Three-step transcriptional priming that drives the commitment of multipotent progenitors toward B cells.

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.

Conversion of T cells to B cells by inactivation of polycomb-mediated epigenetic suppression of the B-lineage program.

In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.

The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells.

It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell-B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells.

The E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to suppress innate variant TFH cell development, thymocyte expansion, and lymphomagenesis.

It is now well established that the E and Id protein axis regulates multiple steps in lymphocyte development. However, it remains unknown how E and Id proteins mechanistically enforce and maintain the naïve T-cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate variant follicular helper T (TFH) cells. Innate variant TFH cells required major histocompatibility complex (MHC) class I-like signaling and were associated with germinal center B cells. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of a TFH transcription signature. We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion. We found that mice depleted for Id2 and Id3 expression developed colitis and αß T-cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc, whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities to genetic deficiencies associated with Burkitt lymphoma. We propose that, in response to antigen receptor and/or cytokine signaling, the E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

The Effect of D-Aspartate on Spermatogenesis in Mouse Testis.

Spermatogenesis is controlled by hormonal secretions from the hypothalamus and pituitary gland, by factors produced locally in the testis, and by direct interaction between germ cells and Sertoli cells in seminiferous tubules. Although the mammalian testis contains high levels of D-aspartate (D-Asp), and D-Asp is known to stimulate the secretion of testosterone in cultured Leydig cells, its role in testis is unclear. We describe here biochemical, immunohistochemical, and flow cytometric studies designed to elucidate developmental changes in testicular D-Asp levels and the direct effect of D-Asp on germ cells. We found that the concentration of D-Asp in mouse testis increased with growth and that fluctuations in D-Asp levels were controlled in part by its degradative enzyme, D-aspartate oxidase expressed in Sertoli cells. In vitro sperm production studies showed that mitosis in premeiotic germ cells was strongly inhibited by the addition of D-Asp to the culture medium. Moreover, immunohistochemical analysis demonstrated that d-Asp accumulated in the differentiated spermatids, indicating either transport of D-Asp to spermatids or its de novo synthesis in these cells. Such compartmentation seems to prevent premeiotic germ cells in mouse testis from being exposed to the excess amount of D-Asp. In concert, our results indicate that in mouse testis, levels of D-Asp are regulated in a spatiotemporal manner and that D-Asp functions as a modulator of spermatogenesis.

Imatinib mesylate directly impairs class switch recombination through down-regulation of AID: its potential efficacy as an AID suppressor.

Activation-induced cytidine deaminase (AID) is essential for class switch recombination and somatic hypermutation. Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that class switch recombination on B-cell activation is apparently inhibited by IM through down-regulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor.

Reciprocal control of G1-phase progression is required for Th-POK/Runx3-mediated CD4/8 thymocyte cell fate decision.

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G(1) cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G(1)-phase extension and represses CD8 expression in a G(1) extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G(1) extension and CD8 repression. Importantly, forced G(1) extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G(1) progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G(1)-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

E2A and CBP/p300 act in synergy to promote chromatin accessibility of the immunoglobulin κ locus.

V(D)J recombination of Ig and TCR genes is strictly regulated in a lineage- and stage-specific manner by the accessibility of target gene chromatin to the recombinases RAG1 and RAG2. It has been shown that enforced expression of the basic helix-loop-helix protein, E2A, together with RAG1/2 in a nonlymphoid cell line BOSC23 can induce V(D)J recombination in endogenous Igκ and TCR loci by increasing chromatin accessibility of target gene segments. In this study, we demonstrate that ectopically expressed E2A proteins in BOSC23 cells have the ability to bind directly to the promoter and recombination signal sequence of Vκ genes and to recruit histone acetyltransferase CBP/p300. Overexpression of CBP/p300 in conjunction with E2A results in enhancement of E2A-induced histone acetylation, germline transcription, and Igκ rearrangement. Conversely, knockdown of endogenous CBP/p300 expression by small interfering RNA leads to a decrease in histone acetylation, germline transcription and Igκ rearrangement. Furthermore, analyses using a mouse pre-B cell line revealed that endogenous E2A proteins also bind to a distinct set of Vκ genes and regulatory regions in the mouse Igκ locus and act to increase histone acetylation by recruiting p300, confirming the similar findings observed with BOSC23 cells. These observations indicate that E2A plays critical roles in inducing Igκ rearrangement by directly binding to and increasing chromatin accessibility at target gene segments.

Claudin-4 induction by E-protein activity in later stages of CD4/8 double-positive thymocytes to increase positive selection efficiency.

Claudins (Clds) are crucial constituents of tight-junction strands in epithelial cells and have a central role in barrier functions. We show that Cld4 is unexpectedly expressed in normal thymic lymphocytes independently of tight junctions. The Cld4 expression was mostly confined to a portion of the CD4/CD8 double-positive (DP) cells. The proportion of Cld4(+) DP cells was markedly increased in MHC-I(-/-) II(-/-) mice but decreased in Rorγ(-/-) mice, and Cld4(+) DP cells contained higher levels of the rearranged Tcra transcripts involving the most distal Va and Ja segments than Cld4(-) DP cells. The Cld4 expression levels were reduced in E47-deficient mice in a gene dose-dependent manner, and ChIP analysis indicated that E2A and HEB were bound to the E-box sites of the putative Cldn4 promoter region. Functionally, Cld4 showed a potent T-cell receptor costimulatory activity by coligation with CD3. The Cld4 was distributed diffusely on the cell surface and associated with CD4/lck independently of CD3 in the resting thymocytes. However, Cld4 was strongly recruited to the immunological synapse on specific T-cell receptor engagement through antigen-presenting cells. In the fetal thymic organ culture, knockdown of Cldn4 resulted in the reduced generation of CD4/CD8 single-positive cells from the DP cells. These results suggest that Cld4 is induced by E-protein activity in the later stages of DP cells to increase the efficiency of positive selection, uncovering a hitherto unrecognized function of a Cld family protein.

Regulation of TCR Vγ2 gene rearrangement by the helix-loop-helix protein, E2A.

V(D)J recombination of Ig and TCR genes is strictly regulated by the accessibility of target gene chromatin in a lineage- and stage-specific manner. In the mouse TCRγ locus, rearrangement of the Vγ2 gene predominates over Vγ3 rearrangement in the adult thymus. This preferential rearrangement is likely due to the differential accessibility of the individual Vγ genes, because the levels of germ line transcription and histone acetylation of the Vγ genes are well correlated with the rearrangement frequency in adult thymocytes. However, factors responsible for the differential regulation of the Vγ gene rearrangement have been largely unknown. In this study, we demonstrated that Vγ2 rearrangement in the adult thymus was substantially reduced in mice deficient for the basic helix-loop-helix protein, E2A. The decreased rearrangement is likely caused by the reduced accessibility of Vγ2 chromatin, since germ line transcription and histone acetylation of the Vγ2 gene were reduced in an E2A dosage-dependent manner. We further showed that E2A bound around the Vγ2 gene in vivo and we identified two canonical E-box sites downstream of Vγ2, to which E2A can bind in vitro. Furthermore, these two E-box sites had the ability to activate transcription upon E2A over-expression. These data suggest that E2A directly binds to and increases accessibility of Vγ2 chromatin, thereby facilitating Vγ2 rearrangement in the adult thymus.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model.

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

The balance between Pax5 and Id2 activities is the key to AID gene expression.

Pax5 activity is enhanced in activated B cells and is essential for class switch recombination (CSR). We show that inhibitor of differentiation (Id)2 suppresses CSR by repressing the gene expression of activation-induced cytidine deaminase (AID), which has been shown to be indispensable for CSR. Furthermore, a putative regulatory region of AID contains E2A- and Pax5-binding sites, and the latter site is indispensable for AID gene expression. Moreover, the DNA-binding activity of Pax5 is decreased in Id2-overexpressing B cells and enhanced in Id2(-/-) B cells. The kinetics of Pax5, but not E2A, occupancy to AID locus is the same as AID expression in primary B cells. Finally, enforced expression of Pax5 induces AID transcription in pro-B cell lines. Our results provide evidence that the balance between Pax5 and Id2 activities has a key role in AID gene expression.

Characterization of tumour-infiltrating lymphocytes in a tumour rejection cynomolgus macaque model.

Immunotherapy has emerged as a promising and effective treatment for cancer, yet the clinical benefit is still variable, in part due to insufficient accumulation of immune effector cells in the tumour microenvironment. Better understanding of tumour-infiltrating lymphocytes (TILs) from nonhuman primate tumours could provide insights into improving effector cell accumulation in tumour tissues during immunotherapy. Here, we characterize TILs in a cynomolgus macaque tumour model in which the tumours were infiltrated with CD4+ and CD8+ T cells and were eventually rejected. The majority of CD4+ and CD8+ TILs exhibited a CD45RA-CCR7- effector memory phenotype, but unlike circulating T cells, they expressed CD69, a marker for tissue-resident memory T (TRM) cells. CD69-expressing CD8+ TILs expressed high levels of the cytotoxic molecule granzyme B and the co-inhibitory receptor PD-1. Consistent with the TRM cell phenotype, CD8+ TILs minimally expressed CX3CR1 but expressed CXCR3 at higher levels than circulating CD8+ T cells. Meanwhile, CXCL9, CXCL10 and CXCL11, chemokine ligands for CXCR3, were expressed at high levels in the tumours, thus attracting CXCR3+CD8+ T cells. These results indicate that tumour-transplanted macaques can be a useful preclinical model for studying and optimizing T cell accumulation in tumours for the development of new immunotherapies.

Regeneration of Tumor-Antigen-Specific Cytotoxic T Lymphocytes from iPSCs Transduced with Exogenous TCR Genes.

In the current adoptive T cell therapy, T cells from a patient are given back to that patient after ex vivo activation, expansion, or genetic manipulation. However, such strategy depends on the quality of the patient's T cells, sometimes leading to treatment failure. It would therefore be ideal to use allogeneic T cells as "off-the-shelf" T cells. To this aim, we have been developing a strategy where potent tumor-antigen-specific cytotoxic T lymphocytes (CTLs) are regenerated from T-cell-derived induced pluripotent stem cells (T-iPSCs). However, certain issues still remain that make it difficult to establish highly potent T-iPSCs: poor reprogramming efficiency of T cells into iPSCs and high variability in the differentiation capability of each T-iPSC clone. To expand the versatility of this approach, we thought of a method to produce iPSCs equivalent to T-iPSCs, namely, iPSCs transduced with exogenous T cell receptor (TCR) genes (TCR-iPSCs). To test this idea, we first cloned TCR genes from WT1-specific CTLs regenerated from T-iPSCs and then established WT1-TCR-iPSCs. We show that the regenerated CTLs from TCR-iPSCs exerted cytotoxic activity comparable to those from T-iPSCs against WT1 peptide-loaded cell line in in vitro model. These results collectively demonstrate the feasibility of the TCR-iPSC strategy.

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model.

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/ß genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors.

Regulation of histone H4 acetylation by transcription factor E2A in Ig gene conversion.

Recent studies implicate the transcription factor E2A in Ig diversification such as somatic hypermutation or gene conversion (GCV). GCV also requires active Ig transcription, expression of the activation-induced deaminase (AID) and a set of homologous recombination factors. We have disrupted the E2A gene in the chicken B-cell line DT40 and found greatly diminished rate of GCV without changes in the levels of transcripts from AID and Ig heavy chain or Ig light chain (IgL) genes. However, chromatin immunoprecipitation analysis revealed that the loss of E2A accompanies drastically reduced acetylation levels of the histone H4 in rearranged IgL locus. Furthermore, the defects in GCV were restored by trichostatin A treatment, which raised H4 acetylation to the normal levels. Thus, E2A may contribute to GCV by maintaining histone acetylation, which could be a prerequisite for targeting or full deaminase function of AID.

The identification of á´ -tryptophan as a bioactive substance for postembryonic ovarian development in the planarian Dugesia ryukyuensis.

Many metazoans start germ cell development during embryogenesis, while some metazoans possessing pluripotent stem cells undergo postembryonic germ cell development. The latter reproduce asexually but develop germ cells from pluripotent stem cells or dormant primordial germ cells when they reproduce sexually. Sexual induction of the planarian Dugesia ryukyuensis is an important model for postembryonic germ cell development. In this experimental system, hermaphroditic reproductive organs are differentiated in presumptive gonadal regions by the administration of a crude extract from sexual planarians to asexual ones. However, the substances involved in the first event during postembryonic germ cell development, i.e., ovarian development, remain unknown. Here, we aimed to identify a bioactive compound associated with postembryonic ovarian development. Bioassay-guided fractionation identified ʟ-tryptophan (Trp) on the basis of electrospray ionization-mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy. Originally masked by a large amount of ʟ-Trp, ᴅ-Trp was detected by reverse-phase high-performance liquid chromatography. The ovary-inducing activity of ᴅ-Trp was 500 times more potent than that of ʟ-Trp. This is the first report describing a role for an intrinsic ᴅ-amino acid in postembryonic germ cell development. Our findings provide a novel insight into the mechanisms of germ cell development regulated by low-molecular weight bioactive compounds.

Direct interaction of NRSF with TBP: chromatin reorganization and core promoter repression for neuron-specific gene transcription.

Neural restrictive silencer factor, NRSF (also known as REST) binds a neuronal cell type selective silencer element to mediate transcriptional repression of neuron-specific genes in non-neuronal cells and neuronal progenitors. Two repression domains (RD-1 and RD-2) occur in its N-terminal and C-terminal regions, respectively. RD-1 recruits mSin3 and HDAC, thereby inhibiting transcription by inducing reorganization of the chromatin structure. However, little is known about how such global repression becomes promoter-specific repression or whether the NRSF-HDAC complex can interact with transcriptional core factors at each specific promoter. Here we show evidence that NRSF interacts with core promoter factors, including TATA-binding protein (TBP). The NRSF-TBP interaction occurred between the linear segments of the N- and C-terminal-most portions of NRSF and the C-terminal half of TBP. A RD-2 mutant of NRSF lost the TBP-binding activity and was unable to repress transcription at an exogenously introduced TGTA promoter. These results indicate that the direct interaction between the NRSF C-terminal domain and TBP is essential for the C-terminal repression mechanism of NRSF. Thus, the RD-1 and RD-2 repression domains of NRSF utilize both chromatin-dependent and chromatin-independent mechanisms, which may be segregated at various stages of neural development and modulation.

Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells.

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status.We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.