Zfat-Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection

The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its physiological roles in T cell development remain poorly understood. Here, we generated Zfat f/f-LckCre mice and demonstrated that T cell-specific Zfat-deletion in Zfat f/f-LckCre mice resulted in a reduction in the number of CD4+CD8+double-positive (DP) cells, CD4+single positive cells and CD8+single positive cells. Indeed, in Zfat f/f-LckCre DP cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of CD3ζ phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection through ERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide deeper insight into T cell development and immune regulation.


Introduction
ZFAT was originally identified as a candidate susceptibility gene for autoimmune thyroid disease [1]. ZFAT contains 18 zinc-finger domains and one AT-hook and is an evolutionally conserved gene from fish to humans, and the Zfat protein is highly expressed in the thymus and the spleen in adult mice [2]. We previously reported that Zfat-deficient (Zfat 2/2 ) mice are embryonic lethal by embryonic day 8.5, and Zfat is a critical transcriptional regulator for primitive hematopoiesis [3], and that ZFAT is functionally involved in the regulation of apoptosis of mouse embryonic fibroblasts and MOLT24 cells [4,5].
Just recently, we generated Zfat f/f -Cd4Cre mice [6], and showed that Zfat-deficient mice exhibited a reduction in the number of peripheral T cells with decreased surface expression of IL-7Ra and T cell antigen receptor (TCR)-stimulation-induced expression of CD25 and IL-2, indicating that Zfat is required for peripheral T cell homeostasis [6]. On the other hand, genetic variants of ZFAT have also been reported to be associated with adult height in Japanese and Korean population [7,8] and several common diseases including hypertension and cancer [9,10]. Of great interest is that a genetic variant of ZFAT is reported to be strongly associated with interferon-b responsiveness in multiple sclerosis [11] and the severity of Hashimoto's disease [12]. However, the exact functions of ZFAT during T cell development remain unknown.
T cells must be reactive to foreign pathogens, but tolerant to self-antigens. These features are formed during T cell development in the thymus [13]. CD4 + CD8 + double-positive (DP) cells expressing complete abTCR complexes undergo positive selection, for differentiation into mature CD4 + single positive (CD4SP) cells or CD8 + single positive (CD8SP) cells [14][15][16]. DP cells that recognize self-peptide and major histocompatibility complex (pMHC) molecules with low affinities receive survival signals and differentiate into mature single positive cells; this process is known as positive selection. Accumulating evidence suggests that mitogenactivated protein kinase (MAPK) signaling pathways and the molecules in this pathway play critical roles in the regulation of the cellular fate during T cell development [17]. Extracellular signalrelated kinase (ERK) is activated by phosphorylation through sequential activation of Ras, Raf and MEK transduced by TCRstimulation, and proper ERK activation is essential for positive selection [18][19][20].
Egr1, Egr2 and Egr3 are zinc-finger transcription factors of the early growth response protein (Egr) family, and they are rapidly induced in response to TCR-stimulation [21][22][23]. Egr1or Egr2deficiency was reported to result in defects of positive selection and survival of T cells [24,25]. Egr3-deficiency was reported to cause the impaired proliferation of thymocytes during transition from DN to DP cells [26]. Thus, TCR-stimulation-induced Egr expression is thought to be essential for the proper progression of T cell development in the thymus.
In this study, we generated Zfat f/f -LckCre mice and showed that they exhibited a loss of CD3f phosphorylation with dysregulation of ERK and Egr activities leading to impaired positive selection. This is the first report demonstrating that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection in the thymus.

Reduction in the Number of Thymic DP, CD4SP and CD8SP cells in Zfat f/f -LckCre Mice
To clarify the physiological roles of Zfat in T cell development in the thymus, we crossed Zfat f/f mice [6] with LckCre transgenic mice. The deletion of Zfat in Zfat f/f -LckCre thymocytes was confirmed by an immunoblot analysis. While Zfat was detected specifically in the nuclear fraction of Zfat f/f thymocytes, the protein was hardly observed in Zfat f/f -LckCre thymocytes ( Figure 1A), indicating the efficient deletion of Zfat in the thymocytes of Zfat f/f -LckCre mice. During the transition of DN stages in the Zfat f/f mice, the Zfat expression levels in the DN1 (CD25 2 CD44 + ) and DN2 (CD25 + CD44 + ) subsets was low, whereas Zfat expression in the DN3 (CD25 + CD44 2 ), DN4 (CD25 2 CD44 2 ) and DP subsets was detected at a higher level ( Figure 1B). In contrast, in Zfat f/f -LckCre mice, Zfat expression in the DN3 subset was slightly decreased compared with that of Zfat f/f mice, whereas Zfat expression in DN4 and DP subsets was apparently decreased compared with that of Zfat f/f mice ( Figure 1B). These results indicated that the Zfat expression in Zfat f/f -LckCre mice was abolished at the DN4 stage.
In Zfat f/f -LckCre mice, the proportions of CD4SP and CD8SP cells, but not DP cells, were remarkably reduced and the total number of thymocytes, DP cells, CD4SP cells and CD8SP cells was significantly decreased compared with that of Zfat f/f mice ( Figure 1C, 1D). On the other hand, the proportion and total number of DN cells in Zfat f/f -LckCre mice seemed to be slightly increased compared with those of Zfat f/f mice, however, the difference of the total number of DN cells was not statistically significant ( Figure 1C, 1D). Consistent with the decreased proportions and total number of CD4SP and CD8SP cells in the Zfat f/f -LckCre thymus, a reduction in the proportion of TCRb + T cells in both the spleen and lymph nodes (LNs) was observed in Zfat f/f -LckCre mice ( Figure 1E). The proportion and the total number of CD4 + or CD8 + T cells in the spleen and LNs were significantly reduced in Zfat f/f -LckCre mice in comparison to those of Zfat f/f mice ( Figure 1E, 1F). These results demonstrated that Zfat-deficiency results in impaired T cell development in the thymus.
In the thymus, a slight reduction in the expression levels of IL-7Ra on CD4SP and CD8SP cells in Zfat f/f -LckCre mice was observed compared with those from Zfat f/f mice ( Figure S1A). However, a difference in the expression levels of Bcl-2, a prosurvival factor induced by IL-7-mediated signaling [27,28], was not observed between the genotypes ( Figure S1B), suggesting that the reduced expression of IL-7Ra did not play a functionally significant role in the reduction of T cells in the thymus of Zfat f/f -LckCre mice.
Zfat-deficiency did not Affect the Transition of thymic DN to DP Cells As the proportion and number of DN cells in Zfat f/f -LckCre mice seemed to be slightly increased compared with those of Zfat f/f mice ( Figure 1C, 1D), we next analyzed whether LckCre-mediated Zfatdeficiency would affect the T cell development at the DN stage. The proportions and total numbers of the DN subsets, i.e., DN1, DN2, DN3 and DN4, in Zfat f/f -LckCre mice did not show significant differences with those of Zfat f/f mice, although the total numbers of DN3 and DN4 subsets seemed to be slightly elevated in Zfat f/f -LckCre mice (Figure 2A, 2B). At the DN3 stage, thymocytes undergo b-selection through the pre-TCR signaling, leading to the transition from DN3a (CD25 + CD44 2 CD27 lo FSC lo ) to DN3b (CD25 + CD44 2 CD27 hi FSC hi ) cells [29,30]. The proportion of DN3b cells was comparable between the genotypes ( Figure 2C), and the expression levels of intracellular TCRb (icTCRb) in Zfat f/f -LckCre mice were comparable to those of Zfat f/f mice during the transition from the DN3 (CD25 + icTCRb + ) to DN4 (CD25 2 icTCRb + ) stage ( Figure 2D), which together indicated that the thymocytes in Zfat f/f -LckCre mice normally passed through b-selection and transition from DN3 to DN4 cells.
We then addressed the transition from the DN4 to DP stage by analyzing an ex vivo culture system using Tst-4/DLL1 cells, which can support T cell development to the DP stage [31]. DN4 cells were sorted and then cultured on a monolayer of Tst-4/DLL1 cells. After 3 days of the culture, we confirmed a decline in Zfat expression in the thymocytes sorted from Zfat f/f -LckCre thymus ( Figure 2E), and observed the comparable proportions of the production of DP cells between Zfat f/f and Zfat f/f -LckCre thymocytes ( Figure 2F), indicating no blockade of the transition of DN4 to DP cells in Zfat f/f -LckCre mice. Taken together, Zfat-deficiency did not affect the transition of thymic DN to DP cells including bselection and transitions from DN3 to DN4 cells, and from DN4 to DP cells. In agreement with a decrease in DP cells in vivo ( Figure 1D), however, the total number of the produced DP cells of Zfat f/f -LckCre mice in ex vivo culture system was significantly reduced compared with that of Zfat f/f mice ( Figure 2G), suggesting that there may be a defect in the proliferation or survival during the transition from DN to DP stage in the Zfat-deficient thymocytes.

Impaired Positive Selection in Thymocytes of
Loss of Zfat did not affect the DN to DP transition, in spite of significant reduction in the number of DP cells. We next analyzed an involvement of Zfat in positive selection of the DP cells. During the positive selection, TCRb int CD69 2 DP cells initially show a TCRb int CD69 + transitional phenotype (P-I; Figure 3A) after the TCR/pMHC interaction. After the positive selection, P-I cells become TCRb hi CD69 + cells (P-II; Figure 3A) and then differentiate into CD4SP or CD8SP cells (TCRb hi CD69 2 ) (P-III; Figure 3A) [20]. A considerable reduction in the proportion of P-I cells in thymocytes (1.4% versus 3.87%, respectively, Figure 3A) and DP cells (1.65% versus 4.35%, respectively, Figure 3B) from Zfat f/f -LckCre mice was observed compared with that of Zfat f/f mice, indicating the existence of impaired positive selection in the Zfat-deficient DP thymocytes. However, no obvious alterations in the rearrangements of TCRachains ( Figure  S2), the surface expression of TCRb ( Figure 3C) and CD45 ( Figure 3D) [32] on DP thymocytes were observed between the genotypes. In addition, the expression levels of CD5, which is known to be correlated with the avidity of TCR/pMHC   interaction [33], on DP, CD4SP and CD8SP cells were comparable between the genotypes ( Figure 3E). These results strongly indicated that the abTCR recombination and TCR/ pMHC avidity are normal during T cell development in To further confirm the impaired positive selection in Zfat f/f -LckCre mice, we crossed ovalbumin (OVA)-specific MHC class Irestricted TCR transgenic (OT-I) [34] or MHC class II-restricted TCR transgenic mice (OT-II) [35] with Zfat f/f -LckCre mice, and examined the developmental fate of thymocytes in OT-I Zfat f/f -LckCre and OT-II Zfat f/f -LckCre mice. It is known that DP cells pass through the CD4 + CD8 int transitional stage before complete differentiation into either CD4SP or CD8SP cells [14]. As expected, not only the proportions but also the total numbers of CD8SP and CD4 + CD8 int cells were significantly decreased in OT-I Zfat f/f -LckCre mice compared with those of OT-I Zfat f/f mice ( Figure 3F), suggesting that MHC class I-restricted positive selection in OT-I Zfat f/f -LckCre mice was impaired. In addtion, MHC class II-restricted positive selection in OT-II Zfat f/f -LckCre mice was also impaired, as both the proportions and the total numbers of CD4SP and CD4 + CD8 int cells were remarkably reduced in OT-II Zfat f/f -LckCre compared with those of OT-II Zfat f/f mice ( Figure 3G). Together, these results confirmed that positive selection is impaired in the Zfat-deficient thymocytes.

Zfat-deficiency Impaired CD3f Phosphorylation with Defects in ERK1/2 Activation
To elucidate mechanisms of the impaired positive selection observed in Zfat f/f -LckCre DP cells, we examined phosphorylation of molecules working at the signaling transduced by TCRstimulation. Phosphorylations of ERK1/2 induced by TCRstimulation, which is known to be critical in the positive selection, were markedly decreased in Zfat f/f -LckCre thymocytes compared with those of Zfat f/f thymocytes (Figure 4). In agreement with the defects in ERK1/2 activation, the phosphorylations of both MEK1/2 and c-Raf, which are located upstream of the ERK signaling pathway, were also reduced in the Zfat f/f -LckCre thymocytes compared with those of Zfat f/f thymocytes (Figure 4). Furthermore, the phosphorylations of Zap70 and PLCc1 were diminished in Zfat f/f -LckCre thymocytes (Figure 4). Finally, TCR stimulation-induced phosphorylation of CD3f was virtually ablated in the Zfat f/f -LckCre thymocytes, and the phosphorylated-CD3f at non-stimulated status was also apparently diminished due to the Zfat-deficiency (Figure 4). However, phosphorylated status at Tyr505 of Lck, which phosphorylates tyrosine residues in ITAMs of CD3f in response to TCR-stimulation [36], was comparable between the genotypes. Taken together, these results indicate TCR signaling cascade is strikingly attenuated in Zfat f/f -LckCre DP cells at CD3f phosphorylation concurrent with ERK1/ 2 phosphorylation. Moreover, the impaired positive selection in Zfat f/f -LckCre DP cells is thought to be attributed to defects in the activation of ERK1/2 induced by TCR-stimulation.
TCR-stimulation-induced Egr Expressions were Impaired in the Zfat-deficient Thymocytes Next, we examined the expression levels of Egr1, Egr2 and Egr3, which are known to be induced by TCR-stimulation. Both the Egr1 and Egr2 expressions were robustly increased in the Zfat f/f DP cells after the stimulation with anti-CD3e and anti-CD28 antibodies in vitro ( Figure 5A), whereas expression levels of both Egr1 and Egr2 in the Zfat f/f -LckCre DP cells after the stimulation were not much induced compared with those of Zfat f/f DP cells. In addition, it appeared that the Egr1 expression level before the stimulation was also decreased in the Zfat f/f -LckCre DP cells compared with that of the Zfat f/f DP cells ( Figure 5A). On the other hand, Egr3 was constitutively expressed and slightly increased in the Zfat f/f DP cells after the stimulation, whereas the expression levels of Egr3 in the Zfat f/f -LckCre DP cells were constitutively lower compared with those of Zfat f/f DP cells and rarely enhanced by TCR-stimulation ( Figure 5A). These results indicated that Zfat-deficiency causes dysregulated expression of Egr protein in the DP cells before and after TCR-stimulation. Similar experiments were performed on the thymocytes from OT-II Zfat f/f -LckCre mice. Phosphorylation of ERK induced by TCR-stimulation in the thymocytes from OT-II Zfat f/f -LckCre mice was decreased compared with that of OT-II Zfat f/f mice ( Figure S3A). Furthermore, the levels of TCR-stimulation-induced Egr1, Egr2 and Egr3 expression in OT-II Zfat f/f -LckCre thymocytes were all decreased compared with those of OT-II Zfat f/f mice ( Figure S3B), and together these findings suggested that the impaired phosphorylation of ERK and Egr expression induced by TCR-stimulation was caused by molecules other than TCR itself.
To address whether MEK activation is essential for ERK mediated Egr inductions under the TCR-stimulated condition, we stimulated DP cells in the presence of U0126, an inhibitor of MEK1/2 [37]. We found that the inductions of Egr1 and Egr2 were considerably reduced by the treatment with U0126 both in Zfat f/f and Zfat f/f -LckCre DP cells ( Figure 5B), indicating that the enhancements of Egr1 and Egr2 expression by TCR-stimulation were essentially regulated by the MEK-ERK axis. On the other hand, the Egr3 induction was just partly reduced by the treatment with U0126 in Zfat f/f DP cells, whereas the Egr3 expression in the Zfat f/f -LckCre DP cells seemed to be slightly increased or unaltered by TCR-stimulation in the presence of U0126 ( Figure 5B). These results suggested the possibility that Egr3 expression may be regulated by other signaling pathways in addition to the MEK-ERK pathway.
While the levels of Egr mRNA expression in the unstimulated DP cells were comparable between the Zfat f/f and Zfat f/f -LckCre genotypes ( Figure 5C), each of the Egr1, Egr2 and Egr3 mRNA inductions by TCR-stimulation were robustly suppressed in Zfat f/f -LckCre DP cells compared with those of Zfat f/f DP cells ( Figure 5D), suggesting that Zfat is required for proper Egr gene expression induced by TCR-stimulation. However, considering the fact that the expressions of Egr proteins were slightly decreased in the unstimulated Zfat f/f -LckCre DP cells ( Figure 5A), Zfat-deficiency might affect the turnover or degradation of Egr proteins as well as the expression of Egr mRNAs. Taken together, these results indicated that impairment of the TCR-stimulation-induced Egr inductions in the Zfat-deficient thymocytes leads to the defects of positive selection.

Discussion
In this study, we demonstrated that Zfat is required for the proper phosphorylation of CD3f, and is a critical regulator for T cell development in the thymus, especially for positive selection. The molecular mechanism by which Zfat-deficiency leads to impaired positive selection would be mainly dependent on the loss of CD3fphosphorylation leading to the impaired ERK activation transduced by TCR-stimulation. Furthermore, activated ERKmediated expression of Egr1 and Egr2, which are critical regulators for positive selection, were also decreased in the Zfat f/ f -LckCre thymocytes. Taken together, these results suggest that Zfat is involved in the proper regulation of the TCR-proximal signalings, which is required for positive selection in the thymus.
Zfat f/f -LckCre mice showed a considerable reduction in the number of DP thymocytes. We could not find apparent defects in T cell development at DN stage or transition of DN to DP cells in Zfat-deficient thymocytes in vivo and ex vivo experiments. Therefore, the reduction in the number of DP cells in Zfat f/f -LckCre mice may be due to the Zfat-deficiency at the DP stage. However, Zfat expression was not completely abolished in the Zfat f/f -LckCre DN3 thymocytes, and thus the possibility that Zfat is necessary for proper development at the DN stage cannot be excluded. Impaired positive selection in Zfat-deficient thymocytes was not restored in the presence of OT-I TCR or OT-II TCR transgene, which promote positive selection and differentiation of DP cells into CD8SP or CD4SP cells. These results strongly indicated that positive selection is impaired in Zfat-deficient mice, and also suggested that the defect of positive selection is caused by the dysregulated signaling downstream of TCR itself. TCR-stimulation triggers activation of the ERK pathway through sequential activation of Ras, Raf and MEK [17]. Activation of ERK is critical in the TCR-mediated signaling cascades and an essential requirement for positive selection in the thymus [19,20].
Both Egr1 and Egr2 expression are critically regulated by activated ERK transduced by TCR-stimulation and play pivotal roles in positive selection and survival of DP cells [24,25,38]. Intriguingly, we identified the defects of TCR-stimulated ERK phosphorylation and Egr inductions in Zfat-deficient DP thymocytes, indicating that impaired Egr induction was at least partially responsible for the defects of positive selection in Zfat f/f -LckCre mice. Moreover, Egr3 was also dysregulated in Zfat-deficient thymocytes. Egr3-deficient mice have been reported to exhibit a defect in the thymocytes proliferation and a partial block in differentiation at the DN3 stage [26]. Therefore, a possibility that Zfat plays an important role in the proliferation of thymocytes during the DN to DP transition through Egr3 induction is not excluded.
Decreased phosphorylation of CD3f in Zfat f/f -LckCre thymocytes induced by TCR-stimulation was observed, indicating that Zfat-deficiency results in impaired activation of TCR signaling at proximal level. Tyrosines in ITAMs of CD3f are phosphorylated by Src family kinase Lck, and then the tyrosine-phosphorylated ITAMs of CD3f serve as docking sites for Zap70 in response to TCR stimulation [36]. However, Zfat-deficiency did not affect phosphorylation status of Lck in the thymocytes, whereas phosphorylation of Zap70 was reduced in Zfat f/f -LckCre thymo- cytes. We have not elucidated how exactly Zfat affects the CD3fphosphorylation in this study. Activation of CD3f is negatively regulated by SHP21 and SHP22 (SH2 domaincontaining phosphatase-1 and -2) through dephosphorylation [39,40]. Moreover, c-Cbl E3 ubiquitin ligase reduces CD3f levels at the plasma membrane by stimulating internalization [39,41]. Considering that Zfat is expected to be a transcriptional regulator in the nucleus [2,3], Zfat might affect the expressions of the genes involved in the regulation of CD3f phosphorylation, such as SHP-1, SHP-2 and c-Cbl. However, elucidation of the precise molecular mechanisms of Zfat function in regulation of TCR signaling should await future studies.
Proper activation of TCR signaling is also required for negative selection in the thymus. Thus, Zfat might be involved in the negative selection since Zfat-deficiency results in the defect in proximal molecules of TCR complex. However, we have not seen obvious differences in the T cell developments between H-Y Zfat f/f and H-Y Zfat f/f -LckCre male mice in preliminarily experiments (data not shown). To establish a role for Zfat in the negative selection, further studies should be required in the future.
In conclusion, we demonstrated that Zfat-deficiency in DP cells results in a loss of CD3f phosphorylation with dysregulation of ERK and Egr activities leading to impaired positive selection in the thymus, suggesting that Zfat is a pivotal molecule in T cell development. As the activation of ERK-Egr pathway was not completely impaired in the Zfat-deficient thymocytes, the possibility that Zfat plays crucial roles in particular signaling pathways other than ERK-mediated pathway does exist. Thus, a full understanding of the roles and precise molecular mechanisms of the transcriptional regulator Zfat will lead to a better understanding of the orchestrated gene expression programs and provide deeper insight into T cell development, immune regulation and a wide variety of diseases.

Mice
Zfat f/f mice were originally generated as described previously [6] and backcrossed to a C57BL/6 background over seven generations. Zfat f/f mice were crossed to Lck-Cre mice from Taconic to generate T-cell-specific Zfat knockout (Zfat f/f -LckCre) mice in the C57BL/6 background. All the animal experiments were approved by the Animal Care and Use Committee of the NCGM Research Institute, and the experiments on mice were carried out under the guidelines of the Institutional Animal Care and Use Committee of Fukuoka University.

Statistical Analysis
Data are presented as the means 6 s.d. and statistical analysis was performed using an unpaired Student's t-test when comparing the means of two groups. Differences of P,0.05 were considered to be statistically significant (*P,0.05; **P,0.01).

Accession Number
GenBank mRNA sequences: Zfat, NT_078782. Figure S1 Surface expression of IL-7Ra and intracellular expression of Bcl-2. Flow cytometry analysis of the expression of surface IL-7Ra (A) and intracellular Bcl-2 (B) in the DP, CD4SP and CD8SP cells from Zfat f/f (gray-filled) and

Supporting Information
Zfat f/f -LckCre (black line) mice at 6 to 7 weeks of age (left). Data of thymocytes from Zfat f/f (black bar) and Zfat f/f -LckCre (white bar) mice were measured as the mean fluorescence intensity (MFI) (right). The fluorophore-conjugated antibodies used for flow cytometry analysis and their specificities were as follows: IL-7Ra (A7R34) and Bcl-2 (10C4; all from Biolegend). Data are representative of three independent experiments. The data are the mean 6 s.d.; * P,0.05; ** P,0.01; n.s., not significant.