipitated with SRC1. SRC1 was transiently expressed in 293T cells along with RORγt, RORγt-DBD, and RORγt-AF2. SRC1 was then immunoprecipitated (IP) with anti-SRC1-specific Ab or isotype control Ab (C). The presence of various RORγt in the immunoprecipitated complexes was detected by Western blot analyses probed with anti-RORγt Ab. Lower panels are the expression levels of SRC1 and various RORγt in lysates (input). d, RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with GRIP1. Similar assays as described in c, but GRIP1 instead of SRC1 were expressed in 293T cells with various RORγt. e, Wild-type GRIP1 but not the GRIP1 with mutated LXXLL motifs (GRIP1m) coimmunoprecipitated with RORγt. RORγt was transiently expressed in 293T cells along with wild-type GRIP1 or GRIP1m. GRIP1 was then immunoprecipitated with anti-GRIP1-specific Ab. The presence of RORγt in the immunoprecipitated complexes was detected by Western blot analyses probed with anti-RORγt Ab. Lower panels are the expression levels of GRIP1 and RORγt in lysates (input). f, 32P-labeled oligonucleotides (probe) containing a RORγt binding site was band shifted by bacterially expressed RORγt but not RORγt-DBD. Data shown are representative of at least three independent experiments.

FIGURE 1.

Identification of an AF2 domain of RORγt responsible for recruiting SRCs. a, Identification of a conserved AF2 in RORγt ligand-binding domain. Conserved sequence (LYKELF) of the AF2 domain among members of the ROR family. b, A model of RORγt ligand-binding domain was predicted by a computer based on the homology to the ligand-binding domains of other nuclear receptors. The AF2 domain is located at helix 12 (H12). The critical tyrosine 479 (Y479) is indicated. c, RORγt and RORγt-DBD but not RORγt-AF2 were coimmunoprecipitated with SRC1. SRC1 was transiently expressed in 293T cells along with RORγt, RORγt-DBD, and RORγt-AF2. SRC1 was then immunoprecipitated (IP) with anti-SRC1-specific Ab or isotype control Ab (C). The presence of various RORγt in the immunoprecipitated complexes was detected by Western blot analyses probed with anti-RORγt Ab. Lower panels are the expression levels of SRC1 and various RORγt in lysates (input). d, RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with GRIP1. Similar assays as described in c, but GRIP1 instead of SRC1 were expressed in 293T cells with various RORγt. e, Wild-type GRIP1 but not the GRIP1 with mutated LXXLL motifs (GRIP1m) coimmunoprecipitated with RORγt. RORγt was transiently expressed in 293T cells along with wild-type GRIP1 or GRIP1m. GRIP1 was then immunoprecipitated with anti-GRIP1-specific Ab. The presence of RORγt in the immunoprecipitated complexes was detected by Western blot analyses probed with anti-RORγt Ab. Lower panels are the expression levels of GRIP1 and RORγt in lysates (input). f, 32P-labeled oligonucleotides (probe) containing a RORγt binding site was band shifted by bacterially expressed RORγt but not RORγt-DBD. Data shown are representative of at least three independent experiments.

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The AF2 domain is a crucial component for recruiting SRC members containing LXXLL motifs (2). To determine whether RORγt associates with SRCs, we conducted immunoprecipitation assays. RORγt and SRCs were transiently expressed in 293T cells and immunoprecipitations using Abs specifically against either SRC1 or GRIP1 were conducted (Fig. 1, c and d). It is clear that RORγt is capable of forming complexes with both SRC1 and GRIP1. To determine whether the AF2 domain is essential for association with SRCs, an AF2 mutant (RORγt-AF2) was created by changing the critical tyrosine 497 in the AF2 domain to phenylalanine (Y479F). As a control, we also created a DNA-binding domain mutant (RORγt-DBD) by changing the two arginines located at the base of the DNA-binding zinc finger to alanine and glycine (R35R36-AG). As expected, the RORγt-DBD failed to bind to the oligonucleotides containing a RORγt binding site in band shift analyses (Fig. 1,f), but still formed a complex with SRC1 or GRIP1 (Fig. 1, c and d), demonstrating that mutation in the RORγt DNA-binding domain did not affect its interaction with SRCs. In contrast to wild-type RORγt and RORγt-DBD, RORγt-AF2 was barely detected in the complexes immunoprecipitated by the specific anti-SRC1 or GRIP1 Ab (Fig. 1, c and d), clearly demonstrating that the AF2 domain is required for RORγt to recruit SRCs.

To determine whether LXXLL motifs of SRCs are required for the interaction with RORγt, we obtained a mutant GRIP1 incapable of binding to nuclear receptors such as thyroid hormone receptor due to the mutations in two of its three LXXLL motifs (25). We generated additional mutations in the third LXXLL motif (LLQLL to LLQAA), so that all three LXXLL motifs were mutated to create GRIP1m. In contrast to wild-type GRIP1, this mutant failed to form a complex with RORγt in the immunoprecipitation assays (Fig. 1 e), suggesting a critical role for LXXLL motifs of SRCs in the interaction with RORγt. Similar results were also obtained from the GRIP1 that had two of the three LXXLL motifs mutated (data not shown), suggesting that mutating two LXXLL motifs is sufficient to disrupt interaction with RORγt. Altogether these results demonstrated that the RORγt-mediated recruitment of SRCs depends on the AF2 and LXXLL motifs.

SRCs are key to enhancing nuclear receptor-mediated transcriptional responses (12, 14). We therefore determined whether recruitment of SRCs regulates RORγt-mediated transcriptional activity. A luciferase reporter driven by a TK promoter with three upstream RORγt binding sites was used to monitor RORγt-mediated transcription. Expression of RORγt or SRC1 alone only slightly enhanced reporter activity, whereas significant enhancement of the transcriptional activity was observed in the presence of SRC1 in combination with RORγt but not RORγt-AF2 (Fig. 2,a), suggesting that SRC1 recruited by the AF2 domain enhances RORγt-mediated transcription. The negative control, RORγt-DBD, failed to stimulate RORγt-DBD activity in the presence of SRC1 as expected, since binding to the target DNA response element is required for a transcription factor to mediate transcription (Fig. 2,a). Similar results were obtained using another SRC member, GRIP1 (Fig. 2,b), suggesting that SRC members regulate RORγt-mediated transcription. Furthermore, wild-type GRIP1, but not GRIP1m incapable of binding to RORγt, stimulated RORγt activity (Fig. 2 c). These results clearly demonstrated that SRCs recruited by the AF2 domain enhance RORγt-mediated transcriptional responses.

FIGURE 2.

Recruitment of SRCs augments RORγt-mediated trans-activation but has no effect on its ability to inhibit NFAT. a, SRC1 facilitates wild-type RORγt but not RORγt-AF2 to stimulate transcription. Expression plasmids for the wild-type RORγt or RORγt-DBD or RORγt-AF2 were transfected to 293T cell alone (striped bars) or in combination with SRC1 expression plasmid (filled bars). RORγt-mediated transcriptional activity was monitored using a luciferase reporter driven by a TK promoter with three upstream RORγt binding sites (reporter). Lower panels are the expression levels of various RORγt and SRC1 detected by Western blot analyses. b, GRIP1 facilitates wild-type RORγt but not RORγt-AF2 to stimulate transcription. Similar experiments as described in b were performed, but GRIP1 expression plasmid replaced SRC1 expression plasmid. c, Wild-type GRIP1 but not GRIP1 containing mutated LXXLL motifs (GRIP1m) stimulated RORγt-mediated transcription. Expression plasmids for wild-type GRIP1 or GRIP1m were transiently expressed in 293T cell alone (□) or along with RORγt expression plasmid (▪). The transcriptional activity was monitored by a RORγt reporter. Right panels are the expression levels of RORγt, GRIP1, and GRIP1m detected by Western blot analysis. d, Both wild-type RORγt and RORγt-AF2 inhibit transcription activity of NFAT. NFAT is transiently expressed in 293T cells alone or with increasing amounts of wild-type RORγt or RORγt-AF2. NFAT-mediated transcription activity was monitored by a NFAT reporter with three upstream NFAT binding sites. NFAT activity is indicated as the fold of stimulation relative to the activity obtained from cells transfected with reporter alone. Lower panels are the expression levels of RORγt or RORγt-AF2 and NFAT. e, Forced expression of SRC1 does not affect RORγt-mediated inhibition of NFAT. Expression plasmid for NFAT was transfected to 293T cells with expression plasmids encoding proteins as indicated. A NFAT reporter was used to monitor NFAT activity. The reporter activity with error bars denoting SD was averaged from at least three independent experiments.

FIGURE 2.

Recruitment of SRCs augments RORγt-mediated trans-activation but has no effect on its ability to inhibit NFAT. a, SRC1 facilitates wild-type RORγt but not RORγt-AF2 to stimulate transcription. Expression plasmids for the wild-type RORγt or RORγt-DBD or RORγt-AF2 were transfected to 293T cell alone (striped bars) or in combination with SRC1 expression plasmid (filled bars). RORγt-mediated transcriptional activity was monitored using a luciferase reporter driven by a TK promoter with three upstream RORγt binding sites (reporter). Lower panels are the expression levels of various RORγt and SRC1 detected by Western blot analyses. b, GRIP1 facilitates wild-type RORγt but not RORγt-AF2 to stimulate transcription. Similar experiments as described in b were performed, but GRIP1 expression plasmid replaced SRC1 expression plasmid. c, Wild-type GRIP1 but not GRIP1 containing mutated LXXLL motifs (GRIP1m) stimulated RORγt-mediated transcription. Expression plasmids for wild-type GRIP1 or GRIP1m were transiently expressed in 293T cell alone (□) or along with RORγt expression plasmid (▪). The transcriptional activity was monitored by a RORγt reporter. Right panels are the expression levels of RORγt, GRIP1, and GRIP1m detected by Western blot analysis. d, Both wild-type RORγt and RORγt-AF2 inhibit transcription activity of NFAT. NFAT is transiently expressed in 293T cells alone or with increasing amounts of wild-type RORγt or RORγt-AF2. NFAT-mediated transcription activity was monitored by a NFAT reporter with three upstream NFAT binding sites. NFAT activity is indicated as the fold of stimulation relative to the activity obtained from cells transfected with reporter alone. Lower panels are the expression levels of RORγt or RORγt-AF2 and NFAT. e, Forced expression of SRC1 does not affect RORγt-mediated inhibition of NFAT. Expression plasmid for NFAT was transfected to 293T cells with expression plasmids encoding proteins as indicated. A NFAT reporter was used to monitor NFAT activity. The reporter activity with error bars denoting SD was averaged from at least three independent experiments.

Close modal

In addition to trans-activation by recruiting SRCs, we found that RORγt was able to inhibit the activity of an important T cell transcription factor, NFAT. The activity of a NFAT reporter was stimulated by forced expression of NFAT as expected (Fig. 2,d). However, the stimulated NFAT activity was inhibited by RORγt in a dose-dependent manner. NFAT is required to stimulate the IL-2 gene during T cell activation (26). Indeed, overexpression of RORγt inhibited IL-2 promoter activity induced by TCR stimulation or PMA/ionomycin treatment (data not shown). One of the common mechanisms for nuclear receptor-mediated inhibition of transcription is the sequestration of coactivators such as SRCs (19). However, overexpression of SRC1 or GRIP1 did not relieve RORγt-mediated inhibition of NFAT (Fig. 2,e, and data not shown). Furthermore, the RORγt-AF2 incapable of binding to SRCs inhibited NFAT activity as efficiently as the wild type (Fig. 2 d). Recruitment of SRCs is thus required for RORγt-mediated trans-activation but not for inhibition of NFAT activity.

To determine the in vivo function of RORγt-mediated recruitment of SRCs, we established Tg mice expressing RORγt (RORγtTg), RORγt-DBD (RORγt-DBDTg), and RORγt-AF2 (RORγt-AF2Tg). Since RORγt is specifically expressed in thymocytes, we used a CD4 promoter that was able to target transgene expression to T cell compartments, including CD4+CD8+ thymocytes (27). The cDNAs encoding various RORγt were cloned downstream of a CD4 promoter along with an internal ribosome entry site (IRES) and an enhanced GFP coding region (Fig. 3,a). The IRES permits both RORγt and GFP to be expressed from a single bicistronic mRNA. The founder mice were screened with Southern blot analysis with RORγt cDNA as a probe (Fig. 3 b). The transgenes were successfully integrated into the genome as indicated by the detection of a 3-kb DNA fragment in BamHI-digested genomic DNA.

FIGURE 3.

Generation of Tg mice expressing wild-type RORγt, RORγt-DBD, and RORγt-AF2. a, Tg constructs. cDNAs encoding various RORγt were cloned between an upstream CD4 promoter and downstream IRES-GFP. NotI is the restriction site used to release the DNA fragment for generating Tg. b, Integration of the transgene into the genome. DNA prepared from the Tg founders was subjected to digestion with restriction enzyme (BamHI) and to Southern blot analyses with RORγt cDNA as a probe. A 3-kb band was detected in mice positive (+) but not negative (−) for transgene. c, Expression of GFP in thymocytes of the Tg mice. Flow cytometric analyses of the expression of CD3 and GFP in wild-type (ROR+), RORγ null (ROR), or mice that integrate the transgene encoding wild-type RORγt (RORγtTg), RORγt-DBD (RORγt-DBDTg), or RORγt-AF2 (RORγt-AF2Tg). Numbers are the percentage of GFP+ cells within the indicated gates. d, Wild-type RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with GRIP1 in thymocytes. GRIP1 in thymocyte lysates was immunoprecipitated (IP) by anti-GRIP1-specific Ab or isotype control Ab (C). The IP complexes were then subjected to Western blot analyses with anti-RORγt Ab (upper panel). Lower panels indicate the expression levels of RORγt and GRIP1 in the lysates used for immunoprecipitation. e, Wild-type RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with SRC1 in thymocytes. Similar assays as described in d, but anti-SRC1 instead of GRIP1 Ab was used. Data shown are representative of at least three independent experiments.

FIGURE 3.

Generation of Tg mice expressing wild-type RORγt, RORγt-DBD, and RORγt-AF2. a, Tg constructs. cDNAs encoding various RORγt were cloned between an upstream CD4 promoter and downstream IRES-GFP. NotI is the restriction site used to release the DNA fragment for generating Tg. b, Integration of the transgene into the genome. DNA prepared from the Tg founders was subjected to digestion with restriction enzyme (BamHI) and to Southern blot analyses with RORγt cDNA as a probe. A 3-kb band was detected in mice positive (+) but not negative (−) for transgene. c, Expression of GFP in thymocytes of the Tg mice. Flow cytometric analyses of the expression of CD3 and GFP in wild-type (ROR+), RORγ null (ROR), or mice that integrate the transgene encoding wild-type RORγt (RORγtTg), RORγt-DBD (RORγt-DBDTg), or RORγt-AF2 (RORγt-AF2Tg). Numbers are the percentage of GFP+ cells within the indicated gates. d, Wild-type RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with GRIP1 in thymocytes. GRIP1 in thymocyte lysates was immunoprecipitated (IP) by anti-GRIP1-specific Ab or isotype control Ab (C). The IP complexes were then subjected to Western blot analyses with anti-RORγt Ab (upper panel). Lower panels indicate the expression levels of RORγt and GRIP1 in the lysates used for immunoprecipitation. e, Wild-type RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with SRC1 in thymocytes. Similar assays as described in d, but anti-SRC1 instead of GRIP1 Ab was used. Data shown are representative of at least three independent experiments.

Close modal

Expression of GFP along with RORγt makes it possible to identify the cells that express transgene. We thus analyzed GFP expression in thymocytes using a flow cytometer (Fig. 3,c). Among the Tg mice identified positive by Southern blot analyses, seven mice displayed GFP expression when gated on CD3+ T cells, whereas GFP was not detected in the mice negative for transgene, suggesting that the transgene was correctly targeted for expression in T cell compartments. Progeny from different lines of Tg mice expressing the same transgene exhibited a similar phenotype, and in this study results from one line are reported. Since not all of the T cells express GFP, presumably due to the variegation effect (28), we were able to compare the GFP+ and GFP T cells obtained from the same Tg mouse as shown later. The GFP expression also allowed us to sort GFP+ cells for analyses, especially in the case of RORγt-AF2Tg mice that only contain a relatively small portion, ∼17%, of GFP+ cells in thymus (Fig. 3 c). The Tg mice were then crossed to the RORγ null mice to eventually obtain the RORγ null Tg expressing wild-type RORγt (RORγ/RORγtTg), DNA-binding mutant (RORγ/RORγt-DBDTg), or AF2 mutant (RORγ/RORγt-AF2Tg).

To confirm whether RORγt and the two mutants expressed from the transgenes exhibit similar interactions with SRCs as observed in in vitro experiments, we conducted immunoprecipitation assays using thymocytes obtained from different genotypes of mice. Anti-SRC1 (Fig. 3,d)- or GRIP1 (Fig. 3,e)-specific Ab coimmunoprecipitated RORγt in thymocytes obtained from wild-type but not mutant mice, confirming that RORγt associates with SRCs in vivo. The immunoprecipitation assays were then performed using GFP+ cells sorted from RORγ-deficient mice expressing various transgenes. These GFP+ cells expressed RORγt only from transgenes, thus excluding the interference from the endogenous RORγt. Similar to the results obtained from transiently transfected cells (Fig. 1), RORγt and RORγt-DBD but not RORγt-AF2 coimmunoprecipitated with GRIP1 and SRC1 (Fig. 3, d and e), suggesting that RORγt interacts with members of SRCs in vivo in an AF2-dependent manner. In addition, our results also indicated that the transgenes encoding the wild-type and mutant RORγt were equivalently expressed (lower panels, Fig. 3, d and e).

To determine the effect of the RORγt transgene on thymocyte survival, we analyzed apoptosis of the thymocytes. As reported earlier, ∼80% of the CD4+CD8+RORγ−/− thymocytes died after 6 h in medium, whereas in wild-type (RORγ+/+) or heterozygous (RORγ+/−) mice <20% of the thymocytes underwent apoptosis (Fig. 4,a). In the RORγ+/− mice, the transgenes encoding either wild-type or mutant RORγt had no significant effect on the survival of thymocytes (data not shown), likely due to the presence of endogenous RORγt. We next examined the effect of transgenes on the survival of RORγ−/− thymocytes by analyzing the apoptosis of CD4+CD8+GFP+ and CD4+CD8+GFP cells sorted from different genotypes of mice. GFP+ cells from RORγ null mice expressing the wild-type RORγt transgene (RORγ/RORγtTg) survived as well as cells from wild-type mice, whereas GFP cells underwent rapid apoptosis similar to the RORγ null mice (Fig. 4,b), suggesting that expression of the RORγt is sufficient to rescue thymocytes from apoptosis. Correspondingly, the reduced thymic cellularity and the antiapoptotic Bcl-xL both returned almost to the wild-type levels (Fig. 4, e and f). We previously observed that RORγ−/− thymocytes displayed abnormal cell cycle progression because of reduced levels of p27kip1 (4). Wild-type RORγtTg prevented down-regulation of p27kip, suggesting that the cell cycle progression was also restored (Fig. 4,f). In contrast to wild-type RORγtTg, RORγt-DBDTg, the negative control, failed to rescue the RORγ−/− thymocytes from apoptosis (Fig. 4,c), confirming that RORγt, a transcription factor, regulates thymocyte survival by DNA-binding-dependent mechanisms. Similar to RORγt-DBDTg, RORγt-AF2Tg also failed to restore the survival of RORγt−/− thymocytes (Fig. 4,d). Accordingly, neither RORγt-DBDTg nor RORγt-AF2Tg restored thymic cellularity (Fig. 4,e) and the levels of Bcl-xL and p27kip1 (Fig. 4,f). We also compared apoptosis of GFP+ and GFP cells in the same mice. In contrast to the RORγ/RORγtTg mice (Fig. 4,b), apoptosis of the GFP cells in RORγ/RORγt-DBDTg and RORγ/RORγt/AF2Tg mice was similar to that of the GFP+ cells (Fig. 4, c and d), strongly suggesting that wild-type RORγt but not RORγt-DBD and RORγt-AF2 supported CD4+CD8+ thymocyte survival. These results clearly demonstrated a critical role of AF2 domain-mediated recruitment of coactivators in vivo.

Flow cytometric analyses of surface expression of CD4, CD8, and TCR on RORγ−/− thymocytes revealed a different expression pattern from that of the wild-type, most likely due to the apoptosis (4). Compared with the wild type (Fig. 5,a), CD4+CD8+ thymocytes in RORγ null mice skewed to CD4low (Fig. 5,b), which were corrected by the wild-type RORγtTg (Fig. 5,c, upper panel) but not by the RORγt-DBDTg (Fig. 5,d, upper panel) and the RORγt-AF2Tg (Fig. 5,e, upper panel). In addition, the CD4 single-positive (SP) cell population was significantly reduced in RORγ null mice (0.81%; Fig. 5,b) compared with the wild-type mice (7.32%; Fig. 5,a). Expression of the RORγtTg but not the RORγt-DBDTg (Fig. 5,d, upper panel) and RORγt-AF2Tg (Fig. 5,e, upper panel) leads to a significant increase in the CD4 SP cells to a level (14.4%; Fig. 5,c, upper panel) much higher than that of the wild-type mice, which is consistent with the increased CD3+ cell population in peripheral blood (Fig. 3,c). The endogenous RORγ gene is turned off during the process of differentiation from double-positive (DP) to SP T cells (7, 8). However, the RORγt transgene driven by a CD4 promoter was not turned off, as shown by the GFP expression in SP T cells both in thymus and peripheral blood (Figs. 3,c and 5), which may be responsible for the increased CD4 cells.

FIGURE 5.

Flow cytometric analyses of the surface expression of CD4 and CD8 on thymocytes obtained from RORγ+ (a), RORγ (b), RORγ/RORγtTg (c), RORγ/RORγt-DBDTg (d), and RORγ/RORγt-AF2Tg (e) when gated on GFP+ cells (upper panels) or GFP cells (lower panels). Numbers are the percentage of CD4 SP cells within the indicated gates. Surface expression of TCR on thymocytes obtained from RORγ+ (f), RORγ (g), RORγ/RORγtTg (h), RORγ/RORγt-DBDTg (i), and RORγ/RORγt-AF2Tg (j) when gated on GFP+ cells (upper panels) or GFP cells (lower panels). Numbers are the percentage of TCRhigh, TCRin, and TCRlow cells within the gated area.

FIGURE 5.

Flow cytometric analyses of the surface expression of CD4 and CD8 on thymocytes obtained from RORγ+ (a), RORγ (b), RORγ/RORγtTg (c), RORγ/RORγt-DBDTg (d), and RORγ/RORγt-AF2Tg (e) when gated on GFP+ cells (upper panels) or GFP cells (lower panels). Numbers are the percentage of CD4 SP cells within the indicated gates. Surface expression of TCR on thymocytes obtained from RORγ+ (f), RORγ (g), RORγ/RORγtTg (h), RORγ/RORγt-DBDTg (i), and RORγ/RORγt-AF2Tg (j) when gated on GFP+ cells (upper panels) or GFP cells (lower panels). Numbers are the percentage of TCRhigh, TCRin, and TCRlow cells within the gated area.

Close modal

With the maturation of T cells, expression of surface TCR increases progressively and the SP cells express the highest levels of TCR. Flow cytometric analyses of TCR revealed that overall TCR levels shifted downward in RORγ null mice (Fig. 5, f and g). If the thymocytes were grouped into three arbitrary populations according to their surface TCR levels, TCRlow, TCR intermediate (TCRin), and TCRhigh, the wild-type mice had ∼30% of TCRlow cells (Fig. 5,f), whereas RORγt null mice had ∼60% of TCRlow cells (Fig. 5,g). RORγtTg reduced the TCRlow population to the wild-type levels in RORγt null mice, ∼30% (Fig. 5,h, upper panel), most likely due to the rescued thymocyte survival. Whereas in RORγ/RORγt-DBDTg (Fig. 5,i, upper panel) and RORγ/RORγt-AF2Tg (Fig. 5,j, upper panel) mice, the TCRlow population remained at the levels similar to that of the RORγ null mice. Interestingly, compared with the RORγ mice, slightly higher TCR levels were observed in the TCRin population of the RORγ/RORγt-DBDTg and RORγ/RORγt-AF2Tg mice. To further confirm the function of the transgenes, we also compared the GFP+ and GFP cells in the same mouse. In mice expressing the RORγtTg, the GFP+ cells (Fig. 5, c and h, upper panel) displayed expression patterns of surface CD4, CD8, and TCR similar to that of the wild-type mice, whereas the GFP cells (Fig. 5, a and h, lower panel) displayed the patterns similar to that of the RORγ−/− thymocytes. In contrast, both GFP+ and GFP cells obtained from the RORγ-/RORγt-DBDTg (Fig. 5, d and i) and RORγt/RORγt-AF2Tg (Fig. 5, e and j) mice had surface CD4, CD8, and TCR expression patterns similar to that of the RORγt null mice, clearly demonstrating that the wild-type RORγt but not RORγt-DBD and RORγt-AF2 is capable of restoring the phenotype of RORγ−/− thymocytes to that seen in the wild-type mice. The above results suggest that the transgene was properly targeted and expressed in thymocytes, and the wild-type RORγt but not the RORγt-DBD and RORγt-AF2 functioned to replace the endogenous RORγ gene for thymocyte maturation. Furthermore, since expression of the wild-type RORγt in T cell compartments was sufficient to restore thymocyte survival, apoptosis of the RORγ−/− thymocytes is most likely due to the absence of RORγt expression in thymus but not due to defects in other somatic tissues.

SRCs are believed to play a critical role in establishing the specific gene expression pattern required for development and differentiation (14). However, it has been a challenge to demonstrate the in vivo function of SRCs due to overlapping functions among its members (14, 22). In vitro transfection analysis of SRC-mediated transcription often indicates overlapping function among SRCs. Such overlapping functions are indicated by the fact that a single nuclear receptor can interact with multiple members of SRCs (14, 29, 30, 31). Therefore, a gene knockout approach is unlikely to be successful in elucidating the entire function of an individual coactivator in vivo due to the functional redundancy. Indeed, mice deficient in either SRC1 or GRIP1 displayed relatively minor defects (14, 21, 22, 23), whereas knockout of both SRC1 and GRIP1 is embryonic lethal (24). Both in transfected cells and in thymocytes, RORγt was coimmunoprecipitated with SRC1 as well as GRIP1 (Figs. 1 and 3). RORγt is thus capable of interacting with more than one member of the SRCs. Moreover, both SRC1 and GRIP1 potentiate RORγt-mediated transcriptional responses (Fig. 2). Such redundancy could explain why knockout of SRC1 or GRIP1 did not lead to defects in thymocyte survival (14). In this study, we created a point mutation in the AF2 domain of RORγt that disrupted the LXXLL motif-based interactions. RORγ null mice display thymocyte apoptosis and are fertile, which allows us to determine the function of AF2 domain-mediated recruitment of coactivators in an in vivo model. We clearly demonstrated here that RORγt-mediated in vivo thymocyte function requires the recruitment of LXXLL motif-containing coactivators.

Nuclear receptors, although capable of interacting with multiple members of SRC, exhibit preferential binding to different SRC family members. For example, androgen receptor binds preferentially to GRIP1 over SRC1 (32). In addition, LXXLL motifs within a given coactivator exhibit binding specificity. The first three LXXLL motifs of SRC1 preferentially mediate the binding to estrogen receptor and progesterone receptor, while the fourth LXXLL motif strongly binds the androgen receptor and glucocorticoid receptor. Using peptides containing various LXXLL motifs, Kurebayashi et al. (33) showed that the binding selectivity of LXXLL peptides for RORγ differs from those for other receptors such as estrogen receptor, RAR, glucocorticoid receptor, and vitamin D receptor (33). Our results do not exclude the possibility that RORγt preferentially binds to one coactivator over the others. We did observe that GRIP1 is more potent than SRC1 in stimulating RORγt-mediated transcription. It is thus very possible that members of the SRCs contribute differentially in the regulation of RORγt-mediated thymocyte survival in vivo.

Thymocytes have developed a mechanism to switch on Bcl-xL specifically at the DP stage to extend their survival. During transition from the double-negative to DP stage, Bcl-xL is significantly up-regulated. Consistently, mice deficient in Bcl-xL exhibit apoptosis specifically at the DP stage (34). Interestingly, Bcl-xL is switched off again during transition from the DP to SP stage. Our previous results demonstrated that in the absence of RORγt, the levels of Bcl-xL mRNA and protein failed to be up-regulated in DP cells, which explains the observed apoptosis (4). RORγt is thus critical for inducing Bcl-xL as cells differentiate into the DP stage. Because the AF2 mutant failed to restore Bcl-xL to wild-type levels (Fig. 4 f), recruitment of SRCs by RORγt is an essential step in switching on the Bcl-xL gene at the DP stage from its relatively inactive state at the earlier double-negative stage. Nuclear receptors are able to regulate cellular function by mechanisms other than DNA-binding-dependent transcriptional regulation (35, 36). For example, Nur77 induces T cell apoptosis by interfering with Bcl-2 function via direct interaction (37). Nur77-mediated apoptosis is thus independent of its DNA-binding activity. Our results with the DNA-binding mutant of RORγt exclude such a possibility. Therefore, SRCs recruited by RORγt likely modify the chromatin structure at the DP stage, allowing up-regulation of Bcl-xL and consequent thymocyte survival.

RORγt has been shown to inhibit up-regulation of the Fas ligand (FasL) and IL-2 production and thus protects T cell hybridoma from activation-induced cell death (7). In this study, we demonstrate that RORγt is a potent inhibitor of NFAT (38). Because transcriptional activation of both FasL and IL-2 requires NFAT activity (26, 39), our results explain why forced expression of RORγt in hybridoma prevents FasL-dependent activation-induced cell death. In addition to regulating FasL, NFAT plays a critical role in multiple T cell functions including apoptosis (26, 40, 41, 42). NFAT4−/− or NFATc1−/− mice have reduced thymic cellularity due to increased apoptosis, suggesting a positive role for both NFAT isoforms in thymocyte survival (40, 41, 42). Surprisingly, thymocytes deficient in both NFATc1 and NFATc2 do not display obvious defects in thymocyte survival (43). A balanced effect among several isoforms of NFAT is thus critical for maintaining optimum thymocyte survival. Although it is not clear whether RORγt inhibits NFAT activity in vivo in thymocytes, our results clearly showed that RORγt has an ability to inhibit NFAT activity in in vitro transfection analysis. We found that RORγt-AF2, which is still capable of inhibiting NFAT activity, failed to support thymocyte survival. Thus, in contrast to AF2 domain-mediated recruitment of coactivators, the ability to inhibit NFAT does not appear to be critical for RORγt-regulated thymocyte survival. However, we do not exclude the possibility that both inhibition of NFAT and recruitment of coactivators are required for thymocyte survival. A mutation that specifically disrupts the inhibitory effect on NFAT, but not the interaction with coactivators, will be useful in examining such a possibility.

Knockout of the RORγ gene disrupted its expression in thymocytes as well as in other somatic tissues (4, 6, 7). It is therefore possible that the observed apoptosis is a secondary effect resulting from the defects in other somatic tissues. We show here that forced expression of the wild-type RORγt in the T cell compartments was sufficient to rescue the defects observed in RORγ−/− thymocytes, including apoptosis and down-regulation of Bcl-xL, CD4, p27kip1, and TCR, strongly suggesting that RORγt functions autonomously in thymocytes. In addition, RORγt-regulated thymocyte survival is likely independent of its function in lymph node genesis, since the transgene encoding the wild-type RORγt did not rescue the lymph node genesis in RORγ null mice (data not shown). Therefore, expression of RORγt in cells other than thymocytes is required for lymph node development. In agreement, Eberl et al. (44) have shown that expression of RORγt in fetal lymphoid inducer cells is critical for lymph node genesis. However, the CD4 promoter may not be active in lymphoid inducer cells, because Cre activity could not be detected in lymphoid inducer cells from CD4-Cre Tg mice (9). It is also possible that cells other than lymphoid inducers are required for lymph node genesis. RORγt thus regulates thymocyte survival and lymph node development differently.

We show here that recruitment of SRCs by RORγt is required to regulate thymocyte survival, presumably by modifying gene expression. RORγt target genes are yet to be identified. Bcl-xL is transcriptionally down-regulated in RORγ null mice (4, 5). However, RORγt does not appear to regulate Bcl-xL transcription directly, because RORγt has no effect on a reporter driven by a Bcl-xL promoter (data not shown). Identification of RORγt target genes will facilitate understanding of the mechanisms responsible for RORγt-regulated functions.

We thank Dr. Michael Stallcup for providing expression plasmids of wild-type and mutant GRIP1; Dr. Jeff Staudinger for expression plasmids encoding SRC1, c-RIP140, and vp16-c-RIP140; Dr. Anthony Means for RORγt reporter; Dr. Dan Littman for CD4 promoter constructs used in generation of Tg mice; and Drs. Prasad Kanteti and Bellur Prabhakar for critically reading this manuscript and helpful discussion.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3

Abbreviations used in this paper: RORγ, retinoic acid-related orphan receptor; RORγt, thymic-specific RORγ; SRC, steroid receptor coactivator; AF2, activation function 2; GRIP, glucocorticoid receptor-interacting protein; Tg, transgenic; IRES, internal ribosome entry site; SP, single positive; DP, double positive; TCRin, TCR intermediate; FasL, Fas ligand.

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