Expression In Thymocytes Research Articles

Differential Chromatin Looping Regulates CD4 Expression in Immature Thymocytes

Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, using looping chromatin immunoprecipitation and chromatin conformation capture assays, we demonstrated that interactions between Runx1 and positive elongation factor b (P-TEFb) appose the silencer and enhancer in CD4-negative thymoma cells and double-negative immature thymocytes. This chromatin loop decoys P-TEFb away from the promoter, thus preventing RNA polymerase II from elongating on the CD4 gene. In the absence of Runx1 on the silencer, P-TEFb interacts with the transcription complex, forming a different chromatin loop between the enhancer and the promoter, which leads to the expression of the CD4 gene in CD4-positive hybridoma cells and double-positive thymocytes. Moreover, the knockdown of CycT1 from P-TEFb abolishes both of these chromatin loops. Finally, the selective removal and restoration of Runx1 causes rapid interchanges between these chromatin loops, which reveals the plasticity of this regulatory circuit. Thus, differential looping and decoying of P-TEFb away from the promoter mediate active repression of the CD4 gene during thymocyte development.

Expression Profiling of Immature Thymocytes Revealed a Novel Homeobox Gene That Regulates Double-Negative Thymocyte Development

Intrathymic development of CD4/CD8 double-negative (DN) thymocytes can be tracked by well-defined chronological subsets of thymocytes, and is an ideal target of gene expression profiling analysis to clarify the genetic basis of mature T cell production, by which differentiation of immature thymocytes is investigated in terms of gene expression profiles. In this study, we show that development of murine DN thymocytes is predominantly regulated by largely repressive rather than inductive activities of transcriptions, where lineage-promiscuous gene expression in immature thymocytes is down-regulated during their differentiation. Functional mapping of genes showing common temporal expression profiles implicates previously uncharacterized gene regulations that may be relevant to early thymocytes development. A small minority of genes is transiently expressed in the CD44(low)CD25(+) subset of DN thymocytes, from which we identified a novel homeobox gene, Duxl, whose expression is up-regulated by Runx1. Duxl promotes the transition from CD44(high)CD25(+) to CD44(low)CD25(+) in DN thymocytes, while constitutive expression of Duxl inhibits expression of TCR beta-chains and leads to impaired beta selection and greatly reduced production of CD4/CD8 double-positive thymocytes, indicating its critical roles in DN thymocyte development.

The in vitro effect of bis(tri- n)butyltin)oxide (TBTO) on gene expression in mouse thymocytes

The in vitro effect of bis(tri- n)butyltin)oxide (TBTO) on gene expression in mouse thymocytes

Notch-induced T cell development requires phosphoinositide-dependent kinase 1

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.

β-Catenin Expression Enhances IL-7 Receptor Signaling in Thymocytes during Positive Selection

Differentiation of CD4+CD8+ double-positive thymocytes into CD8+ single-positive (SP) thymocytes is regulated by TCR and cytokine receptor signals. Previously, we have shown that expression of stabilized beta-catenin, the major transcriptional cofactor of T cell factor, results in increase in both CD4SP and CD8SP thymocytes with a preferential effect on CD8SP thymocytes. In this report, using mice expressing stabilized beta-catenin and mice with T cell specific deletion of beta-catenin, we show that beta-catenin expression augments IL-7Ralpha-chain expression and down-regulates suppressor of cytokine signaling-1 expression in thymocytes undergoing positive selection. Consequently, beta-catenin expression augments IL-7R signaling in thymocytes during positive selection and promotes the development of CD8SP thymocytes.

Redundant Role for Early Growth Response Transcriptional Regulators in Thymocyte Differentiation and Survival

The early growth response (Egr) family of transcriptional regulators consists of four proteins that share highly conserved DNA-binding domains. In many cell types, they are coexpressed and appear to have cooperative roles in regulating gene expression during growth and differentiation. Three Egr proteins, Egr1, Egr2, and Egr3, are induced during thymocyte differentiation in response to pre-TCR signaling, suggesting they may be critical for some aspects of pre-TCR-mediated differentiation. Indeed, enforced expression of Egr proteins in developing thymocytes can recapitulate some aspects of pre-TCR signaling, but the mechanisms by which they contribute to beta-selection are still poorly understood. Egr3 stimulates proliferation of beta-selected thymocytes, and Egr3-deficient mice have hypocellular thymuses, defects in proliferation, and impaired progression from double-negative 3 to double-negative 4. Surprisingly, Egr1-deficient mice exhibit normal beta-selection, indicating that the functions of Egr1 during beta-selection are likely compensated by other Egr proteins. In this study, we show that mice lacking both Egr1 and Egr3 exhibit a more severe thymic atrophy and impairment of thymocyte differentiation than mice lacking either Egr1 or Egr3. This is due to a proliferation defect and cell-autonomous increase in apoptosis, indicating that Egr1 and Egr3 cooperate to promote thymocyte survival. Microarray analysis of deregulated gene expression in immature thymocytes lacking both Egr1 and Egr3 revealed a previously unknown role for Egr proteins in the maintenance of cellular metabolism, providing new insight into the function of these molecules during T cell development.

Germline transcription from T-cell receptor Vβ gene is uncoupled from allelic exclusion

Allelic exclusion operates in B and T lymphocytes to ensure clonal expression of antigen receptors after V(D)J recombination. Germline transcription, which proceeds V(D)J recombination, has been postulated to provide an instructive signal for allelic exclusion. Here, we use a genetic marker to track germline transcription from a Vbeta gene within the TCRbeta locus. We find that developing thymocytes exhibit uniformed, bi-allelic activation of the Vbeta gene before V-DJ recombination, a process subject to allelic exclusion. We further show that V-DJ rearrangement promotes activation rather than silencing of germline transcription from the remaining Vbeta genes on either the functionally or non-functionally rearranged chromosome. Results presented here suggest that germline transcription, although necessary for V(D)J recombination, is not sufficient to instruct allelic exclusion.

Oncogenic role of Pax5 in the T-lymphoid lineage upon ectopic expression from the immunoglobulin heavy-chain locus

AbstractFour of 9 PAX transcription factor genes have been associated with chromosomal translocations in human tumors, although their oncogenic potential has not yet been demonstrated in transgenic mouse models. The B-lymphoidPAX5 gene participates in the generation of the t(9;14)(p13;q32) translocation in germinal center B cells, which leads to deregulated PAX5 expression under the control of the immunoglobulin heavy-chain (IgH) locus in a subset of B-cell non-Hodgkin lymphomas. Here we reconstructed a human t(9;14) translocation in a knock-in mouse by inserting a PAX5 minigene into the IgH locus. The IgHP5ki allele, which corresponds to a germline rather than somatic mutation, is activated in multipotent hematopoietic progenitors and is subsequently expressed in dendritic cells (DCs) and in natural killer (NK), T, and B cells. Ectopic Pax5 expression interferes with normal T-cell development and causes immature T-lymphoblastic lymphomas in IgHP5ki/+ and IgHP5ki/P5ki mice. Aggressive T-cell lymphomas develop even faster in IkPax5/+ mice expressing Pax5 from the Ikaros locus. Pax5 expression in thymocytes activates B-cell–specific genes and represses T-lymphoid genes, suggesting that Pax5 contributes to lymphomagenesis by deregulating the T-cell gene-expression program. These data identify Pax5 as a potent oncogene and demonstrate that the T-lymphoid lineage is particularly sensitive to the oncogenic action of Pax5.

Effect of monosodium glutamate on apoptosis and Bcl-2/Bax protein level in rat thymocyte culture

Monosodium glutamate (MSG), the sodium salt of glutamate, is commonly used as a flavor enhancer in modern nutrition. Recent studies have shown the existence of glutamate receptors on lymphocytes, thymocytes and thymic stromal cells. In this study, we evaluated the in vitro effect of different MSG concentrations on rat thymocyte apoptosis and expression of two apoptosis-related proteins, Bcl-2 and Bax. Rat thymocytes, obtained from male Wistar rats, were exposed to increasing concentrations of MSG (ranging from 1 mM to 100 mM) for 24 h. Apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit and cells were analyzed using a flow cytometer. Expression of Bcl-2 and Bax proteins were determined with flow cytometry using respective monoclonal antibodies. Exposure to MSG resulted in a dose-dependent decrease in cell survival (as determined by trypan blue exclusion method). Annexin V-FITC/PI also confirmed that MSG increased, in a dose-dependent manner, apoptotic cell death in rat thymocyte cultures. MSG treatment induced downregulation of Bcl-2 protein, while Bax protein levels were not significantly changed. Our data showed that MSG significantly modulates thymocyte apoptosis rate in cultures. The temporal profile of Bcl-2 and Bax expression after MSG treatment suggests that downregulation of Bcl-2 protein and the resulting change of Bcl-2/Bax protein ratio may be an important event in thymocyte apoptosis triggered by MSG.

Control of thymocyte development and recombination-activating gene expression by the zinc finger protein Zfp608

The products of recombination-activating gene 1 (Rag1) and Rag2 are required for T cell receptor gene assembly and thymocyte maturation, yet their transcriptional control mechanisms remain unclear. A congenic strain (called 'ZORI' here) with defects in Rag1 and Rag2 expression, thymocyte maturation and peripheral T cell homeostasis has been developed. Here, we mapped the mutation in this strain to a chromosome 18 locus containing a single known gene encoding the zinc finger protein Zfp608. This gene (Zfp608) was highly expressed in neonatal thymus but was extinguished thereafter. In contrast to wild-type mice, ZORI mice had sustained thymocyte expression of Zfp608 throughout life. The ZORI mutation produced a thymocyte-intrinsic developmental defect. Overexpression of Zfp608 in BALB/c thymocytes substantially impaired Rag1 and Rag2 expression, indicating the underlying mechanism for the defect in ZORI thymocyte development. Thus, the normal function of Zfp608 may be to prevent Rag1 and Rag2 expression in utero.

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.

Unexpected features of acute T lymphoblastic lymphomas in Notch1IC transgenic rats

Dysregulated Notch signaling accounts for the majority of acute T lymphoblastic leukemia/lymphoma (T-ALL) cases in humans. Here, we characterize lymphomas from Notch1IC transgenic rats, which develop T-ALL shortly after weaning, and show that they display a number of previously undocumented features. Starting from monoclonal thymic tumors, the CD4(+)CD8alphaalpha(+) lymphoma cells infiltrate the bone marrow and then spread to secondary lymphoid and non-lymphoid organs. However, major hallmarks of T-ALL cells in other murine models and human patients, such as constitutive NF-kappaB activity and increased levels of anti-apoptotic proteins, are remarkably absent in Notch1IC lymphomas. In contrast, CD30, a classic marker of Hodgkin lymphomas, is overexpressed in these tumors. Intriguingly, enforced Notch1 signaling up-regulates expression of Notch3, which has also been implicated in the pathogenesis of T-ALL. By blocking endogenous Notch signaling, we could demonstrate that Notch1IC is sufficient to induce sustained preTCR expression in transgenic thymocytes but not for their progression to the double-positive stage. This suggests that other Notch activities may also contribute to the phenotype of the transgenic rats. In summary, we anticipate this new animal model will help to further elucidate the role of Notch1 in the pathogenesis of T-ALL.

Golli Protein Negatively Regulates Store Depletion-Induced Calcium Influx in T Cells

Calcium influx is crucial for T cell activation and differentiation. The detailed regulation of this process remains unclear. We report here that golli protein, an alternatively spliced product of the myelin basic protein gene, plays a critical role in regulating calcium influx in T cells. Golli-deficient T cells were hyperproliferative and showed enhanced calcium entry upon T cell receptor stimulation. We further found that golli regulates calcium influx in T cells through the inhibition of the store depletion-induced calcium influx. Mutation of the myristoylation site on golli disrupted its association with the plasma membrane and reversed its inhibitory action on Ca2+ influx, indicating that membrane association of golli was essential for its inhibitory action. These results indicate that golli functions in a unique way to regulate T cell activation through a mechanism involving the modulation of the calcium homeostasis.

Transcription factor Pax5 (BSAP) transactivates the RAG-mediated VH-to-DJH rearrangement of immunoglobulin genes

Immunoglobulin rearrangement from variable heavy chain (V(H)) to diversity (D)-joining heavy chain (J(H)), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage-specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of D(H)-proximal V(H)7183 genes. In exploring the mechanism for Pax5 regulation of V(H)-to-DJ(H) recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse V(H) gene segments. Pax5 bound to those sites in vitro and occupied V(H) genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)-RAG2 complex to enhance RAG-mediated V(H) recombination signal sequence cleavage and recombination of a V(H) gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin V(H)-to-DJ(H) recombination.

Loss of a gimap/ ian gene leads to activation of NF-κB through a MAPK-dependent pathway

The diabetes-prone biobreeding (BB-DP) rat contains the lyp mutation which results in lymphopenia and promotes the progression of a T cell-mediated autoimmune attack of the pancreas in certain rat strains. This mutation has been mapped to a gene which bears homology to human Gimap5/Ian5 and results in the truncation and loss of activity of this protein. The lymphopenic state induced by the loss of this protein has led to the proposal that Gimap5 has an anti-apoptotic function. Previously we described an additional phenotype of incomplete activation mediated by the loss of Gimap5 function. Here we further characterize this incomplete activation phenotype and map a potential signal transduction pathway leading to activation. We show that CD5 expression on peripheral T cells is elevated in Gimap5 animals, while thymocyte expression remains similar between the two strains. Additionally, we show that NF-κB but not NFAT is activated in unstimulated Gimap5 mutant T cells as compared to unstimulated wild type T cells. Mapping this activation to its upstream source we show that activation of NF-κB is correlated with an activation of IKK. Using a variety of kinase inhibitors we further map this increase in IKK to an increase in MEK activation. Finally, to counter the possibility that activation is an indirect consequence of the lymphopenic environment, we created bone marrow chimeras in which Gimap5 mutant T cells developed in a normal environment and show that these cells retain their activated phenotype. Together, we interpret these data as demonstrating that the activation caused by loss of Gimap5 is a cell intrinsic phenomenon caused, in part, by a MEK-dependent activation of IKK. This, in turn, would suggest that Gimap5 functions to promote both T cell survival and quiescence and that these pathways are biochemically linked.

Regulation of intrathymic T‐cell development by Lunatic Fringe– Notch1 interactions

Intrathymic Notch1 signaling critically regulates T-lineage specification and commitment as well as T-cell progenitor survival and differentiation. Notch1 activation is continuously required during progression of early CD4/CD8-double-negative thymocytes to the CD4/CD8-double-positive stage. This developmental transition occurs as thymocytes migrate from the corticomedullary junction (CMJ) to the outer subcapsular zone (SCZ) of the thymus. Members of two families of structurally distinct Notch ligands, Delta-like 1 and Jagged-1, are expressed by cortical thymic epithelial cells, but it is not known which ligands are functionally required within the CMJ and SCZ microenvironmental niches. Our laboratory has investigated this question by genetically manipulating thymocyte expression of Lunatic Fringe (L-Fng), a glycosyltransferase that enhances sensitivity of Notch receptors to Delta-like ligands. This approach has revealed that low-threshold intrathymic Notch1 signals instruct multipotent thymus-seeding progenitors to suppress their B-cell potential and choose the T-cell fate. This strategy has also revealed that Delta-like Notch ligands are functionally limiting in both the CMJ and SCZ microenvironmental niches. Finally, we discuss our recent demonstration that L-Fng-mediated competition for Delta-like ligands is an important mechanism for regulating thymus size.

A function for interleukin 2 in Foxp3-expressing regulatory T cells

Regulatory T cells (T(reg) cells) expressing the forkhead family transcription factor Foxp3 are critical mediators of dominant immune tolerance to self. Most T(reg) cells constitutively express the high-affinity interleukin 2 (IL-2) receptor alpha-chain (CD25); however, the precise function of IL-2 in T(reg) cell biology has remained controversial. To directly assess the effect of IL-2 signaling on T(reg) cell development and function, we analyzed mice containing the Foxp3(gfp) knock-in allele that were genetically deficient in either IL-2 (Il2(-/-)) or CD25 (Il2ra(-/-)). We found that IL-2 signaling was dispensable for the induction of Foxp3 expression in thymocytes from these mice, which indicated that IL-2 signaling does not have a nonredundant function in the development of T(reg) cells. Unexpectedly, Il2(-/-) and Il2ra(-/-) T(reg) cells were fully able to suppress T cell proliferation in vitro. In contrast, Foxp3 was not expressed in thymocytes or peripheral T cells from Il2rg(-/-) mice. Gene expression analysis showed that IL-2 signaling was required for maintenance of the expression of genes involved in the regulation of cell growth and metabolism. Thus, IL-2 signaling seems to be critically required for maintaining the homeostasis and competitive fitness of T(reg) cells in vivo.

Nuclear Matrix Binding Regulates SATB1-mediated Transcriptional Repression

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

Impairment of Thymocyte Development by Dominant-Negative Kuzbanian (ADAM-10) Is Rescued by the Notch Ligand, Delta-1

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study we show that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRbeta expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components.

Enforced Expression of Spi-B Reverses T Lineage Commitment and Blocks β-Selection

The molecular changes that restrict multipotent murine thymocytes to the T cell lineage and render them responsive to Ag receptor signals remain poorly understood. In this study, we report our analysis of the role of the Ets transcription factor, Spi-B, in this process. Spi-B expression is acutely induced coincident with T cell lineage commitment at the CD4(-)CD8(-)CD44(-)CD25(+) (DN3) stage of thymocyte development and is then down-regulated as thymocytes respond to pre-TCR signals and develop beyond the beta-selection checkpoint to the CD4(-)CD8(-)CD44(-)CD25(-) (DN4) stage. We found that dysregulation of Spi-B expression in DN3 thymocytes resulted in a dose-dependent perturbation of thymocyte development. Indeed, DN3 thymocytes expressing approximately five times the endogenous level of Spi-B were arrested at the beta-selection checkpoint, due to impaired induction of Egr proteins, which are important molecular effectors of the beta-selection checkpoint. T lineage-committed DN3 thymocytes expressing even higher levels of Spi-B were diverted to the dendritic cell lineage. Thus, we demonstrate that the prescribed modulation of Spi-B expression is important for T lineage commitment and differentiation beyond the beta-selection checkpoint; and we provide insight into the mechanism underlying perturbation of development when that expression pattern is disrupted.