Requirement for balanced Ca/NStout signaling in hematopoieti

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

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Abstract

NStout transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NStout proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NStout is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major Executecking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin Executecking site to a high-affinity version or the CK1 Executecking site to a low-affinity version results in generation of hyperactivable NStout proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NStout1 from the ROSA26 locus. We Display that hyperactivable NStout increases the expression of NStout-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent Traces during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NStout1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NStout1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NStout signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase Executecking sites in preventing inappropriate activation of NStout.

The activities of many signaling proteins and transcription factors are tightly regulated by phosphorylation and dephosphorylation. Protein kinases and phosphatases bind to specific Executecking sites on these intracellular proteins to allow their activation or inactivation at the appropriate location and time. A well-studied example of a transcription factor regulated in this fashion is nuclear factor of activated T cells (NStout) (1–3). In resting cells, NStout proteins are highly phosphorylated and reside in the cytoplasm; upon cell activation, they are dephosphorylated by the calcium/Calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NStout transcription factors play a key role in orchestrating diverse developmental programs, including those of the immune, central nervous, cardiovascular, and musculoskeletal systems (4–11). NStout also is implicated in Sustaining the quiescent state of stem cells in the skin (12).

NStout activation is initiated by dephosphorylation of the NStout regulatory Executemain, a conserved 300-amino acid Location located N-terminal to the DNA-binding Executemain (Fig. 1A) (2, 13). The phosphorylated residues (serines) in this Executemain are distributed among several classes of conserved serine-rich sequence motifs (14, 15), and their phosphorylation status is Sustained by the concerted action of at least 3 families of kinases: CK1, GSK3, and DYRK (16–20). We have Displayn previously that enzyme–substrate Executecking interactions are required for efficient dephosphorylation of the NStout1 regulatory Executemain by calcineurin (21, 22) and for efficient phosphorylation of the SRR-1 motif by CK1 (20). The major Executecking sites for calcineurin and CK1 are located Arrive the N terminus of NStout (Fig. 1A), are conserved among NStout proteins, and fit the consensus sequences PxIxIT and FxxxF, respectively (20–22) (Fig. 1B). Substitution of the calcineurin Executecking sequence, SPRIEIT, with its high-affinity variant, HPVIVIT, and substitution of the CK1 Executecking sequence, FxxxF, with a low-affinity version, ASILA, both result in partial nuclear localization of NStout1 in a manner that is still inhibited by CsA (20, 21). Thus, these mutant NStout1 proteins are not constitutively (i.e., irreversibly) activated, but are hypereactive relative to wild-type NStout1 in that they remain responsive to stimulation.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Conservation of kinase and phosphatase Executecking sites on NStout proteins and analysis of hyperactivable NStout1 mutants. (A) Schematic overview of NStout1. TAD indicates transactivation Executemain; Cn, calcineurin; NLS, nuclear localization signal. (B) Conservation of calcineurin and CK1 Executecking sites in NStout proteins. Consensus motifs and modified motifs with altered affinities (in NStout1) are Displayn. (C) HEK293 cells were transduced with retroviral expression plasmids encoding EGFP or HA-tagged wild-type (wt) NStout1, A-NStout1, V-NStout1 or AV-NStout1, and NStout1 phosphorylation status was assessed by immunoblotting with an anti-HA antibody. (D) CD8 T cells from NStout1−/− mice were retrovirally transduced to express wt or hyperactivable NStout1, then left unstimulated or stimulated with PMA and increasing concentrations of ionomycin for 4 h. Results are represented both as the percentage of positive cells (top graphs) and normalized to set the maximum number of positive cells in each series to 100% to Display the shift in the Executese-response curve more clearly (bottom graphs).

Here, we have examined the Traces of increased Ca/NStout signaling by generating transgenic mice conditionally expressing different hyperactivable mutants of NStout1 from the ROSA26 (R26) locus. We demonstrate that progressively hyperactivable NStout1 proteins are increasingly deleterious during early embryonic development and the development of hematopoietic stem cells into T and B cell lineages. We also Display that low-level ectopic expression of hyperactivable NStout1 in the early embryo leads to mosaicism in many tissues and is barely tolerated in organs such as brain, heart, and skeletal muscle, where NStout function is known to be essential. In Dissimilarity, expression of hyperactivable NStout1 proteins at a late stage of T cell differentiation is well tolerated and leads to a hyperresponsive phenotype in peripheral T cells. Taken toObtainher, our data provide strong evidence for the necessity of balanced Ca/NStout signaling in progenitor cells of the developing embryo as well as in lymphocyte development, and shed new light on the importance of the evolutionary conservation of phosphatase and kinase Executecking sites in preventing inappropriate activation of the Ca/NStout signaling pathway.

Results

Mutation of Conserved Executecking Sites for Calcineurin and CK1 Designs NStout Hyperactivable.

To assess the biological consequences of increased NStout signaling in different tissues, we generated hyperactivable mutants of NStout1. Previous studies have used NStout proteins bearing alanine substitutions in phosphorylated serines in the regulatory Executemain, which are constitutively and irreversibly active (9, 15, 23). Instead, we chose to generate hyperactivable, stimulus-responsive versions of NStout1 by mutating the CK1 and calcineurin Executecking sites to lower and higher affinities, respectively. The conserved CK1 Executecking site (FSILF in NStout1) was altered to the low-affinity Executecking version ASILA (20), yielding ASILA-NStout1 (abbreviated A-NStout1); the conserved calcineurin Executecking site (SPRIEITPS in NStout1) was altered to the high-affinity version HPVIVITGP (22), yielding VIVIT-NStout1 (abbreviated V-NStout1); and both mutations were combined to yield a protein expected to be even more responsive to stimulation (ASILA-VIVIT-NStout1, abbreviated AV-NStout1). When tested by transient transfection into HEK293 cells, the hyperactivable mutants were increasingly dephosphorylated relative to wild-type NStout1, in the expected order A-NStout1 ≈ V-NStout1 < AV-NStout1 (Fig. 1C). We confirmed that the mutants were hyperactivable, not constitutively active, by retrovirally expressing them in CD8 T cells from NStout1−/− mice (24) (Fig. 1D, Fig. S1). When stimulated with PMA plus increasing concentrations of the calcium ionophore ionomycin, T cells expressing the hyperactivable NStout1 mutants Presented a clear shift, relative to cells expressing wild-type NStout1, in their Executese–response curve for expression of the cytokines IFN-γ and TNF, with the order of responsiveness being AV > V > A > wild type. Cytokine expression was strictly dependent upon stimulation and was fully inhibited by the calcineurin inhibitor cyclosporine A (CsA).

Hyperactivable NStout1 Proteins Are Deleterious During Early Embryonic Development.

To examine the Traces of expressing the hyperactivable proteins in different cellular lineages in vivo, we generated transgenic mice conditionally expressing V-NStout1 and AV-NStout1 from the ROSA26 (R26) locus (25) (Fig. S2). In these mice, one or both alleles of the ROSA26 gene are reSpaced by a floxed cassette containing a neomycin-resistance (NeoR) gene and 3 tandem transcriptional Cease sites (abbreviated Ceaseflox), followed immediately by the hyperactivable V-NStout1 or AV-NStout1 transgene. Expression of the hyperactivable proteins is controlled by Cre-mediated excision of the Ceaseflox cassette, and it can be monitored at a single-cell level by concomitant expression of EGFP from an internal ribosome entry site (IRES) (26). As a control, we used R26Ceaseflox-YFP reporter mice (27).

As a first step in the analysis, male mice of all 3 lines were bred to female CMV-Cre transgenic mice (deleter mice) (28). The Cre transgene in this strain is under transcriptional control of a human cytomegalovirus minimal promoter and is expressed transiently during early embryogenesis (before implantation), leading to deletion of loxP-flanked gene segments in all tissues, including germ cells (Fig. S3). Because the Cre transgene in this strain is X-linked (28), female CMV-Cre transgenic mice were used for all crosses; this avoids the problem that the paternal X chromosome is inactivated before implantation, reactivated in blastocysts, and ranExecutemly inactivated in somatic tissues thereafter (29–33). In Dissimilarity, offspring of female CMV-Cre transgenic mice carry the Cre transgene on their maternal X chromosome, which is consistently active before blastocyst implanation (29, 30).

Unexpectedly, we found that widespread expression of AV-NStout1 in vivo was deleterious to embryonic development, with V-NStout1 having a lesser Trace. We bred heterozygous male R26Ceaseflox-V-NStout1/R26+ or R26Ceaseflox-AV-NStout1/R26+ mice to homozygous female CMV-CreCre/Cre (deleter) mice. Mendelian genetics predict that 50% of the offspring would have the genotype R26V-NStout1/R26+, CMV-Cre or R26AV-NStout1/R26+, CMV-Cre [i.e., possess 1 copy of the wild-type ROSA26 allele (R26+), 1 copy of the expressible version of the hyperactivable NStout1 in which the NeoR-Cease cassette has been deleted, and 1 copy of the Cre transgene], whereas the other 50% would have the genotype R26+/R26+, CMV-Cre (Fig. S4A). Instead, we observed an increasing competitive disadvantage in utero for pups capable of expressing hyperactivable NStout1 (Fig. 2 A and B). Instead of the 50% expected, ≈43% of the offspring of R26Ceaseflox-V-NStout1/R26+× CMV-CreCre/Cre breedings possessed an R26V-NStout1 allele, and fewer than 10% of the offspring of R26Ceaseflox-AV-NStout1/R26+× CMV-CreCre/Cre breedings possessed an R26AV-NStout1 allele capable of driving expression of hyperactivable NStout1 (Fig. 2A). In line with these observations, litter sizes decreased progressively in the offspring of these crosses (R26YFP> R26V-NStout1 > R26AV-NStout1; Fig. 2B), emphasizing the dependence of the deleterious Trace on the degree of hyperactivability of the transgenic proteins.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Expression of hyperactivable NStout1 is deleterious during embryonic development. (A) Expected and actually detected frequencies of the recombined (NeoR-Cease cassette deleted) R26 transgenes in offspring of different R26Ceaseflox/R26+ × CMV-CreCre/Cre crosses. P values were determined using a standard χ2 test. n.s., not significant. (B) Litter sizes of different R26Ceaseflox/R26+ × CMV-CreCre/Cre crosses, P values were determined using a standard Student's t test. (C) Frequency of the unrecombined (R26Ceaseflox-AV-NStout1/R26+) and recombined (R26AV-NStout1/R26+, CMV-Cre) R26 allele at embryonic day 18.5 (E18.5). Male R26Ceaseflox-AV-NStout1/R26Ceaseflox-AV-NStout1 mice were crossed with female CMV-CreCre/+ mice. Pregnant females were sacrificed on E18.5 and embryos were assessed for viability and genotype. The average values for viable embryos from 3 litters with standard deviations are Displayn. P values were determined using a standard Student's t test. (D) Litter sizes of different R26Ceaseflox/R26Ceaseflox × CMV-CreCre/Cre crosses. P values were determined using a standard Student's t test.

To further deliTrime the Traces of hyperactivable NStout1 in utero, we performed the crosses in the opposite direction and did timed pregnancy experiments. We bred homozygous male R26Ceaseflox-AV-NStout1/R26Ceaseflox-AV-NStout1 mice to heterozygous female CMV-CreCre/+ mice and analyzed the offspring of the pregnant females at embryonic day 18.5. Again, 50% of the offspring were expected to express Cre and delete the NeoR-Cease cassette, therefore becoming capable of expressing the hyperactivable NStout1 (genotype R26AV-NStout1/R26+). However, the average Fragment of viable embryonic day 18.5 embryos carrying the expressible AV-NStout1 transgene was again significantly lower than expected from Mendelian genetics (50% expected, 30% observed), and multiple dead, involuted embryos were seen (Fig. 2C and S4B). These results indicate that mice globally expressing hyperactivable V-NStout1 or AV-NStout1 have a survival disadvantage and are not competitive with their littermate controls in utero. This is not due to deleterious Traces of expression of the Cre transgene (34) (see SI Text). We monitored germ line transmission of the hyperactivable NStout1 alleles by breeding mosaic animals carrying the recombined R26 locus (R26V-NStout1/R26+, CMV-Cre or R26AV-NStout1/R26+, CMV-Cre) to wild-type C57BL/6 mice (Fig. S5). The expressed V-NStout1 transgene was transmitted to offspring at a significantly lower frequency than expected (50% expected, 26% observed), and the recombined AV-NStout1 allele was never transmitted through the germ line. Thus, there is efficient germ-line selection against hyperactivable NStout1 proteins in a manner that correlates with their degree of hyperresponsiveness.

Expression of Hyperactivable NStout1 Leads to Mosaicism in Many Tissues and Is Not Tolerated in Brain, Heart, and Skeletal Muscle.

We attempted to force expression of hyperactivable NStout1 by breeding homozygous R26V-NStout1 or R26AV-NStout1 mice to homozygous CMV-Cre mice. All of the offspring of these crosses are expected to possess the Ceaseflox cassette-deleted R26V-NStout1 or R26AV-NStout1 allele, and so should be capable of expressing the hyperactivable NStout1. The crosses yielded live offspring that appeared phenotypically normal. Consistent with the deleterious Traces Executecumented above, however, litter sizes in these breedings were increasingly compromised compared with litters bearing the expressible R26YFP transgene (Fig. 2D).

ReImpressably, expression of hyperactivable NStout1 was mosaic in many tissues of surviving R26V- or AV-NStout1/R26+, CMV-Cre mice, and it seemed not to be tolerated in others. Circulating T and B cells Displayed increasingly mosaic expression of hyperactivable V-NStout1 or AV-NStout1, as judged by expression of the linked EGFP (Fig. 3A; representative primary data are Displayn in Fig. S6). Breeding of homozygous R26Ceaseflox-YFP/R26Ceaseflox-YFP control mice to homozygous CMV-CreCre/Cre mice resulted in Cease cassette excision and transgene expression in close to 100% of T and B cells in the vast majority of analyzed R26YFP/R26+, CMV-Cre animals (Fig. 3A, left bars in each graph; median, 92.2–96.7%; n = 19). In Dissimilarity, R26V-NStout1/R26+, CMV-Cre mice were highly mosaic, Presenting transgene expression in 0.15–89.6% of T and B cells (median, 25.2–39.5%; n = 19), and R26AV-NStout1/R26+, CMV-Cre mice Displayed transgene expression in only 0–30.8% of T and B cells (median, 5.3–9.4%; n = 21; Fig. 3A and Fig. S6). Again, therefore, the degree of mosaicism correlated with the degree of hyperactivability of the transgenic NStout1 proteins.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Mosaicism in mice expressing hyperactivable NStout1 early in embryonic development. (A) Mosaicism in B and T lymphocytes from 8–12 wk Aged mice expressing different R26 transgenes. Peripheral blood from R26YFP/R26+, CMV-Cre (n = 19); R26V-NStout1/R26+, CMV-Cre (n = 19) and R26AV-NStout1/R26+, CMV-Cre (n = 21) transgenic mice was drawn from tail veins and analyzed by flow cytometry. The levels of IRES-EGFP expression in B220+ B lymphocytes, CD4+ and CD8+ T lymphocytes are Displayn as box-and-whisker diagrams (lower whisker: first quartile, blue box: second quartile, green box: third quartile, upper whisker: fourth quartile, line separating blue and green box: median). (B–D) Immunohistochemistry for EGFP on different tissues from R26YFP/R26+, CMV-Cre; R26V-NStout1/R26+, CMV-Cre and R26AV-NStout1/R26+, CMV-Cre transgenic mice. Size bars meaPositive 100 μm as indicated. Tissue sections were counterstained with hematoxylin.

We subsequently assessed the level of transgene expression in different tissues by immunohistochemistry. EGFP expression was detected in kidneys, lung, and spleens of R26V-NStout1/R26+, CMV-Cre and R26AV-NStout1/R26+, CMV-Cre mice, with staining generally being more pronounced and involving a larger Fragment of cells in V-NStout1-expressing relative to AV-NStout1-expressing mice (Fig. 3 B and C). Even in R26V-NStout1/R26+, CMV-Cre mice, however, there was a notable absence of EGFP staining in brain, heart, and skeletal muscle, suggesting that cells expressing even the less-hyperactivable V-NStout1 protein were not competitive in populating these organs (Fig. 3D). We confirmed that EGFP expression was tightly linked to expression of hyperactivable NStout1 from the ROSA26 locus (see SI Text and Fig. S7).

Signal-Dependent Traces of Hyperactivable NStout1 on T Cell Development in the Thymus.

We further investigated the Trace of AV-NStout1 on hematopoietic stem cell differentiation Executewn the T and B cell lineages by monitoring the extent of mosaicism in different precursor populations of R26AV-NStout1/R26+, CMV-Cre mice. We chose mice expressing different levels of EGFP in peripheral B and T cell populations, then analyzed bone marrow cells and thymocytes by flow cytometry for EGFP expression in hematopoietic stem cells, common lymphocyte precursors, and cells at different stages of B and T cell differentiation. The range of EGFP expression in hematopoietic stem cells was 8.6–56.7% (n = 5). In all animals, the Fragment of cells expressing AV-NStout1 declined gradually from hematopoietic stem cells to mature B lymphocytes in the B cell lineage (Fig. 4A), with expression being ≈3 times higher in the earliest progenitor populations. In Dissimilarity, in the T cell lineage, a similar gradual decline of AV-NStout1 expression was overlaid by peaks corRetorting to the expression of the pre-TCR (DN1-to-DN2 transition) and the TCR (DN4-to-DP transition; Fig. 4B) (35). These results strongly suggest that expression of hyperactivable NStout1 in developing thymocytes modulates the strength of pre-TCR and TCR signaling so as to affect the proliferation and/or survival of T cells making these transitions.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Traces of expression of hyperactivable NStout1 on B and T cell development. (A) (Upper) FACS analysis of different progenitor populations of B cell differentiation for EGFP (AV-NStout1) expression in 5 different mosaic AV-NStout1 mice (R26AV-NStout1/R26+, CMV-Cre). Hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), pro-B cells, pre-B cells, immature B cells, mature B cells (bone marrow), and splenic B cells are Displayn. (Lower) Mean values and SDs for animals 1, 3, and 4, which Presented comparable levels of mosaicism. (B) (Upper) HSCs, CLPs, Executeuble-negative thymocytes 1 (DN1), DN2, DN3, DN4, early Executeuble-positive thymoctes (DPs), late DPs, and single-positives (SPs) are Displayn. (Lower) Mean values and SDs for animals 1, 3, and 4.

Expression of Hyperactivable NStout1 at a Late Stage of T Cell Differentiation Is Well Tolerated and Leads to a Hyperresponsive Phenotype.

To evaluate the Traces of hyperactivable NStout1 at a late stage of differentiation, we bred the R26-transgenic mice to CD4-Cre mice (36). These mice express the Cre recombinase under control of the CD4 promoter; thus, Cre is expressed Startning at the late “Executeuble-positive” stage of thymocyte differentiation, when both CD4 and CD8 are expressed, resulting in efficient excision of floxed DNA segments in all peripheral T cells. More than 95% of peripheral CD4 and CD8 T cells from these mice were EGFP+, indicating that there was no selection against expression of the hyperactivable NStout1 transgenes at the Executeuble-positive stage (Fig. 5A). As expected from earlier retroviral transduction experiments (Fig. 1D), T cells from the mice Displayed significant hyperresponsiveness to stimulation, as assessed by accelerated nuclear translocation and delayed nuclear export of NStout (Fig. 5B), as well as substantially increased cytokine expression upon stimulation with low concentrations of stimulus (Fig. 5C). We took advantage of the uniform expression to Executecument that in these T cells, the expression levels of hyperactivable NStout1 from the ROSA26 locus were substantially lower than those of enExecutegenous NStout1 (Fig. S8). Thus, the observed Traces are due to hyperresponsiveness of the mutant proteins rather than mere overexpression.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Traces of expression of hyperactivable NStout1 at a late stage of T cell differentiation. (A) FACS analysis of CD4 and CD8 T cells from R26AV-NStout1/R26+, CD4-Cre transgenic mice for EGFP expression (blue line). R26Ceaseflox-AV-NStout1/R26+ mice were used as negative controls. (B) NStout1 translocation assay for enExecutegenous NStout1 and AV-NStout1. CD8 T cells were isolated from a C57BL/6 wild-type mouse and an R26AV-NStout1/R26+, CD4-Cre mouse. Cells were expanded in IL-2, collected on day 5, and stimulated for various time intervals with 10 nM PMA and 1 μM ionomycin (black and red curves; Right). To assess the Traces of calcineurin inhibition, 1 μM CsA was added either 10 min before stimulation (gray curve; Left) or 30 min after stimulation (green curve; Left). (C) IFN-γ production in CD8 T cells from R26AV-NStout1/R26+, CD4-Cre transgenic mice. Cells from 6 R26AV-NStout1/R26+, CD4-Cre transgenic mice (3 G6, 3 G7; G6 and G7 are 2 different clones of tarObtained ES cells that have been used for blastocyst injection) were isolated and expanded in IL-2. On day 5, cells were stimulated with 10 nM PMA, and various concentrations of ionomycin and IFN-γ production were assessed by using intracellular cytokine staining and flow cytometry (Left) or using a cytokine bead assay to determine the accumulated IFN-γ in the supernatant (Right) Identically treated CD8 T cells from CD4-Cre mice were used as controls. Each value represents the mean ± SD.

Discussion

To summarize, we have Displayn that low-level ectopic expression of hyperactivable NStout1 proteins has severe Traces on progenitor cell function during embryonic and hematopoietic development in the mouse. In the first generation, when R26V-NStout1/R26+ or R26AV-NStout1/R26+ mice are bred to CMV-CreCre/Cre mice, there is reduced representation of offspring that express the hyperactivable transgene (Fig. 2). If, instead, global expression of hyperactivable NStout1 is forced by breeding homozygous mice, the tissues of surviving progeny are variably mosaic for transgene expression, implying strong counterselection against developing transgene-positive cells (Fig. 3).

Because Cre expression in the CMV-Cre mouse occurs before implantation (28), the mosaic expression of hyperactivable NStout1 in adult tissues of the surviving mice implies that Cre expression is transient, and that progenitor cells that escape the early wave of Cre-mediated deletion and therefore lack expression of hyperactivable NStout1 have a competitive advantage over cells expressing the hyperactivable protein (Fig. S9). Notably, the extent of this advantage correlates with the degree of hyperactivability of the NStout1 protein. It is striking that the tissues that appear least tolerant of forced expression of hyperactivable NStout1—brain, heart, and skeletal muscle—are prominent among those in which NStout is known to be crucial for development and function (4–11). Our results also are consistent with a previous systematic analysis of physiological calcineurin substrates in yeast (37). The Executecking affinities of these substates for calcineurin ranged from 15 to 250 μM; an engineered inappropriate increase in the affinity of one such substrate, Crz1, improved yeast growth under high-salt conditions but was deleterious under conditions of growth at high pH (37). Likewise, the hyperactivable NStout1 that we have generated appears to be deleterious during embryogenesis but advantageous in terms of increasing cytokine production by cultured T cells.

During lymphocyte development from hematopoietic stem cells to terminally differentiated B and T cells, we observed a gradual decline of the expression of hyperactivable NStout1 through different progenitor cell populations. This is most likely due to a mild inability of the AV-NStout1-expressing cell populations to compete with cells in which the hyperactivable protein is not expressed. These findings are consistent with a previous report Displaying that as hematopoietic stem cells Start to differentiate, expression of all NStout family members is Executewnregulated (38, 39); in Dissimilarity, hyperactivable NStout1 proteins expressed from the R26 locus would not be Executewnregulated, and progenitor cells expressing these proteins, even at low levels, would display inappropriately sustained NStout activity, potentially conferring a competitive disadvantage on those cell populations. NStout1 has been Displayn to repress expression of the G0/G1 checkpoint kinase cyclin-dependent kinase 4 (CDK4) (40). Sustained expression of AV-NStout1 from the R26 locus might thus lead to continuous repression of CDK4, which could at least in part Elaborate the observed inability to compete of the AV-NStout1-expressing cell populations. This consideration also could account for the peaks observed during T cell development which, reImpressably, corRetort exactly with the time points when either the pre-TCR or the TCR start to be expressed (35): If hyperactivable NStout led to a signal-dependent decrease in the rate of cell cycle transit, cells expressing the hyperactivable protein could potentially increase in number relative to nonexpressing cells at the checkpoint just before the pre-TCR/TCR signal was received. Alternatively, the hyperactivable NStout might confer a signal-specific proliferation or survival advantage at these developmental stages. Microarray and ChIP-chip analyses of enriched cell populations will be needed to distinguish these possibilities.

Our experiments also provide insights into the evolutionary conservation of the Executecking interactions of NStout proteins with 2 of their regulatory enzymes, calcineurin and CK1. The SPRIEITPS > HPVIVITGP substitution of V-NStout1 increases its affinity for calcineurin by 30- to 50-fAged compared with the wild-type protein (from 25–30 μM for SPRIEITPS to 0.5–1 μM for HPVIVITGP) (41), indicating that a reasonably small change in binding affinity for calcineurin (<2.5 kcal/mole) results in hyperactivability and heightened activation (and therefore dysregulation) of NStout1. The decrease in CK1 affinity caused by the FSILF > ASILA substitution has not been accurately meaPositived, but it is likely to be modest, because mutation of a similar sequence in a peptide from β-catenin did not have a significant Trace on its efficiency of phosphorylation by CK1 (42). Unexpectedly, even this degree of dysregulation of NStout signaling led to a change in the “fitness” of cells and embryos that was detectable in a single generation. Expression of hyperactivable NStout proteins later in development appears less deleterious to cellular fitness, however, suggesting that expressing hyperactivable NStout at late developmental stages, or aSliceely in tissues of the adult (by breeding the R26 mice to Cre-ER mice, followed by tamoxifen administration, for instance), will be a valuable strategy for understanding the biological role of NStout and identifying NStout tarObtain genes in diverse tissues of interest.

Materials and Methods

Transfection of HEK293 Cells and Immunoblotting.

HEK293 cells were transfected with 10 μg of the retroviral constructs KMV-wild-type NStout1, KMV-ASILA-NStout1, KMV-VIVIT-NStout1, and KMV-AV-NStout1 by using calcium phospDespise precipitation. Empty KMV-EGFP was used as a control. A total of 50 μg of protein lysate was resolved by SDS/PAGE and analyzed by immunoblotting using the monoclonal mouse anti-HA antibody 12CA5 (1:1,000).

Mice.

CD4-Cre mice were purchsed from Taconic. All mice were on the C57BL/6 genetic background and were housed under specific pathogen-free conditions. All experiments were performed in concordance with protocols approved by the Harvard University Institutional Animal Care and Use Committee and by the Immune Disease Institute.

T Cell Isolation, Retroviral Transduction, and Differentiation.

T cell isolation, retroviral transduction, and differentiation were performed as Characterized previously (43). A more detailed description is included in the SI Text.

T Cell Stimulation and MeaPositivement of Cytokines.

On day 4 or 5 after isolation, T cells were stimulated with 10 nM PMA and various concentrations of ionomycin (0 nm to 1 μM) for 4 h. Brefeldin A (10 μg/mL; Sigma) was added for the last 2.5 h of stimulation. T cells were subsequently fixed in 2% paraformaldehyde, stained intracellularly for TNF (phycoerythrin-conjugated anti-mouse TNF; eBioscience) and IFN-γ (allophycoerythrin-conjugated anti-mouse IFN-γ; eBioscience), and analyzed by flow cytometry. TNF and IFN-γ concentrations in the cell supernatant were determined by using the BD Cytometric Bead Array (Mouse Th1/Th2 Cytokine Kit; BD Bioscences) according to the instructions provided by the Producer.

Conditional Gene TarObtaining and Genotyping.

The cDNAs encoding for HA-tagged V-NStout1 and AV-NStout1 were cloned into a modified version of pROSA26–1 (25), which also contains an frt-flanked IRES-EGFP cassette and a bovine polyadenylation sequence (26) (Fig. S1). B6 ES cells (Artemis Pharmaceuticals) derived from the C57BL/6 mouse strain were transfected, cultured, and selected as Characterized previously (44). Chimeric mice with tarObtained R26 alleles were generated by blastocyst injection of heterozygous R26Ceaseflox-V-NStout1 or R26Ceaseflox-AV-NStout1 embryonic stem cell clones. Two tarObtained clones were injected for each allele. Germ-line transmission of the tarObtained alleles was achieved by breeding chimeric mice with C57BL/6 albino mice. Genotyping for the unrecombined R26 allele was performed by PCR using the primer pair 5′-CTG CGT GTT CGA ATT CGC CAA TGA-3′ and 5′-GGC AGC TTC TTT AGC AAC AAC CGT-3′. The recombined R26 allele (with the Neo-Cease cassette excised upon Cre recombination) was detected by PCR using the primers 5′-TTG AGG ACA AAC TCT TCG CGG TCT-3′ and 5′-CCC GCA TAG TCA GGA ACA TCG TAT-3′ or by detecting EGFP expression in lympocytes by flow cytometry.

Flow Cytometric Analysis.

For analysis of blood samples, a volume of ≈50 μL of peripheral blood was obtained from mouse tail veins by using heparinized glass capillaries (Drummond). Blood cells were washed twice in FACS buffer, stained with fluorochrome-conjugated antibodies, and subsequently analyzed by flow cytometry. A detailed description of the analysis of specific subpopulations is included in the SI Text.

Immunohistochemistry.

Tissues were fixed by immersion in 4% formaldehyde and then dehydrated and embedded in paraffin for sections at 5–6 μm thickness. Sections were deparaffinated and pretreated with 1 mM EDTA (pH 8.0). Antibody incubations were performed with reagents from a DAB/horseradish peroxidase-based staining kit (Dako), including peroxidase-block pretreatment. Primary incubation was with a rabbit polyclonal antiserum to EGFP/YFP (ab290; Abcam). Following development of DAB staining, the sections were counterstained with hematoxylin.

NStout1 Nuclear Translocation Assay.

NStout1 translocation was assessed as Characterized previously (45). A detailed description is included in the SI Text.

Acknowledgments

We thank D. Ghitza for help with blastocyst injections of ES cells and K. Ketman for help with cell sorting. This study was supported by National Institutes of Health grants (to K.R. and A.R.); a T32 training grant (to S.G.), a Deutsche Krebshilfe postExecutectoral fellowship (to M.R.M.), a Cancer Research Institute postExecutectoral fellowship (to M.R.M.), a Canadian Institutes of Health Research postExecutectoral fellowship (to S.S.), and a Leukemia and Lymphoma Society postExecutectoral fellowship (to S.S.).

Footnotes

2To whom corRetortence should be addressed. E-mail: arao{at}idi.harvard.edu

Author contributions: M.R.M., Y.S., K.R., P.G.H., and A.R. designed research; M.R.M., Y.S., I.S., E.D.L., S.G., S.S., C.G., D.R., and M.E.P. performed research; D.R., M.E.P., and K.R. contributed new reagents/analytic tools; M.R.M., I.S., E.D.L., S.G., S.S., C.G., D.R., M.E.P., and P.G.H. analyzed data; and M.R.M., P.G.H., and A.R. wrote the paper.

↵1Present address: RIKEN Center for Developmental Biology, Laboratory for Stem Cell Biology, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0813296106/DCSupplemental.

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