Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2014 Sep 8;111(38):13930–13935. doi: 10.1073/pnas.1310118111

Novel INHAT Repressor (NIR) is required for early lymphocyte development

Chi A Ma a, Antonia Pusso a, Liming Wu a, Yongge Zhao a, Victoria Hoffmann b, Luigi D Notarangelo c, B J Fowlkes d, Ashish Jain a,1
PMCID: PMC4183277  PMID: 25201955

Significance

Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor that can bind to p53 at promoters and suppress p53-transcriptional activity by inhibiting histone acetylation. We found that lymphoid-restricted deletion of NIR resulted in the absence of mature B and T lymphocytes, which is partially, but not completely, rescued by the combined deletion of p53 with NIR. Thus, NIR cooperates with p53 to impose a checkpoint for the generation of mature B and T lymphocytes in vivo. Further delineation of additional protein interactions with NIR may lead to the better understanding of the mechanisms that regulate cell-cycle regulation, apoptosis, and lymphocyte differentiation.

Keywords: cell cycle, apoptosis, transcription

Abstract

Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor with inhibitor of histone acetyltransferase activity and is a potent suppressor of p53. Although NIR deficiency in mice leads to early embryonic lethality, lymphoid-restricted deletion resulted in the absence of double-positive CD4+CD8+ thymocytes, whereas bone-marrow-derived B cells were arrested at the B220+CD19 pro–B-cell stage. V(D)J recombination was preserved in NIR-deficient DN3 double-negative thymocytes, suggesting that NIR does not affect p53 function in response to physiologic DNA breaks. Nevertheless, the combined deficiency of NIR and p53 provided rescue of DN3L double-negative thymocytes and their further differentiation to double- and single-positive thymocytes, whereas B cells in the marrow further developed to the B220+CD19+ pro–B-cell stage. Our results show that NIR cooperate with p53 to impose checkpoint for the generation of mature B and T lymphocytes.


The tumor suppressor p53 protein is a potent inhibitor of cell growth and, in response to DNA damage, activates the apoptotic machinery leading to cell death (1). These various functions of p53 are of critical importance for tumor suppression in humans and mice (13). Unlike other members of the p53 family, p53 function is not absolutely necessary for normal cell growth and differentiation, and embryonic development is largely unaffected by the loss of p53 (2). However, aberrant activation of p53 function is lethal to the developing embryo (4, 5). In contrast, suppressed p53 activity is necessary to enhance bacterial killing in neutrophils (6). In B and T lymphocytes, p53 must be temporarily inactivated upon the occurrence of physiologic DNA breaks generated by V(D)J recombination to allow cells to enter the cell cycle (79), thus indicating that strict temporal repression of p53 activity is of paramount importance in host defense against infection. Several levels of regulation of p53 have been described, including control of transcription and translation, as well as control of the stability of the p53 protein (1). However, the exact contribution of these individual mechanisms is not known.

Histone acetyltransferase (HAT) transfers acetyl groups onto lysine tails of histones to weaken histone–DNA binding and facilitate gene transcription. Inhibitors of HAT (INHATs) repress HAT activity and block target gene transcription. Novel INHAT repressor (NIR; also called NOC2L) is a transcriptional corepressor with INHAT activity (10). Conventional inhibitors of histone deacetylases (HDACs) do not suppress NIR activity, suggesting that NIR does not function by attracting HDACs to its target promoters (10). Instead, NIR directly associates with p53, nucleosomes, and core histones to prevent acetylation by HATs, such as p300/CBP (also called EP300 or E1A binding protein/CREB binding protein) and p/CAF (also called p300/CBP-associated factor) (10). NIR can block acetylation of p53 and inhibit p53 function (11). Additionally, NIR suppresses p53 transactivation activity by functioning as a bridge between Aurora B and p53, allowing Aurora B to phosphorylate the p53 DNA binding domain and inhibit p53 activity (12, 13). NIR is expressed in most tissue types, but its precise role in vivo remains to be addressed.

We previously found that the expression of NIR and genes related to nonhomologous recombination was attenuated in stimulated B lymphocytes of patients diagnosed with anhidrotic ectodermal dysplasia with immunodeficiency (EDI) (14). EDI patients have hypomorphic mutations of NF-κB essential modulator (NEMO) (1315) that impair NF-κB signaling and result in a developmental disorder with severe immune deficiency. Immunologic assessments of these patients reveal defective development and function of mature lymphocytes. To delineate the role of NIR in lymphocyte development, we examined the outcome of conditional deletion of the NIR gene specifically in CD2-expressing B- and T-cell compartments in mice.

Results

Generation of Systemic and CD2–Cre-Driven NIR Conditional Deficient Mice.

NIR (NOC2L) protein is encoded by the Noc2l gene. We made mice in which Noc2l exons 3 and 4 were flanked by loxP sites (Noc2l+/fl) (SI Appendix, Fig. S1A). Homozygous Noc2lfl/fl mice were born healthy with normal Mendelian ratios. We first examined the systemic NIR-deficient (Noc2lfl/fl ACTBCre) mice. The knockout mice died in utero, and the embryos were not detected beyond embryonic day 10.5 (SI Appendix, Fig. S2), indicating that NIR was essential for embryonic development. To bypass this embryonic lethality, Noc2lfl/fl mice were bred with CD2–Cre transgenic mice (16) to create Noc2lfl/fl CD2Cre conditional knockout mice (referred to as NIR-CKO in this work) (SI Appendix, Fig. S1 BE).

Defective Thymocyte Development in NIR-CKO Mice.

Although NIR-CKO mice were born healthy and were fertile with normal body weights, histological examination of thymic tissue sections from 5- to 6-wk-old mice showed a dramatic defect in thymic development. Severe early thymus involution with a significant reduction in size was observed (Fig. 1A, Left). Thymi from NIR-CKO mice also showed a dramatically reduced cellularity compared with littermate controls (Fig. 1A, Right). Thymic structure in control mice showed a typical distinct border between the dark H&E-stained cortex and the light H&E-stained medulla (Fig. 1B, Left). In contrast, thymi from NIR-CKO mice were much smaller and lacked distinct cortex–medulla compartmentalization (Fig. 1B, Right).

Fig. 1.

Fig. 1.

Defects in early T lymphocyte development in NIR-CKO mice. (A) Significant decrease in thymic size (Left) and cellularity (Right) (n = 8, mean ± SEM) in NIR-CKO mice. ****P < 0.0001. (B) H&E staining of the thymic section shows dramatic involution and decompartmentalization between regions of cortex and medulla in NIR-CKO mice (n = 3), (C) Flow cytometric analysis of the CD4 and CD8 populations from thymi reveals the absence of DP (CD4+CD8+) T cells and a dramatic increase in DN (CD4CD8) T cells in NIR-CKO mice. Numbers in the plots indicate the percent of gated cells of three mice per group with similar results.

Further flow-cytometric analysis of NIR-CKO thymocytes revealed defects in T-cell development with a marked reduction in single-positive CD4+ and CD8+ cells and almost complete absence of double-positive (DP; CD4+CD8+) cells (Fig. 1C). Additionally, ∼98% of cells recovered from NIR-CKO thymi were double-negative (DN; CD4CD8) (Fig. 1C). We observed homeostatic proliferation under this lymphopenic condition in splenic T-cells of 6-wk-old NIR-CKO mice (SI Appendix, Fig. S6A). However, the homeostatic proliferation was drastically reduced, and the NIR-deficient defects were more striking in 5-d neonates (SI Appendix, Fig. S6B). Taken together, these results suggests that NIR plays an important role in very early T lymphocyte development.

Thymocyte DN3 to DN4 Transition Is Abolished in NIR-Deficient Mice.

The significant increase in the percentage of DN T cells in the thymi of NIR-CKO mice (Fig. 1C) led us to look more closely at early DN T-cell developmental stages. Thymocytes were first stained with lineage-specific antibodies to remove non-DN T cells (Lin+) by flow cytometry cell sorting. Lineage-negative (Lin) cells were then stained with specific antibodies for CD44 and CD25. Compared with littermate controls, NIR-CKO DN thymocytes exhibited an absence of CD44/CD25 DN4 cells along with a highly retained CD44/CD25+ DN3 cell population (Fig. 2 A and B and SI Appendix, Fig. S8). These results strongly indicated a complete developmental blockage at the transition between these two stages. Furthermore, in control mice, NIR protein levels were up-regulated approximately threefold in the DN3 stage compared with DN1 and plateaued at DN4, suggesting that NIR regulation is important for DN cell development (Fig. 2C).

Fig. 2.

Fig. 2.

Complete blockage of DN3-to-DN4 transition in the thymi of NIR-CKO mice. (A, Left) Schematic presentation of the four DN subpopulations (DN1, DN2, DN3, and DN4) of Lin DN thymocytes distinguished by CD44 and CD25 surface markers. (Center and Right) Flow-cytometric analysis of the four DN populations from thymi shows the absence of DN4 T cells and an increase in DN3 T cells in NIR-CKO mice. (B) Percentage of DN subpopulations (DN1, DN2, DN3, and DN4) from A (n = 3, mean ± SEM). ***P = 0.0003; ****P < 0.0001. (C) NIR protein expression in FACS-sorted Lin wild-type DN thymocytes shows up-regulation of NIR protein in the DN3 stage (Upper). Band intensities were determined by scanning and analysis using ImageJ software. Numbers are expressed as the fold change of NIR protein relative to DN1 cells after normalization with β-actin.

Increased Apoptosis, Cell-Cycle Arrest, and p53 Target Gene Expression in NIR-CKO DN3 Thymocytes.

To further characterize the defect in DN3–DN4 transition (17), we performed TUNEL staining of thymic sections. DN3 thymocytes are generated at the cortex of the thymus (18), and, in comparison with control mice, NIR-CKO thymic sections showed increased nuclear TUNEL staining in this region (Fig. 3A). We next stained isolated DN3 thymocytes with Annexin V and found that NIR-deficient DN3 cells contained a higher proportion of apoptotic cells (Fig. 3B). These results indicate that NIR expression in DN3 cells is important for their survival.

Fig. 3.

Fig. 3.

Increased apoptosis, cell-cycle arrest, and enhanced expression of p53 target genes in DN3 thymocytes of NIR-CKO mice. (A) Increased nuclear TUNEL staining in the NIR-CKO thymi (n = 3, mean ± SEM). **P = 0.0092. (B) Annexin V positive cells gated on DN populations show increased apoptosis in NIR-CKO DN3 thymocytes ex vivo (n = 4, mean ± SEM). ***P = 0.003. (C) NIR-CKO DN3L development is significantly impaired. (Left and Center) DN3E and DN3L are separated by forward and side scattering in gated DN3 cells. (Right) Percentage of DN3E and DN3L thymocytes (n = 4, mean ± SEM). ***P = 0.0001. (D) G1 cell-cycle arrest in the DN3 population of NIR-CKO thymocytes. DNA content was measured in sorted DN3 population by DAPI staining (SI Appendix, SI Materials and Methods). (Left) Histograms show DNA contents from three independent experiments with similar results. (Right) Percentage of cells in each stage of cell cycle of sorted DN3 cells (n = 3, mean ± SEM). *P = 0.0351; **P = 0.0075; ***P = 0.0002. (E) NIR levels were determined by Western blot analysis of cellular lysates prepared from control DN3E and DN3L cells (forward scatter/side scatter). β-actin was used as a loading control. (F) Annexin V-positive cells in gated DN3E vs. DN3L populations (n = 4, mean ± SEM). *P = 0.0481. (G) Western blot analysis shows increased expression of p53 target genes responsible for cell-cycle arrest (p21) and apoptosis (Bax). β-actin was used as a loading control.

DN3 cells can be further subdivided into DN3E and DN3L. DN3L cells are larger cycling cells in S and G2/M phase with an in-frame β-chain configuration. Pre–T-cell receptor (pre-TCR) signaling in DN3L cells promotes their proliferation and further differentiation (17, 19). In contrast, DN3E cells are small resting G1 cells, and the percentage of DN3E cells that have successfully completed β-chain rearrangement is markedly reduced compared with DN3L cells (20). We examined NIR-deficient DN3E and DN3L cells by flow cytometry using forward and side scatters. NIR-deficient DN3 cells showed a remarkable decrease in DN3L cells, but had a preserved DN3E population (Fig. 3C). This result was further supported by the nearly complete absence of S-phase cycling cells in the NIR-CKO DN3 population and an increased number of cells in G1 phase (Fig. 3D and SI Appendix, Fig. S10 A and B). These findings indicate that NIR deficiency is associated with a block at the G1/S phase of the cell cycle. Furthermore, the NIR protein level was elevated at the DN3L stage in wild-type cells (Fig. 3E), and increased apoptosis was observed in DN3L cells deficient in NIR (Fig. 3F). Collectively, these data suggest that, in the absence of NIR, DN3L cells were not able to proceed into the cell cycle and failed to survive the transition to the DN4 stage.

Previous reports demonstrated that NIR could repress the expression of p53 target genes, and that a lack of NIR may lead to p53-dependent cell-cycle arrest and apoptosis (10, 12, 13). Indeed, expression levels of the p53 target genes p21, an inhibitor of cyclin-dependent kinase responsible for cell-cycle arrest, and Bax, a proapoptotic protein, were highly elevated in DN3 cells of the NIR-CKO mice (Fig. 3G). Real-time RT-PCR confirmed that the expressions of p21 and Bax messages were increased in the FACS-sorted NIR-CKO’s DN3E thymocytes (SI Appendix, Fig. S7).

TCR-β–Expressing Thymocytes Are Decreased in NIR-CKO Mice.

The majority of pre-TCR is not detectable at the cell surface and probably does not recognize a ligand, but instead signals through oligomerization of intracellular intermediates similar to those triggered by TCR complexes in mature T cells (21, 22). Intracellular staining of TCR-β in DN3 cells showed a significantly reduced number of TCR-β–producing cells in NIR-CKO mice compared with littermate controls (Fig. 4A), and intracellular staining of TCR-β was significantly lower in the DN3L population of NIR-CKO thymus compared with the control (Fig. 4B and SI Appendix, Fig. S8B). Interestingly, surface TCR-γδ–positive thymocytes expressing NIR were not affected by NIR deficiency (Fig. 4C and SI Appendix, Table S2 and Fig. S11). Development of TCR-γδ cells requires V(D)J recombination and common progenitors (DN1 and DN2) of TCR-αβ cells (23), but, in contrast to TCR-αβ, TCR-γδ cell fate determination does not require pre-TCR–dependent β-selection (20, 23, 24). In support of the hypothesis that V(D)J recombination is retained in NIR-CKO DN cells, PCR and Southern blot analyses of the Dβ2–Jβ2 joining fragments revealed a decreased, but notable, level of nonclonal successful V(D)J rearrangements in NIR-CKO DN thymocytes (Fig. 4D). Clonalilty analysis by high-throughput DNA sequencing of the thymocytes in NIR-CKO mouse revealed productive in-frame DJ joining of the β-locus (SI Appendix, Fig. S3), indicating that V(D)J recombination was functional and unaffected by NIR deficiency. These results suggest that NIR deficiency is not likely to directly affect V(D)J recombination, but instead plays a role in regulating the survival of rearranged TCR-β–expressing cells.

Fig. 4.

Fig. 4.

Reduction of intracellular TCR-β–expressing DN3 thymocytes in NIR-CKO mice. (A) Flow-cytometric analysis of expression of intracellular TCR-β protein in DN3 thymocytes (n = 4, mean ± SEM). **P = 0.0001. (B) Intracellular TCR-β expression by flow cytometry on gated DN3E and DN3L populations. Results are representative of four mice per group (n = 4, mean ± SEM). **P = 0.0058. (C) Flow-cytometric analysis of surface expressions of TCR-γδ and -β on thymocytes with three independent experiments with similar results. (D) PCR–Southern blot analyses of V(D)J recombination determined by Dβ2–Jβ2 rearrangements in the TCR-β locus shows a decrease in, but not absence of, rearranged Dβ2–Jβ2 NIR-CKO thymocytes. The Cre gene was amplified as a loading reference for the Southern blot.

TCR Transgene Fails to Rescue Thymocyte Development in NIR-CKO Mice.

To further test our hypothesis that V(D)J recombination and pre-TCR signaling were not a cause of the impaired β-selection in the NIR-CKO mice, we generated a NIR-CKO/TCR (NIRfl/fl TCRVβ3 CD2Cre) hybrid mouse by crossing NIR-CKO mice with mice harboring a TCR-β transgene (TCR–Vβ3). Constitutive expression of the fully rearranged TCR-β transgene suppresses physiologic DNA breaks at the endogenous receptor loci and restores DP thymocytes in mice with a RAG-deficient background (25) (SI Appendix, Fig. S4). Although NIR–CKO/TCR mice showed significantly increased surface TCR-β expression compared with NIR-CKO thymocytes (Fig. 5A), they did not show recovery of DP differentiation (Fig. 5B and SI Appendix, Table S3). The increase of CD4+ single positive (SP) cells in the NIR-CKO/TCR thymus may be due to sustained constitutive TCR signaling that has been reported to suppress CD8 expression, causing a bias toward the CD4+ SP phenotype (23). In addition, the TCR transgene was not able to rescue the loss of the DN4 population in NIR-CKO mice (Fig. 5C and SI Appendix, Table S4). Thus, the decrease in DN3L cells is best explained by poor survival of the completely rearranged TCR-β cells. Surface TCR-β–negative cells show absence of SP in both NIR-CKO and NIR-CKO/TCR’s thymi (SI Appendix, Fig. S9).

Fig. 5.

Fig. 5.

Expression of the TCR-β transgene cannot restore the defects of thymocyte development in NIR-CKO mice. (A) Flow-cytometry analysis of surface TCR-β expression in the thymus of NIR-CKO and NIR-CKO mice harboring the TCR–Vβ3 transgene (NIR-CKO/TCR). (B) Flow-cytometric analysis of CD4 and CD8 expression on the surface of thymocytes of control mice expressing the TCR transgene (Noc2l+/fl CD2Cre/TCR), NIR-CKO, and NIR-CKO/TCR mice. The cells were gated on the thymocyte population expressing surface TCR-β. (C) Flow-cytometric analysis of CD44 and CD25 expressions gated on Lin DN thymocytes. Results are representative of two mice per group with similar results.

Together, our data strongly suggest that the developmental block in producing DN4 cells is caused by the loss of pre-TCR–expressing cells that fail to survive β-selection (Fig. 4), which likely resulted from aberrant p53 activation during DN3L differentiation in the absence of NIR. This lack of p53 regulation could lead to cell-cycle arrest, apoptosis, and a subsequent depletion of the DN4 and DP cells that produce pre–TCR-β.

NIR-CKO Splenocytes and Bone Marrow Exhibit Impairment of B-Lymphopoiesis in Early Progenitor Cells.

NIR-CKO mice showed a significant reduction in cellularity in the spleen compared with littermate controls (Fig. 6A). Notably, besides the T-cell abnormalities, splenocytes in the NIR-CKO mice exhibited a significant reduction in the proportion of B220+IgM+ mature B cells in the periphery (Fig. 6B). To trace the earlier B-cell development defect in the NIR-CKO mice, we examined subpopulations of early progenitor cells in the bone marrow. Consistent with the splenic data, NIR-CKO mice showed a dramatic reduction in IgM+ B220+ immature B cells in the bone marrow compared with littermate controls (Fig. 6C and SI Appendix, Table S5). In contrast, the myeloid compartment represented by surface markers CD11b (Mac-1) and Gr-1 remained intact (Fig. 6D and SI Appendix, Table S6). To further delineate the developmental block preceding the B220+ IgM+ stage, pre- and pro-B cells in bone marrow were compared based on their distinct expression of CD43 (26, 27). Flow-cytometric analysis revealed a significant decrease of the B220+CD43 pre-B population (Fig. 6E and SI Appendix, Fig. S8C), indicating an early developmental block between the pro-B (B220+CD43+) and pre-B (B220+CD43) cells in NIR-CKO mice. Further look into the pro-B (B220+CD43+IgM) population revealed a developmental block between very early pro-B cells (B220+CD43+CD19) and later pro-B (B220+CD43+CD19+) stage (Fig. 6F and SI Appendix, Fig. S8D). In summary, loss of NIR resulted in a very early defect in bone marrow B220 positive cells, which failed to develop into CD19+-committed B-lymphocytes.

Fig. 6.

Fig. 6.

Defective early B-lymphocyte development in NIR-CKO mice. (A) Cellularity of splenocytes from NIR-CKO mice and littermate controls (n = 8, mean ± SEM). ***P = 0.0002. (B) Flow-cytometric analysis of IgM and B220 populations from control and NIR-CKO splenocytes. Results are representative of five mice per group with similar results. (C) Flow-cytometric analysis of IgM and B220 populations from control and NIR-CKO bone marrow cells. Results are representative of five mice per group with similar results. (D) Flow-cytometric analysis of CD11b (Mac-1) and Gr-1 expression in the myeloid populations of bone marrow cells. Results are representative of three mice per group with similar results. (E) Flow-cytometric analysis of pro–B- to pre–B-cell transition in B220 and CD43 populations of control and NIR-CKO bone marrow cells (n = 5, mean ± SEM). ***P < 0.0001 (pre-B); ***P = 0.0002 (pro-B). (F) Flow-cytometric analysis of CD19 and HSA surface expressions in pro-B (B220+CD43+IgM) cells in control and NIR-CKO bone marrow [Fr. A (CD19B220+CD43+HSAlo CD25), Fr. B/C (CD19+ B220+CD43+HSA+ CD25), and Fr. C’ (CD19+ B220+CD43+HSAhi CD25+)] (26). Results are representative of three mice per group (n = 3, mean ± SEM). *P = 0.0002; **P = 0.0002 (Fr. A); **P = 0.0002 (Fr. B/C).

Defective Thymocyte Development in NIR-CKO Can Be Rescued by p53 Deficiency.

To examine whether NIR deficiency had a direct impact on the p53-mediated defects, we generated a double-conditional NIR- and p53-deficient mouse under the same CD2–Cre background (NIR/p53 CKO, NIRfl/flp53fl/fl CD2Cre). Introduction of the p53-conditional allele into NIR-CKO rescued the CD4+CD8+ DP thymocyte developmental defect present in the NIR-deficient mouse: A significant increase in DP cells was observed in the NIR/p53–CKO double-mutant mouse compared with the absence of DP thymocyte differentiation in NIR-CKO (Fig. 7A). In addition, NIR/p53 CKO mice lacking both NIR and p53 recuperate notable numbers of CD4+ and CD8+ SP thymocytes that were not present in the NIR-deficient mice (Fig. 7A).

Fig. 7.

Fig. 7.

p53 deficiency rescues NIR-deficient phenotypes. (A) Flow-cytometric analysis of CD4 and CD8 expressions in thymocytes from littermate control, NIR-CKO, and NIR/p53–CKO mice. (B) NIR-CKO DN3L is rescued by p53-deficiency in NIR/p53–CKO mice. (C) Pro-B CD43+ bone marrow cells were gated, and the expressions of B220 and CD19 were assessed by flow cytometry. (D) Pre-B CD43 bone marrow cells were gated, and B220 and IgM expression were assessed by flow cytometry. Results are representative of two independent experiments.

In support of our hypothesis that reduction of DN3L cells was due to a lack of regulation of p53 by NIR, the NIR/p53 CKO DN3 population showed significant recovery of DN3L cells close to the levels in control mice (Fig. 7B). Our findings therefore strongly indicate that the impairment caused by NIR deficiency was p53-dependent. Together, these data emphasize that the developmental block is due to a deletion of pre-TCR–expressing cells undergoing β-selection in the absence of NIR, which likely resulted in aberrant p53 activation causing cell-cycle arrest and subsequent depletion of DN4 and DP cells.

To examine whether p53 deficiency could also restore the early B-cell defects observed in the NIR-CKO mice, bone marrow cells from NIR/p53–CKO, NIR-CKO, and littermate control mice were gated on the CD43+ pro-B population. The B220+CD19+ cells were then compared. Interestingly, the NIR/p53–CKO mouse showed approximately fourfold higher levels of the late pro-B (CD43+B220+CD19+) compared with NIR-CKO (Fig. 7C). However, in contrast to T-cell rescue, p53 deficiency failed to rescue the development of the more mature pre-B (IgM+B220+CD19+CD43) cells (Fig. 7D). These findings suggest that NIR plays an additional p53-independent role in early B-cell development.

Discussion

Activity of p53 is important for tumor suppression, and the general function of p53 is to induce cell-cycle arrest and apoptosis when aberrant DNA breaks occur (1). However, in certain biological conditions, p53 activity needs to be repressed to achieve cell-cycle progression and obtain proper cell differentiation and homeostasis. Inhibition of p53 is sufficient to facilitate generation of CD4+CD8+ DP thymocytes in the absence of TCR rearrangement in RAG2−/− mice (28). Although the necessity of down-regulation of p53 activity has been recognized (7, 9), a basal level of p53 can be detected in highly proliferating cell stages, such as in GC centroblasts (29) and the DN3L (30, 31), suggesting the presence of additional factors that suppress p53 activity in these cells. It has been proposed that elevated cdk1/cdc2 kinase activity induced by pre-TCR signaling could phosphorylate p53 and lead to its degradation in DN3L cells (17, 32). Nevertheless, the factors that inactivate p53 in immune regulation have not been clearly defined, and those involved in the DN3L stage remain elusive.

Based on the findings that EDI immune-deficient NEMO-mutated patients have impaired NIR expression, it is likely that NIR could be an NF-κB target gene that regulates lymphocyte differentiation to overcome p53-mediated checkpoints. Lymphopenic NIR-CKO mice lack DN4/DP/SP thymocytes as a result of defective DN3E-to-DN3L transition undergoing β-selection, an NF-κB–regulated process (19). Elevated expressions of p53 target genes p21 (33) and Bax (1, 33) cause a marked cell-cycle arrest and increased apoptosis in the DN3L stage, which results in a dramatic reduction of TCR-β–expressing cells in NIR-deficient thymocytes. These defects lead to abolition of the ensuing DN4 population and, not surprisingly, the absence of DP thymocytes. Although the number of DP thymocytes of NIR/p53–CKO mice did not fully recover to the wild-type level, NIR/p53–CKO mice showed an approximate 50-fold recovery of DP thymocytes compared with the NIR-CKO mouse. Thus the impairment in thymocyte development observed in NIR-CKO mice was at least in part mediated by p53. Because inhibition of p53 activity is important for proliferation and survival of the DN3L cells to complete β-selection (7, 17), our results suggest that a major role of NIR is to suppress p53 activity in this transition of early T-cell development.

The presence of intracellular and surface TCR-β on NIR-CKO thymocytes, together with in-frame TCR-β–rearranged DJ DNA sequences, suggested that NIR is unlikely to be directly involved in the V(D)J recombination machinery. In addition, introduction of a TCR-β transgene fails to rescue the NIR-CKO phenotypes, suggesting that the defect in the NIR-CKO mice is not caused by TCR signaling and is independent of V(D)J recombination and DNA DSBs. Instead, our findings indicated that NIR/p53 is an important checkpoint and an essential survival factor for the development of DN thymocytes. A model of NIR function in early T-cell development is displayed in SI Appendix, Fig. S5.

Besides the early thymocyte defects, early B-cell development in the bone marrow was blocked between the pro- and pre-B stages in NIR-CKO mice. There was a dramatic reduction in CD43B220+ pre-B cells and immature IgM+/B220+ cells in the bone marrow, as well as a lack of splenic immature IgM+/B220+ cells. Additionally, p53 deficiency could only rescue NIR-CKO B-cell phenotypes up to the pro-B (CD43+B220+CD19+) stage, indicating that NIR may have additional p53-independent roles in later pre-B (IgM+CD43) cell development. Finally, our data supported the previously reported notion that distinct mechanisms underlie early T- and B-cell development (3, 34). Study of the p53-independent function of NIR could help to identify important factors that distinguish between early T- and B-cell differentiation.

Because NIR is an INHAT, we cannot exclude the possibility of NIR having a broader role for regulating gene transcription in lymphocytes. In addition, NIR may have other functions that regulate lymphocyte differentiation, such as ribosome biogenesis (11) or the function of the inner centrosome protein complex through Aurora B kinase (13, 35). These possible functions of NIR await further investigation (SI Appendix, Table S1). Nevertheless, our data demonstrate that epigenetic regulation of p53 activity by NIR is important for the development of early B and T lymphocytes.

Materials and Methods

All experiments with mice were approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee and were consistent with local, state, and federal guidelines. The animals were maintained in a sterile environment. A detailed of summary of research methods, reagents, and statistical analysis is available in the SI Appendix, SI Materials and Methods.

Supplementary Material

Supplementary File

Acknowledgments

We thank Warren Strober, Rémy Bosselut, David Allman, and Ronald Germain for their input and suggestions; Joshua Milner for support; Donna Butcher, Miriam Anver, Angela Thornton, Mathew Sebastian, Andrea Carpenter, Atsushi Kitani, and Ivan Fuss for technical assistance; and Mary Derry for editorial assistance. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases/National Institutes of Health.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1310118111/-/DCSupplemental.

References

  • 1.Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000;408(6810):307–310. doi: 10.1038/35042675. [DOI] [PubMed] [Google Scholar]
  • 2.Donehower LA, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992;356(6366):215–221. doi: 10.1038/356215a0. [DOI] [PubMed] [Google Scholar]
  • 3.Nacht M, et al. Mutations in the p53 and SCID genes cooperate in tumorigenesis. Genes Dev. 1996;10(16):2055–2066. doi: 10.1101/gad.10.16.2055. [DOI] [PubMed] [Google Scholar]
  • 4.Jones SN, Roe AE, Donehower LA, Bradley A. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature. 1995;378(6553):206–208. doi: 10.1038/378206a0. [DOI] [PubMed] [Google Scholar]
  • 5.Montes de Oca Luna R, Wagner DS, Lozano G. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature. 1995;378(6553):203–206. doi: 10.1038/378203a0. [DOI] [PubMed] [Google Scholar]
  • 6.Madenspacher JH, et al. p53 Integrates host defense and cell fate during bacterial pneumonia. J Exp Med. 2013;210(5):891–904. doi: 10.1084/jem.20121674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Haks MC, Krimpenfort P, van den Brakel JH, Kruisbeek AM. Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways in pre-T cells. Immunity. 1999;11(1):91–101. doi: 10.1016/s1074-7613(00)80084-9. [DOI] [PubMed] [Google Scholar]
  • 8.Lu L, Lejtenyi D, Osmond DG. Regulation of cell survival during B lymphopoiesis: Suppressed apoptosis of pro-B cells in P53-deficient mouse bone marrow. Eur J Immunol. 1999;29(8):2484–2490. doi: 10.1002/(SICI)1521-4141(199908)29:08<2484::AID-IMMU2484>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  • 9.Phan RT, Dalla-Favera R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature. 2004;432(7017):635–639. doi: 10.1038/nature03147. [DOI] [PubMed] [Google Scholar]
  • 10.Hublitz P, et al. NIR is a novel INHAT repressor that modulates the transcriptional activity of p53. Genes Dev. 2005;19(23):2912–2924. doi: 10.1101/gad.351205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Wu J, et al. Transcriptional repressor NIR functions in the ribosome RNA processing of both 40S and 60S subunits. PLoS ONE. 2012;7(2):e31692. doi: 10.1371/journal.pone.0031692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wu L, Ma CA, Jain A. When Aurora B met p53: Newly revealed regulatory phosphorylation in an old protein. Cell Cycle. 2011;10(2):171–172. doi: 10.4161/cc.10.2.14349. [DOI] [PubMed] [Google Scholar]
  • 13.Wu L, Ma CA, Zhao Y, Jain A. Aurora B interacts with NIR-p53, leading to p53 phosphorylation in its DNA-binding domain and subsequent functional suppression. J Biol Chem. 2011;286(3):2236–2244. doi: 10.1074/jbc.M110.174755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Jain A, et al. Specific NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal differentiation. J Clin Invest. 2004;114(11):1593–1602. doi: 10.1172/JCI21345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Jain A, et al. Specific missense mutations in NEMO result in hyper-IgM syndrome with hypohydrotic ectodermal dysplasia. Nat Immunol. 2001;2(3):223–228. doi: 10.1038/85277. [DOI] [PubMed] [Google Scholar]
  • 16.de Boer J, et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol. 2003;33(2):314–325. doi: 10.1002/immu.200310005. [DOI] [PubMed] [Google Scholar]
  • 17.Hoffman ES, et al. Productive T-cell receptor beta-chain gene rearrangement: Coincident regulation of cell cycle and clonality during development in vivo. Genes Dev. 1996;10(8):948–962. doi: 10.1101/gad.10.8.948. [DOI] [PubMed] [Google Scholar]
  • 18.Anderson G, Lane PJ, Jenkinson EJ. Generating intrathymic microenvironments to establish T-cell tolerance. Nat Rev Immunol. 2007;7(12):954–963. doi: 10.1038/nri2187. [DOI] [PubMed] [Google Scholar]
  • 19.Voll RE, et al. NF-kappa B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development. Immunity. 2000;13(5):677–689. doi: 10.1016/s1074-7613(00)00067-4. [DOI] [PubMed] [Google Scholar]
  • 20.Fehling HJ, Krotkova A, Saint-Ruf C, von Boehmer H. Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells. Nature. 1995;375(6534):795–798. doi: 10.1038/375795a0. [DOI] [PubMed] [Google Scholar]
  • 21.von Boehmer H, et al. Crucial function of the pre-T-cell receptor (TCR) in TCR beta selection, TCR beta allelic exclusion and alpha beta versus gamma delta lineage commitment. Immunol Rev. 1998;165:111–119. doi: 10.1111/j.1600-065x.1998.tb01234.x. [DOI] [PubMed] [Google Scholar]
  • 22.Yamasaki S, et al. Mechanistic basis of pre-T cell receptor-mediated autonomous signaling critical for thymocyte development. Nat Immunol. 2006;7(1):67–75. doi: 10.1038/ni1290. [DOI] [PubMed] [Google Scholar]
  • 23.Carpenter AC, Bosselut R. Decision checkpoints in the thymus. Nat Immunol. 2010;11(8):666–673. doi: 10.1038/ni.1887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Aifantis I, et al. On the role of the pre-T cell receptor in alphabeta versus gammadelta T lineage commitment. Immunity. 1998;9(5):649–655. doi: 10.1016/s1074-7613(00)80662-7. [DOI] [PubMed] [Google Scholar]
  • 25.Shinkai Y, et al. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes. Science. 1993;259(5096):822–825. doi: 10.1126/science.8430336. [DOI] [PubMed] [Google Scholar]
  • 26.Hardy RR, Hayakawa K. B cell development pathways. Annu Rev Immunol. 2001;19:595–621. doi: 10.1146/annurev.immunol.19.1.595. [DOI] [PubMed] [Google Scholar]
  • 27.Osmond DG, Rolink A, Melchers F. Murine B lymphopoiesis: Towards a unified model. Immunol Today. 1998;19(2):65–68. doi: 10.1016/s0167-5699(97)01203-6. [DOI] [PubMed] [Google Scholar]
  • 28.Jiang D, Lenardo MJ, Zúñiga-Pflücker JC. p53 prevents maturation to the CD4+CD8+ stage of thymocyte differentiation in the absence of T cell receptor rearrangement. J Exp Med. 1996;183(4):1923–1928. doi: 10.1084/jem.183.4.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Martinez-Valdez H, et al. Human germinal center B cells express the apoptosis-inducing genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2. J Exp Med. 1996;183(3):971–977. doi: 10.1084/jem.183.3.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pedraza-Alva G, et al. Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint. EMBO J. 2006;25(4):763–773. doi: 10.1038/sj.emboj.7600972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Okada H, et al. Survivin loss in thymocytes triggers p53-mediated growth arrest and p53-independent cell death. J Exp Med. 2004;199(3):399–410. doi: 10.1084/jem.20032092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lin WC, Desiderio S. Regulation of V(D)J recombination activator protein RAG-2 by phosphorylation. Science. 1993;260(5110):953–959. doi: 10.1126/science.8493533. [DOI] [PubMed] [Google Scholar]
  • 33.Bunz F, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science. 1998;282(5393):1497–1501. doi: 10.1126/science.282.5393.1497. [DOI] [PubMed] [Google Scholar]
  • 34.Danska JS, et al. Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents. Science. 1994;266(5184):450–455. doi: 10.1126/science.7524150. [DOI] [PubMed] [Google Scholar]
  • 35.Song J, Salek-Ardakani S, So T, Croft M. The kinases aurora B and mTOR regulate the G1-S cell cycle progression of T lymphocytes. Nat Immunol. 2007;8(1):64–73. doi: 10.1038/ni1413. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary File

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES