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The Journal of Immunology, 2007, 179, 4694-4703
Copyright © 2007 by The American Association of Immunologists, Inc.
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Early Growth Response 1 and NF-ATc1 Act in Concert to Promote Thymocyte Development beyond the beta-Selection Checkpoint1

Ekaterina K. Koltsova*, Maria Ciofani{dagger}, Robert Benezra{ddagger}, Toru Miyazaki§, Neil Clipstone, Juan Carlos Zúñiga-Pflücker{dagger} and David L. Wiest2,*

* Division of Basic Sciences, Immunobiology Working Group, Fox Chase Cancer Center, Philadelphia, PA 19111; {dagger} Department of Immunology, University of Toronto, Sunnybrook Research Institute, Toronto, Ontario, Canada; {ddagger} Cancer Biology and Genetics Program, Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; § Division of Molecular Biomedicine for Pathogenesis, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and Department of Pharmacology, Loyola University Chicago, Maywood, IL 60153


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Development of immature T cell precursors beyond the beta-selection checkpoint is regulated by signals transduced by the pre-TCR complex. The pre-TCR-induced differentiation program is orchestrated by a network of transcription factors that serve to integrate this signaling information. Among these transcription factors are those of the early growth response (Egr) and NF-AT families. In this study, we demonstrate that Egr1 and NF-ATc1 act together to promote development of T cell precursors beyond the beta-selection checkpoint to the CD8 immature single-positive and CD4+CD8+ double-positive stages. Moreover, we find that Egr1 and NF-AT cooperatively induce the expression of inhibitor of DNA binding 3 (Id3), a regulatory factor known to play an important role in positive selection of thymocytes, but not previously demonstrated to be required for beta-selection. Importantly, we show in this study that Id3 deficiency abrogates the ability of ectopically expressed Egr1 to promote traversal of the beta-selection checkpoint. Id3 is presumably essential for traversal of the beta-selection checkpoint in this context because of the inability of other inhibitor of DNA binding family members to compensate, since transgenic Egr1 does not induce expression of inhibitor of DNA binding 1 (Id1) or 2 (Id2). Taken together, these data demonstrate that Id3 is a cooperatively induced target that is important for Egr-mediated promotion of development beyond the beta-selection checkpoint. Moreover, these data indicate that the ERK and calcium signaling pathways may converge during beta-selection through the concerted action of Egr1 and NF-ATc1, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Development of thymocytes from the CD4–CD8– (double-negative (DN))3 to the CD4+CD8+ (double-positive (DP)) stage is controlled by a critical checkpoint termed beta-selection, which ensures that only those precursors that have productively rearranged the TCRbeta locus will undergo further maturation (1). beta-selection occurs within the DN population of thymocytes, which can be further subdivided into four subsets based upon differences in the expression of CD44 and CD25 (DN1, DN2, DN3, and DN4) (2). During maturation between the DN2 and DN3 stages, rearrangement of the TCRbeta locus is initiated, and if the rearrangement is productive, TCRbeta protein is expressed (1, 3, 4). Expression of a functional TCRbeta subunit alerts the developing thymocyte that TCRbeta rearrangement has been productive through assembly of the TCRbeta protein with pre-TCR{alpha} and CD3 chains, thereby forming the pre-TCR complex (5, 6). Pre-TCR complex assembly results in its activation via a recently described self-oligomerization process that is dependent upon charged amino acids in the pre-T{alpha} ectodomain (7). Pre-TCR signals, in turn, produce a number of biological outcomes such as entry into the cell cycle, rescue from apoptosis, allelic exclusion at the TCRbeta locus, and differentiation beyond DN3 to the DP stage (8).

Pre-TCR signaling induces the ensuing differentiation program through a battery of transcription factors, including NF-{kappa}B, early growth response (Egr) family members, members of the NF-AT family, E proteins, beta-catenin/Tcf-1/Lef-1, Myb, and GATA-3 (9, 10, 11, 12, 13, 14, 15, 16, 17). Among these transcriptional regulators, the Egr family is particularly interesting because its activity has been shown to be both necessary and sufficient for traversal of the beta-selection checkpoint (18, 19, 20, 21). The Egr family comprises four members as follows: Egr1 (Krox24), Egr2 (Krox20), Egr3, and Egr4. These family members are thought to play largely overlapping roles in early thymocyte development because mice singly deficient for Egr family members do not exhibit a profound impairment at the beta-selection checkpoint (19, 20). However, simultaneous inhibition of the activity of all family members is able to impair development beyond the beta-selection checkpoint (18). Moreover, we and others have shown that enforced expression of Egr proteins (particularly Egr1 and Egr3) is sufficient to promote development of thymocytes beyond the beta-selection checkpoint even in the absence of pre-TCR signals (18, 21, 22). Although these observations strongly support the notion that Egr proteins play an important role in development beyond the beta-selection checkpoint, it remains unclear how Egr proteins cooperate with other signaling pathways and the regulators of gene expression that those pathways control.

NF-AT proteins are transcription factors that are activated by pre-TCR signaling and are important in executing the ensuing beta-selection differentiation program (10). The NF-AT family consists of five members as follows: NF-AT1 (NF-ATc2/NF-ATp), NF-AT2 (NF-ATc1/NF-ATc), NF-AT3 (NF-ATc4), NF-AT4 (NF-ATc3/NF-ATx), and NF-AT5. Four family members (except NF-AT3) are expressed in the immune system (23, 24, 25). Studies of animals singly or multiply deficient in NF-AT proteins have revealed both distinct and overlapping roles of NF-AT family members in T cell development and differentiation (26, 27, 28, 29, 30, 31). NF-AT2 (NF-ATc1) in particular has been implicated as an important regulator of the DN to DP transition (27, 30). Moreover, it was recently reported that NF-ATc1 physically interacts with Egr1 (32). Other reports have indicated that Egr1 and NF-AT proteins also functionally cooperate to transactivate a number of different gene targets, including the proinflammatory cytokines IL-2 and TNF, CD154, and Fas ligand (32, 33, 34, 35). Consequently, we sought to investigate whether Egr1 and NF-ATc1 act together to promote development of immature thymocytes beyond the beta-selection checkpoint.

Indeed, we report in this study that Egr1 and NF-ATc1 act together to induce both transformed and normal cells to develop beyond the beta-selection checkpoint. We demonstrate that enforced expression of Egr1 and NF-ATc1 in a thymic lymphoma differentiation model leads to enhanced down-modulation of CD25, a hallmark of beta-selection. Moreover, enforced coexpression of Egr1 and NF-ATc1 in normal thymocytes is far more effective at promoting the development of pre-TCR-deficient thymocytes beyond the beta-selection checkpoint than is expression of either factor alone. Egr1 requires both its N-terminal activation domain and C-terminal DNA-binding domain (DBD) for optimal cooperation with NF-ATc1 in promoting the development of T cell precursors. Finally, Egr1 and NF-ATc1 act together synergistically to induce expression of the helix-loop-helix (HLH) factor inhibitor of DNA binding 3 (Id3), which we demonstrate is essential for Egr1 to promote differentiation of Rag2-deficient (Rag2–/–) thymocytes beyond the beta-selection checkpoint to the DP stage. Taken together, these data suggest that Egr1 and NF-ATc1 may serve as an important point of convergence of the ERK and calcium signaling pathways during early thymocyte development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Egr1 transgenic (Tg) mice (21) were bred to Rag2–/– mice obtained from Taconic Farms, and to Id3–/– mice (36). All mice were maintained in the American Association of Laboratory Animal Care-accredited animal colony of the Fox Chase Cancer Center and were handled in compliance with guidelines established by the Institutional Animal Care and Use Committee.

Cell lines

Scid.adh thymic lymphoma cells expressing the TAC:CD3{epsilon} signaling chimera (human IL-2R{alpha} exo and transmembrane domains fused to the cytoplasmic domain of CD3{epsilon}) were cultured and stimulated with plate-bound anti-TAC mAb, as described (37). The anti-TAC mAb-producing hybridoma hd245/332 was obtained from American Type Culture Collection with the permission of T. Waldman (National Institutes of Health, Bethesda, MD). As indicated, stimulation cultures were supplemented with vehicle control (DMSO) or the following pharmacologic inhibitors titrated to establish the most effective dose that was not cytotoxic: 1) cyclosporin A (CsA), calcineurin inhibitor (2 µM) (38); 2) PP2, src kinase inhibitor (1 µM) (39); 3) UO126, MEK inhibitor (5 µM) (40, 41); and 4) SB203580, p38 inhibitor (25 µM) (40). Cells were stimulated for 24 h, following which the extent of differentiation was assessed by monitoring CD25 down-regulation by flow cytometry. Cells were stained with commercially prepared fluorochrome-coupled mAb from BD Pharmingen and analyzed on either BD LSRII or FACSVantageSE cytometers (BD Pharmingen) using propidium iodide gating to exclude dead cells.

Plasmids

The MSCV-GFP-NF-ATc1 encoding a full-length cDNA of NF-ATc1 was described previously (42). Egr1 was PCR cloned using standard methodologies into LZRS-YFP, a retroviral vector that contains an internal ribosomal entry site (IRES) that allows cap-independent translation of the yellow fluorescent protein (YFP) marker. Likewise, Flag-tagged constructs of mouse Egr1 as well as all mutants described in this study were cloned into MSCV-IRES-YFP (provided by D. Vignali, St. Jude Children’s Research Hospital, Nashville, TN) by PCR using standard methodologies and sequence verified. DYRK1A cDNA was PCR cloned using standard methodologies into MSCV-IRES-GFP retroviral vector (43). Cloning details will be provided upon request. Miev-RasV12 was described previously (44).

Retrovirus production and transduction

Phoenix-E retroviral packaging cells (provided by G. Nolan, Stanford University, Stanford, CA) were transfected with retroviral vectors using the calcium phosphate transfection method, as described (45). Transfection efficiencies were evaluated by determining the percentage of GFP- or YFP-positive Phoenix-E using flow cytometry. All cell lines were maintained in complete Iscove’s medium containing 10% FBS and supplemented, as described (18). Scid.adh-TAC:CD3{epsilon} cells at a concentration 1 x 106/ml were spin infected for 45 min at 30°C with retroviral supernatant treated with 8 µg/ml polybrene, as described (46). At 30 h postinfection, cells were analyzed by flow cytometry or isolated by flow cytometry for analysis of changes in gene expression.

Measurement of changes in gene expression

Expression of NF-AT family members in Scid.adh cells was assessed by RT-PCR and ethidium bromide staining. Total RNA was isolated using the RNA easy RNA purification system (Qiagen) and treated with DNase I (Invitrogen Life Technologies) before first strand cDNA synthesis using the Superscript II system, according to the random priming protocol (Invitrogen Life Technologies). Titrated amounts of cDNA were amplified by PCR with the following primers: 1) NF-AT1, forward, 5'-ATCACTGGGAAAACGGTCACC-3' and reverse, 5'-TTAGGCTGGCTCTTGTCTTTAATCC-3'; 2) NF-AT2, forward, 5'-CCAAGTCTCTTTCCCCGACATC-3' and reverse, 5'-TCAGCCGTCCCAATGAACAG-3'; 3) NF-AT3, forward, 5'-GGATTACTGGCAAGATGGTGGC-3' and reverse, 5'-AGTCTGGCAGGAAGTTGGAACC-3'; 4) NF-AT4, forward, 5'-CAGGGAAAAATGTCAAGGGGC-3' and reverse, 5'-CAACTGTGGCAAATGGGTGGAG-3'; 5) actin, forward, 5'-CCTAAGGCCAACCGTGAAAAG-3' and reverse, 5'-TCTTCATGGTGCTAGGAGCCA-3'. Gene expression was analyzed by TaqMan real-time PCR using the following commercially prepared primers and probes (Applied Biosystems): actin, Egr1, Egr2, Egr3, inhibitor of DNA binding 1 (Id1), inhibitor of DNA binding 2 (Id2), Id3, and TCR{alpha}. Reactions were performed in triplicate for each gene, and expression was normalized to beta-actin expression.

OP9-DL1 cultures

Hemopoietic precursor cells (HPC) were enriched from embryonic day 13.5 (E13.5) livers by pelleting onto a cushion of lympholyte M (Cedarlane Laboratories), at 2800 rpm for 45 min at 30°C. HPC (5 x 104) were induced to differentiate by culture on OP9-DL1 monolayers in {alpha}MEM medium containing the following supplements (47): 20% FCS, 10 mM HEPES (pH 7.0), sodium pyruvate, gentamicin, penicillin/streptomycin, L-glutamine, 2-ME, 1.0 ng/ml IL-7, and 5 ng/ml Flt3-ligand. Cultures were passaged to fresh monolayers every 4 days. HPC were retrovirally transduced on day 4 of culture and analyzed by FACS on day 16. For cultures using adult thymocytes, DN3 thymocytes were isolated by flow cytometry, immediately spin infected, and then seeded onto OP9-DL1 monolayers in 24-well plates at 100,000 cells/well. Cultures of adult cells were supplemented as above, except that the dose of IL-7 (R&D Systems and Shenandoah BioTechnology) was increased to 5 ng/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Blockade of the calcineurin/NF-AT pathway completely inhibits the differentiation of Scid.adh cells, but not Egr1 induction

The Egr proteins are critical molecular effectors of the pre-TCR-induced beta-selection differentiation program in that their function is both necessary and sufficient for traversal of that checkpoint (18, 19, 20, 21). They are among the few transcription factors whose enforced expression is capable of promoting development of thymocytes beyond the beta-selection checkpoint in the absence of pre-TCR signals (18, 21). However, the signaling pathways and transcriptional regulators that act in concert with Egr proteins in controlling early thymocyte development remain largely unexplored.

To investigate this question, we used the Scid.adh thymic lymphoma model, whose differentiation in vitro mimics that of normal thymocytes undergoing beta-selection in vivo (37). Indeed, stimulation of Scid.adh through a chimera that mimics pre-TCR signaling (i.e., the TAC:CD3{epsilon} chimera) induces a battery of phenotypic changes that closely resemble the differentiation of normal thymocytes in vivo, including down-regulation of CD25 expression, a phenotypic hallmark of beta-selection (Fig. 1A) (48). Consequently, we used this model to investigate signaling pathways that might cooperate with Egr proteins in promoting the differentiation of T cell precursors beyond the beta-selection checkpoint. To do so, we first used pharmacologic inhibitors of candidate signaling pathways to identify those that impair differentiation in response to pre-TCR signaling, but not the induction of Egr family members. We found that pharmacologic blockade of the calcineurin (CsA), src (PP2), and MEK (UO126) pathways completely blocked Scid.adh differentiation, as assessed by inhibition of CD25 down-modulation (Fig. 1A). In contrast, inhibition of phospholipase C{gamma}1, PI3K, p38 kinase (SB203580), mammalian target of rapamycin, protein kinase C, and JNK did not impair differentiation at doses that were not cytotoxic (data not shown). Therefore, the calcineurin, src, and MEK signaling pathways are critical for thymocyte development. Importantly, of the inhibitors that impaired differentiation, only CsA, an inhibitor of calcineurin activity, impaired differentiation while allowing induction of the Egr family member, Egr1 (Fig. 1B). Consistent with previous reports in DP thymocytes (49), CsA treatment completely blocked induction of Egr2 and Egr3; however, it only partially inhibited induction of Egr1 (Fig. 1B). Unlike CsA, treatment with inhibitors of MEK (UO126) and src (PP2) blocked both differentiation and Egr1 induction (Fig. 1B; data not shown). The observation that blockade of the calcineurin/NF-AT pathway completely blocked differentiation, while allowing substantial induction of Egr1, led us to consider the possibility that Egr1 might act in concert with the calcineurin/NF-AT signaling pathway in promoting the differentiation of Scid.adh cells.


Figure 1
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  		FIGURE 1. CsA completely inhibits differentiation, but not Egr1 induction. A, Pharmacologic inhibition of calcineurin, src kinases, and MEK kinase blocks Scid.adh differentiation. TAC:CD3{epsilon}-expressing SCID.adh cells were stimulated for 24 h on plate-bound anti-TAC (10 µg/ml) in medium containing vehicle control (DMSO) or the indicated inhibitor as follows: CsA (2 µM), PP2 (1 µM), UO126 (5 µM), and SB (25 µM). CD25 expression was measured by flow cytometry on viable cells defined by propidium iodide exclusion. B, CsA only partially inhibits Egr1 induction. Expression of the indicated genes was measured by real-time PCR on cells stimulated as described in A. Gene expression in stimulated cell populations was normalized to that of beta-actin and expressed as fold induction relative to unstimulated cells that were defined as 1. Graphs represent mean ± SD of triplicate samples.

 
Egr1 and NF-ATc1 act together to promote differentiation beyond the beta-selection checkpoint

To test the possibility that Egr1 might interact functionally with the calcineurin signaling pathway, we first determined which of the calcineurin-regulated NF-AT family members (NF-AT1–4) were expressed in Scid.adh (24, 50). RT-PCR analysis revealed the presence of mRNA encoding NF-AT1, NF-AT2, and NF-AT4 (Fig. 2A), among which NF-AT2 (which we will refer to as NF-ATc1) in particular has been implicated in the DN to DP transition of thymocyte development (27, 30). Accordingly, we examined whether NF-ATc1 could function in concert with Egr1 in promoting the differentiation of Scid.adh. To do so, we ectopically coexpressed Egr1 and NF-ATc1 using retroviral vectors distinguished by YFP and GFP, respectively (Fig. 2, B and C). Expression of NF-ATc1 alone induced partial down-regulation of CD25 in most cells, whereas enforced expression of Egr1 induced full down-regulation of CD25 in a subpopulation (Fig. 2C, left and middle panels). Interestingly, ectopic coexpression of Egr1 and NF-ATc1 induced extensive down-modulation of CD25 (a hallmark of differentiation beyond the beta-selection checkpoint) in most cells (Fig. 2C). Taken together, these data suggest that Egr1 and NF-ATc1 act in concert to induce the differentiation of Scid.adh lymphoma cells in vitro.


Figure 2
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  		FIGURE 2. NF-AT acts together with Egr1 in promoting Scid.adh differentiation. A, Scid.adh cells express NF-AT1, NF-AT2, and NF-AT4. Total RNA from Scid.adh cells was reverse transcribed, following which the presence of the indicated genes was assessed by RT-PCR and ethidium bromide staining on titrated input cDNA. Template cDNA for NF-AT1, NF-AT2, NF-AT3, NF-AT4, and beta-actin was serially diluted 1/1, 1/3, and 1/6. B, Cotransduction of Scid.adh cells with LZRS-YFP-Egr1 and MSCV-GFP-NFAT. A two-color histogram of the GFP and YFP at 30 h postinfection is displayed as surrogate markers for expression of the NF-AT and Egr1 protein products, respectively. C, Coexpression of Egr1 and NF-ATc1 produces greater CD25 down-modulation than expression of either protein alone. At 30 h postinfection, CD25 expression was assessed by flow cytometry on electronically gated cells expressing Egr1 only (YFP+; C), NF-ATc1 only (GFP+; A), or both Egr1 and NF-ATc1 (GFP+/YFP+; B). CD25 levels on the indicated populations were compared with the equivalent population transduced with empty vector.

 
To test the physiological relevance of this observation, we next wished to determine whether Egr1 and NF-AT could act in concert to promote the development of primary thymocytes beyond the beta-selection checkpoint. To do so, we used Egr1 Tg Rag2-deficient (Egr1 Tg Rag2–/–) mice in which the Egr1 Tg promotes development of pre-TCR-deficient DN thymocytes to the DP stage (21). Dpc 13.5 fetal liver precursors from Egr1 Tg Rag2–/– and Rag2–/– fetuses were cultured on OP9-DL1 monolayers, which support development of hemopoietic precursors into T cells (51). Following retroviral transduction of the cultured precursors with either vector control or NF-ATc1, we assessed their differentiation by flow cytometry (Fig. 3). Interestingly, whereas the Egr1 Tg promotes development of Rag2–/– thymocytes beyond the beta-selection checkpoint in adult mice (see Fig. 7), this was not evident in the OP9-DL1 stromal cultures (Fig. 3A, right panels, third from top) perhaps because the cells whose development is promoted by the Egr1 Tg are quickly diluted by the proliferation of undifferentiated cells, a phenomenon not observed in vivo. In contrast, ectopic expression of NF-ATc1 in Rag2–/– precursors did induce a discernible increase in DP thymocytes, suggesting that overexpression of NF-ATc1 alone can promote some development beyond the beta-selection checkpoint. Importantly, however, as we observed in Scid.adh cells, coexpression of Egr1 and NF-ATc1 produced greater differentiation beyond the beta-selection checkpoint to the CD8 immature single-positive (ISP) and DP stage (Fig. 3A, lower right panel) than did expression of either Egr1 or NF-ATc1 alone (Fig. 3, A and B). This was also the case when purified DN3 thymocytes from adult mice were used (Fig. 3, C and D). Indeed, infection of Egr1 Tg Rag2–/– DN3 thymocytes with NF-ATc1 increased the proportion of cells that had matured to the CD8 ISP or DP stage both earlier (as early as day 4 postinfection) and to a greater extent than did infection of Rag2–/– DN3 thymocytes (Fig. 3C). Moreover, this was particularly striking regarding the absolute number of cells produced (Fig. 3D). NF-ATc1 infection of Egr1 Tg Rag2–/– DN3 thymocytes induced 9-fold more cells to differentiate beyond the DN stage to the CD8 ISP or DP stage than did transduction of Rag2–/– DN3 thymocytes (Fig. 3D). Importantly, this increase was only slightly less than that observed following transduction with activated Ras (RasV12) (Fig. 3D) (44). Taken together, our data suggest that Egr1 and NF-ATc1 act together to promote differentiation beyond the beta-selection checkpoint in both the Scid.adh cell line model and primary thymocytes.


Figure 3
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  		FIGURE 3. Egr1 and NF-ATc1 act together to promote traversal of the beta-selection checkpoint by pre-TCR-deficient thymocytes. A–D, Coexpression of Egr1 and NF-ATc1 promotes development of pre-TCR-deficient thymocytes to the CD8 ISP and DP stage. A, E13.5 HPC from Egr1 Tg Rag2–/– and Rag2–/– fetal livers were cultured on OP9-DL1 monolayers for 4 days, retrovirally transduced with MSCV-GFP-NF-AT or empty vector (MSCV-GFP), and then analyzed by flow cytometry on day 16. Histograms of electronically gated GFP– and GFP+ cells are shown. B, Graphic depiction of the absolute number of DP and CD8 ISP. Mean ± SD of the absolute number of cells from three experiments are shown. C, Adult DN3 thymocytes from mice of the indicated genotypes were transduced, cultured, and analyzed as in A. Histograms of CD4 and CD8 staining on electronically gated GFP+ populations at the indicated time postinfection are depicted. D, The absolute number of retrovirally transduced GFP+ CD8 ISP+DP thymocytes is represented graphically. Data are representative of two experiments performed.

 

Figure 7
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  		FIGURE 7. Id3 is required for Egr1-mediated promotion of differentiation. A and B, Explanted thymocytes from Rag2–/–, Egr1 Tg Rag2–/–, and Egr1 Tg Id3–/–Rag2–/– mice were stained with the indicated Ab. A and B, Two-color histograms of CD4/CD8 staining on total thymocytes (A) and CD44/CD25 staining on gated DN thymocytes (B) are depicted. Graphic representation of the absolute number of cells in the indicated subsets is displayed to the right of the two-color histograms. Data are representative of three experiments, three mice per genotype in each experiment. C, Enforced expression of Egr1 induces Id3, but not Id1 and Id2. Real-time PCR analysis of Id1–3 mRNA expression was performed on thymocytes from Egr1 Tg Rag2–/– thymocytes. Results of triplicate analysis were normalized to beta-actin and represented graphically as fold induction relative to that in Rag2–/– thymocytes.

 
Egr1 activation domain and DBD are required to optimally promote development with NF-ATc1

To determine which domains of Egr1 are functionally required to promote development when coexpressed with NF-ATc1, we produced several mutants of the conserved functional domains of Egr1 (Fig. 4A). These include the following: 1) DBD, H417->L mutation that destroys DNA binding (52); 2) {Delta}P, deletion of PMIPDY, a motif of unknown function that is conserved among Egr family members; 3) {Delta}112, deletion of the proximal N-terminal activation subdomain (53); and 4) {Delta}N, deletion of the entire N-terminal activation domain. To assess the effect of those mutations on induction of Scid.adh differentiation, these constructs were retrovirally coexpressed with NF-ATc1 in Scid.adh cells as in Fig. 2 (Fig. 4, B and C). Deletion of the PMIPDY motif ({Delta}P) resulted in a modest impairment in the ability of Egr1 to promote Scid.adh differentiation when expressed either alone or together with NF-ATc1. Mutation of the proximal N-terminal activation domain ({Delta}112), the entire N-terminal activation domain ({Delta}N), and the DBD resulted in progressively more pronounced interference with the ability of Egr1 to promote differentiation either alone or together with NF-ATc1 (Fig. 4, B and C). Taken together, these data demonstrate that both the N-terminal activation domain and DBD of Egr1 are required to optimally promote differentiation together with NF-ATc1.


Figure 4
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  		FIGURE 4. Both the activation domain and DBD of Egr1 are required for optimal promotion of development with NF-ATc1. A, Schematic of Egr1 mutant constructs. B, Mutation of the activation domain of Egr1 impairs its ability to cooperate with NF-ATc1. TAC:CD3{epsilon}-expressing SCID.adh cells were retrovirally transduced with NF-ATc1 (GFP) and wild-type Egr1, or the following mutant constructs in MSCV-IRES-YFP: 1) {Delta}P, PMIPDY motif deletion; 2) {Delta}112, aa1–112 deleted; and 3) {Delta}N, activation domain deleted. CD25 expression was assessed by flow cytometry at 30 h postinfection on electronically gated cells as in Fig. 2, B and C. C, Mutation of the DBD of Egr1 impairs its ability to cooperate with NF-ATc1. TAC:CD3{epsilon}-expressing SCID.adh cells were retrovirally transduced and analyzed as in B with an Egr1 mutant lacking DNA-binding ability.

 
Egr1 requires the NF-AT signaling pathway to promote T cell development

Because Egr1 exhibited the ability to act together with NF-ATc1 in promoting development of thymocytes beyond the beta-selection checkpoint, we next investigated whether Egr1 required NF-AT activity to promote thymocyte differentiation. Indeed, Scid.adh cells transduced with Egr1 already express significant amounts of endogenous NF-AT, which may be necessary for the action of Egr1 in promoting development. Because NF-AT1, NF-AT2, and NF-AT4 are expressed in Scid.adh cells (Fig. 2A), we were not able to use a straightforward genetic approach to analyze the effects of complete NF-AT deficiency on Egr1-mediated effects. Consequently, we used a pharmacologic approach using CsA, which is able to inhibit the activity of all of the NF-AT family members expressed by Scid.adh cells (Fig. 2A). Egr1-transduced cells were treated with CsA to determine whether inhibition of NF-AT activity impaired the ability of Egr1 to promote Scid.adh differentiation. Indeed, CsA treatment, but not treatment with an inhibitor of p38 kinase SB203580, was able to substantially inhibit the ability of ectopically expressed Egr1 to promote Scid.adh differentiation (Fig. 5, A and B). Moreover, this was not an artifact of drug treatment, because cotransduction of Egr1 with DYRK1A, a kinase recently reported to phosphorylate and inhibit NF-ATc1 activity, impaired the ability of Egr1 to promote CD25 down-modulation to a similar degree as did CsA treatment (Fig. 5C) (43). Taken together, these results suggest that the ability of ectopically expressed Egr1 to promote differentiation depends at least in part on the activity of the calcineurin/NF-AT signaling pathway.


Figure 5
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  		FIGURE 5. The ability of Egr1 to promote differentiation is impaired by inhibition of NF-AT acitvity. A–C, Inhibition of calciuneurin, but not p38 kinase, impairs Egr1-induced differentiation. TAC:CD3{epsilon}-expressing SCID.adh cells were transduced with LZRS-YFP-Egr1 or empty vector (shaded) and cultured in the presence of the indicated pharmacological inhibitors for 24 h, following which CD25 expression was measured by flow cytometry. A, Single-color histograms of CD25 expression are depicted. B, Graphic depiction of the extent to which CD25 down-modulation is inhibited relative to vehicle control (DMSO). CD25 down-modulation in untreated control cells was defined as complete down-modulation, and the impairment by drug treatment was expressed as a percentage. Egr1 expression in CsA-treated cells was determined by real-time PCR to be 88% of that in cells treated with DMSO (data not shown). C, Graphic depiction of the effect of cotransduction with DYRK1A kinase on the ability of Egr1 to induce Scid.adh differentiation. Results from two experiments are shown. Cotransduction of DYRK1A with Egr1 did not affect Egr1 expression (data not shown).

 
NF-ATc1 and Egr1 act cooperatively in regulating expression of downstream genes

To investigate the molecular basis whereby Egr1 and NF-ATc1 act in concert to promote differentiation beyond the beta-selection checkpoint, we sought to identify gene targets whose expression was cooperatively modulated in our Scid.adh differentiation model. In doing so, we focused on gene targets known to be modulated during beta-selection and whose function is known, or predicted, to be important in traversal of this checkpoint. Interestingly, among the gene targets analyzed, we identified Rag2 and Id3 as being cooperatively regulated by Egr1 and NF-ATc1. In the case of Rag2, we observed that coexpression of Egr1 and NF-ATc1 resulted in a significant suppression of Rag2 gene expression, compared with the effects of either Egr1 or NF-ATc1 alone (Fig. 6). Id3 mRNA levels were also altered more significantly by coexpression of Egr1 and NF-ATc1 than by expression of either factor alone; however, unlike Rag2, whose expression was decreased, that of Id3 was markedly increased (Fig. 6). One potential explanation for the cooperative effects of NF-ATc1 and Egr1 on gene expression could have been through NF-AT-mediated induction of Egr2 and 3, because NF-AT has been shown to induce Egr2 and 3 expression in mature T cells (33). However, this was not the case in Scid.adh cells coexpressing Egr1 and NF-ATc1 (data not shown). These findings indicate that Egr1 and NF-ATc1 cooperate in the regulation of gene targets that are known to play important roles in thymocyte development (54, 55).


Figure 6
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  		FIGURE 6. NF-AT and Egr1 cooperate in modulation of target gene expression. TAC:CD3{epsilon}-expressing SCID.adh cells were retrovirally transduced with Egr1, NF-ATc1, or both, or vector controls, as in Fig. 2. At 30 h postinfection, the indicated cell populations were isolated by flow cytometry. Isolated RNA was reverse transcribed, and, following expression of the indicated genes, was measured in triplicate by real-time PCR using Taqman probes. Expression of specified genes was normalized to beta-actin and expressed as a fold change relative to cells transduced with empty vector. The data are representative of three experiments performed. The extent to which Egr1 and NF-AT suppress Rag2 mRNA and induce Id3 mRNA is greater than the sum of the individual effects of Egr1 and NF-AT (p < 0.05).

 
Id3 is a critical downstream target through which Egr1 induces development beyond the beta-selection checkpoint

Id3 is a HLH factor known to antagonize E protein activity (56). The suppression of E protein activity has been shown through gene targeting to be important for traversal of the beta-selection checkpoint (54, 57). Nonetheless, Id3-mediated suppression of E protein activity has not, to date, been shown to be important for traversal of the beta-selection checkpoint (54, 55). To determine whether the induction of Id3 is important for promotion of development beyond the beta-selection checkpoint by Egr1 and NF-ATc1, we bred Egr1 Tg mice to a background doubly deficient for Rag2, to prevent pre-TCR expression, and for Id3 (21, 58). Importantly, as reported previously, enforced expression of Egr1 was sufficient to promote development of pre-TCR-deficient thymocytes beyond the beta-selection checkpoint (Fig. 7, A and B) (18, 21). In fact, in Egr1 Tg Rag2–/– mice, we observed strong promotion of the DN3 to DN4 transition, as well as development of CD8 ISP and DP thymocytes. In contrast, the ability of the Egr1 Tg to promote development of pre-TCR-deficient (Rag2–/–) thymocytes beyond the beta-selection checkpoint was blocked by ablation of the Id3 gene (Fig. 7, A and B). Indeed, in the Id3-deficient background, Egr1-induced development of DN3 thymocytes to the DN4 stage and beyond (i.e., CD8 ISP and DP stages) was completely blocked (Fig. 7, A and B). Therefore, collectively, our data demonstrate that Id3 is a cooperatively induced target that is critical for Egr-mediated promotion of development beyond the beta-selection checkpoint. Importantly, Id3 deficiency has not previously been reported to interfere with differentiation beyond the beta-selection, perhaps because of compensation by other inhibitor of DNA binding family members. This does not occur during Egr1-induced differentiation beyond the beta-selection checkpoint because Id1 and Id2 are not induced by Egr1 (Fig. 7C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Activation of the pre-TCR complex triggers a diverse array of signaling cascades that orchestrate the beta-selection differentiation program. One way in which information from distinct signaling cascades can be integrated is through the concerted action of transcription factors, such as has been observed for NF-AT and the AP-1 complex or Foxp3 (25, 59, 60). Nevertheless, it is difficult to identify the individual components through which signals are integrated due to the complexity and redundancy of transcription factor usage during thymocyte differentiation. By using a model in which traversal of the beta-selection checkpoint is induced by Egr1, we have revealed that the concerted action of Egr1 and NF-ATc1 is important for traversal of the beta-selection checkpoint. Indeed, we demonstrate that Egr1 and NF-ATc1 act together to promote the differentiation of both transformed and primary immature thymocyte precursors beyond the beta-selection checkpoint. Egr1 requires both its transcriptional activation domain and DBD to promote development either alone or together with NF-ATc1. Moreover, the ability of Egr1 to induce pre-TCR-deficient thymocytes to differentiate beyond the beta-selection checkpoint is impaired by inhibition of either calcineurin or NF-AT function, suggesting that the ability of Egr1 to promote differentiation is dependent, at least in part, upon the activity of the calcineurin/NF-AT signaling pathway. Finally, we have shown that Egr1 and NF-ATc1 act cooperatively to induce expression of the HLH factor Id3, whose activity we have now demonstrated is required for Egr1-mediated promotion of thymocyte development. Taken together, these data indicate that Egr1 and NF-ATc1 can act together to promote differentiation beyond the beta-selection checkpoint and provide the first demonstration that Id3 function is required in this context.

Previously, we and others have demonstrated that Egr proteins are both necessary and sufficient for development of thymocytes beyond the beta-selection checkpoint (18, 19, 21). Indeed, whereas mice singly deficient for Egr family members do not exhibit profound impairment of development beyond the beta-selection checkpoint, dominant-negative mutants that impair the collective function of all Egr family members do impair this transition, suggesting functional redundancy among family members (19, 20). Moreover, enforced expression of Egr family members, including Egr1, is sufficient to substitute for the pre-TCR complex, at least in part, and to promote maturation of pre-TCR-deficient thymocyte beyond the beta-selection checkpoint (18, 21). Nevertheless, the functional interaction of Egr proteins with other transcription factor families in regulating early thymocyte development has not been previously explored.

NF-AT family members have also been implicated as important molecular effectors of pre-TCR signaling, particularly NF-ATc1, whose ablation has previously been shown to impair the DN to DP transition (27, 30). Moreover, recent reports have indicated that NF-AT proteins are able to cooperate with Egr1 in other cellular contexts in regulating the expression of IL-2, TNF-{alpha}, CD154, and a membrane type 1 matrix metalloproteinase (32, 35, 61). Importantly, however, the current report is the first description of Egr1 and NF-ATc1 acting together to promote development beyond the beta-selection checkpoint, a transition normally controlled by pre-TCR signaling. Indeed, we demonstrate that the enforced expression of both Egr1 and NF-ATc1 more effectively promotes the differentiation of the Scid.adh thymic lymphoma in vitro than either Egr1 or NF-ATc1 alone. Interestingly, the ability of Egr1 to promote this differentiation may actually depend upon NF-AT activity, because Egr1-mediated differentiation of Scid.adh is antagonized by both pharmacologic and genetic inhibition of calcineurin-mediated NF-AT activation. Taken together, we propose the following model incorporating our findings. Enforced expression of NF-AT in Scid.adh cells is able to induce partial CD25 down-regulation (Fig. 2C), which we propose represents a partial pre-TCR signal. Similar effects have been observed following Tg expression of a stabilized beta-catenin in pre-TCR-deficient thymocytes in vivo (62). Enforced expression of Egr1, by contrast, induces near complete CD25 down-modulation (Fig. 2C), but only in a subpopulation (~30–40%) of Egr1-transduced cells. Because Egr1-mediated differentiation is at least partially dependent upon NF-AT activity, we hypothesize that those cells that differentiate fully in response to enforced expression of Egr1 do so because of limited cooperativity between Egr1 and endogenous NF-AT. This presumably occurs in only a subset of cells due to differences in bioavailability of endogenous NF-AT, perhaps because fluctuations in intracellular calcium levels sequester more NF-AT in the cytosol of some cells than others. Accordingly, enforced expression of both Egr1 and NF-AT may ensure that there is sufficient bioavailable NF-AT to cooperate with Egr1 and drive the differentiation of a large fraction of the cell population.

Egr1 and NF-ATc1 also act in concert to induce traversal of the beta-selection checkpoint by normal thymocytes. Using OP9-DL1 monolayers, we observed a profound induction of development of pre-TCR-deficient thymocytes to the CD8 ISP and DP stages when Egr1 and NF-ATc1 were coexpressed. This presumably entails increases in both proliferation and survival because coexpression of Egr1 and NF-AT increased the absolute number of NF-AT-transduced cells by >1000-fold over the 11-day culture period (Fig. 3D). Although coexpression of Egr1 and NF-AT induces an impressive increase in cell number, this increase is ~10- to 15-fold less than that induced by activated Ras (RasV12), suggesting that Egr1 and NF-AT are unable to replicate a full pre-TCR signal.

What is the mechanistic basis for the concerted action of Egr1 and NF-ATc1? Evidence in support of several different mechanisms has been reported. Egr1 has been reported to directly interact with NF-ATc1 and mediate activation of IL-2 and TNF-{alpha} promoters (32). Another potential basis for the concerted action of Egr1 and NF-ATc1 is through binding to composite sites such as have been reported to be involved in cooperative activation of the CD95 ligand promoter (63). Evidence has also been reported that Egr1 and NF-AT can cooperate by binding to different sites on the promoter positioned in close proximity to each other, as in the case of the CD154 promoter (35). Because of the numerous target genes whose expression is regulated by Egr1 and NF-ATc1, several alternative mechanisms could be involved in their concerted promotion of development beyond the beta-selection checkpoint.

We have specifically explored the possibility that Egr1 and NF-ATc1 promote development beyond the beta-selection checkpoint through cooperative modulation of target genes. Two potential target genes were identified, Rag2 and Id3. Id3 is a HLH factor that has been shown to heterodimerize with the E proteins, HEB and E2A, and block their binding to DNA (56, 64). Moreover, the suppression of E protein activity has been shown to be important for traversal of the beta-selection checkpoint. Hence, it is predicted that Id3 and related products are likely to play an important role in regulating differentiation beyond the beta-selection checkpoint. It is somewhat surprising then that Id3 deficiency does not markedly disrupt early thymocyte development (54, 65). The lack of an effect of Id3 deficiency on pre-TCR-induced traversal of the beta-selection checkpoint may result from compensation by other inhibitor of DNA binding family members. Indeed, Id2 has been shown to be a target of myc, which is reportedly induced by pre-TCR signaling (66, 67). The fact that Id3 is essential in our Egr1 Tg system presumably reflects the simplified experimental system in which Egr1 provides a developmental stimulus that does not induce expression of potentially compensatory inhibitor of DNA binding family members (Fig. 7). This allows us to define a role for Id3 in Egr-mediated traversal of the beta-selection checkpoint. Accordingly, we propose a model whereby Egr1-NF-ATc1 cooperatively induces Id3 expression, which in turn promotes traversal of the beta-selection checkpoint by suppressing the activity of E proteins (54, 56, 68). We recognize that our model system represents a simplified version of the differentiation process normally induced by pre-TCR signaling and that there are likely to be additional target genes whose expression is modulated by Egr1 and NF-ATc1, which are also important effectors of the pre-TCR-induced differentiation cascade. Efforts to identify these targets are in progress.

Because Id3 is an important downstream target of Egr1 and NF-ATc1, we bioinformatically examined the sequence of the Id3 promoter reported to support normal Id3 regulation in myoblasts (69). This promoter element contained no composite Egr1/NF-ATc1 or independent NF-AT sites; however, it did contain a consensus Egr binding site. This suggests that cooperative induction of Id3 by binding of Egr1 and NF-ATc1 to composite or independent binding sites is unlikely to be responsible for Id3 induction. Nevertheless, it is possible that the regulation of Id3 expression in thymocytes requires yet-to-be-identified upstream elements at which these mechanisms might be active.

Egr1 and NF-ATc1 are independently activated by ERK-MAPK and calcium signaling, respectively. The concerted action of NF-ATc1 and Egr1 during beta-selection, in turn, may act to integrate that signaling information. This type of integration may serve not only to amplify inductive signals, but also to provide specificity.


   Acknowledgments
 
We thanks Drs. K. Campbell and D. Kappes for critical reading of the manuscript and helpful discussion, and Dr. Y. Zhuang for permission to use the Id3-deficient mice. Finally, we gratefully acknowledge the assistance of the following core facilities of the Fox Chase Cancer Center: Cell Culture, DNA Sequencing, DNA Synthesis, Flow Cytometry, and Laboratory Animal.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grants CA73656, CA87407, and CA100144; National Institutes of Health Core Grant P01CA06927; Center Grant P30-DK-50306; and an appropriation from the Commonwealth of Pennsylvania. Back

2 Address correspondence and reprint requests to Dr. David L. Wiest, Fox Chase Cancer Center, Division of Basic Sciences, Immunobiology Working Group, R390, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: DL_Wiest{at}fccc.edu Back

3 Abbreviations used in this paper: DN, double negative; CsA, cyclosporin A; DBD, DNA-binding domain; DP, double positive; Egr, early growth response; HLH, helix-loop-helix; HPC, hemopoietic precursor cell; Id1, inhibitor of DNA binding 1; Id2, inhibitor of DNA binding 2; Id3, inhibitor of DNA binding 3; IRES, internal ribosomal entry site; ISP, immature single positive; Tg, transgenic; YFP, yellow fluorescent protein. Back

Received for publication November 16, 2006. Accepted for publication July 12, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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