A mutant BCL11B-N440K protein interferes with BCL11A function during T lymphocyte and neuronal development.

Genetic studies in mice have shown that the zinc finger transcription factor BCL11B has an essential role in regulating early T cell development and neurogenesis. A de novo heterozygous missense BCL11B variant, BCL11BN441K, was isolated from a patient with T cell deficiency and neurological disorders. Here, we show that mice harboring the corresponding Bcl11bN440K mutation show the emergence of natural killer (NK)/group 1 innate lymphoid cell (ILC1)-like NKp46+ cells in the thymus and reduction in TBR1+ neurons in the neocortex, which are observed with loss of Bcl11a but not Bcl11b. Thus, the mutant BCL11B-N440K protein interferes with BCL11A function upon heterodimerization. Mechanistically, the Bcl11bN440K mutation dampens the interaction of BCL11B with T cell factor 1 (TCF1) in thymocytes, resulting in weakened antagonism against TCF1 activity that supports the differentiation of NK/ILC1-like cells. Collectively, our results shed new light on the function of BCL11A in suppressing non-T lymphoid developmental potential and uncover the pathogenic mechanism by which BCL11B-N440K interferes with partner BCL11 family proteins.

A variant in human AIOLOS impairs adaptive immunity by interfering with IKAROS.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

The Majority of the Serine/Threonine Phosphorylation Sites in Bcl11b Protein Are Dispensable for the Differentiation of T Cells.

Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.

Clonal conversion of B lymphoid leukemia reveals cross-lineage transfer of malignant states.

Even though leukemia is considered to be confined to one specific hematopoietic cell type, cases of acute leukemia of ambiguous lineage and patients relapsing in phenotypically altered disease suggest that a malignant state may be transferred between lineages. Because B-cell leukemia is associated with mutations in transcription factors of importance for stable preservation of lineage identity, we here investigated the potential lineage plasticity of leukemic cells. We report that primary pro-B leukemia cells from mice carrying heterozygous mutations in either or both the Pax5 and Ebf1 genes, commonly mutated in human leukemia, can be converted into T lineage leukemia cells. Even though the conversion process involved global changes in gene expression and lineage-restricted epigenetic reconfiguration, the malignant phenotype of the cells was preserved, enabling them to expand as T lineage leukemia cells in vivo. Furthermore, while the transformed pro-B cells displayed plasticity toward myeloid lineages, the converted cells failed to cause myeloid leukemia after transplantation. These data provide evidence that a malignant phenotype can be transferred between hematopoietic lineages. This has important implications for modern cancer medicine because lineage targeted treatment of leukemia patients can be predicted to provoke the emergence of phenotypically altered subclones, causing clinical relapse.

EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene.

Genes encoding B lineage-restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is early B-cell factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-sequencing, chromatin immunoprecipitation-sequencing, and reporter gene assays, six EBF1-responsive enhancer elements were identified within the Myc locus. CRISPR-Cas9-mediated targeting of EBF1-binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, chromatin immunoprecipitation-sequencing analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1-deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro-B-cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-cell acute lymphoblastic leukemia cell line indicates that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.

B Lymphocyte Specification Is Preceded by Extensive Epigenetic Priming in Multipotent Progenitors.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

PAX5 is part of a functional transcription factor network targeted in lymphoid leukemia.

One of the most frequently mutated proteins in human B-lineage leukemia is the transcription factor PAX5. These mutations often result in partial rather than complete loss of function of the transcription factor. While the functional dose of PAX5 has a clear connection to human malignancy, there is limited evidence for that heterozygote loss of PAX5 have a dramatic effect on the development and function of B-cell progenitors. One possible explanation comes from the finding that PAX5 mutated B-ALL often display complex karyotypes and additional mutations. Thus, PAX5 might be one component of a larger transcription factor network targeted in B-ALL. To investigate the functional network associated with PAX5 we used BioID technology to isolate proteins associated with this transcription factor in the living cell. This identified 239 proteins out of which several could be found mutated in human B-ALL. Most prominently we identified the commonly mutated IKZF1 and RUNX1, involved in the formation of ETV6-AML1 fusion protein, among the interaction partners. ChIP- as well as PLAC-seq analysis supported the idea that these factors share a multitude of target genes in human B-ALL cells. Gene expression analysis of mouse models and primary human leukemia suggested that reduced function of PAX5 increased the ability of an oncogenic form of IKZF1 or ETV6-AML to modulate gene expression. Our data reveals that PAX5 belong to a regulatory network frequently targeted by multiple mutations in B-ALL shedding light on the molecular interplay in leukemia cells.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Novel functional small RNAs are selectively loaded onto mammalian Ago1.

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1-4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein-Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1-4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors.

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

A Bcl11bN797K variant isolated from an immunodeficient patient inhibits early thymocyte development in mice.

BCL11B is a transcription factor with six C2H2-type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11BN441K and BCL11BN807K. To elucidate the pathogenesis of the BCL11BN807K variant, we generated a mouse model of BCL11BN807K by inserting the corresponding mutation, Bcl11bN797K, into the mouse genome. In Bcl11b+/N797K mice, the proportion of immature CD4-CD8+ single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11bN797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11bN797K/N797K fetal liver cells nearly lacked CD4+CD8+ double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11bN797K/N797K fetal liver progenitors. Interestingly, Bcl11bN797K/N797K progenitors gave rise to aberrant c-Kit+ and CD44+ cells both in vivo and in vitro. The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11bN797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11bN797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4-CD8- into CD4+CD8+ thymocytes.

Correction: Anti-schistosomal activity and ADMET properties of 1,2,5-oxadiazinane-containing compound synthesized by visible-light photoredox catalysis.

[This corrects the article DOI: 10.1039/D4MD00599F.].

Anti-Schistosomal activity and ADMET properties of 1,2,5-oxadiazinane-containing compound synthesized by visible-light photoredox catalysis.

The incorporation of saturated nitrogen-containing heterocycle 1,2,5-oxadiazinane into small molecules represents a compelling avenue in drug discovery due to its unexplored behavior within biological systems and incomplete protocols for synthesis. In this study, we present 1,2,5-oxadiazinane, an innovative heterocyclic bioisostere of piperizin-2-one and novel chemotype of the anti-schistosomal drug praziquantel (PZQ), which has been the only clinical drug available for three decades. PZQ is associated with significant drawbacks, including poor solubility, a bitter taste, and low metabolic stability. Therefore, the discovery of a new class of anti-schistosomal agents is imperative. To address this challenge, we introduce a pioneering method for the synthesis of 1,2,5-oxadiazinane derivatives through the cycloaddition of nitrones with N,N,N',N'-tetraalkyldiaminomethane in the presence of an IrIII complex photosensitizer. This transformative reaction offers a streamlined route to various kinds of 1,2,5-oxadiazinanes that is characterized by mild reaction conditions and broad substrate scope. Mechanistic investigations suggest that the photoredox pathway underlies the [3 + 3] photocycloaddition process. Thus, based on bioisosteric replacement, we identified a remarkable molecule as a new chemotype of a potent anti-schistosomal compound that not only exhibits superior solubility, but also retains the potent biological activity inherent to PZQ.

CCR7-independent transport of skin antigens occurs in the dermis.

Under homeostatic conditions, skin DCs migrate to regional LNs transporting self-antigens (self-Ags). The transport of self-Ags is considered to be critical for maintaining peripheral tolerance. Although the chemokine receptor CCR7 potently induces the migration of skin DCs to regional LNs, Ccr7(-/-) (Ccr7-KO) mice do not show skin auto-immune diseases. To resolve this inconsistency, we examined Ccr7-KO epidermis- or dermis-hyperpigmented transgenic (Tg) mice, in which the transport of skin self-Ags is traceable by melanin granules (MGs). Under CCR7-deficient conditions, the transport of epidermal MGs to regional LNs was impaired at 7 weeks of age. However, epidermal MGs could be transported when they had accumulated in the dermis. Ccr7-KO-dermis-pigmented Tg mice confirmed the presence of CCR7-independent transport from the dermis. Compared with WT-dermis-pigmented Tg mice, the amount of transported melanin and number of MG-laden CD11c(+) cells were both approximately 40% of the WT levels, while the number of MG-laden CD205(+) or CD207(+) cells decreased to about 10% in skin regional LNs of Ccr7-KO-dermis-pigmented Tg mice. Cell sorting highlighted the involvement of CD11c(+) cells in the CCR7-independent transport. Here, we show that CCR7-independent transport of skin self-Ags occurs in the dermis. This system might contribute to the continuous transport of self-Ags, and maintain peripheral tolerance.

A checkpoint in B-lymphopoiesis related to Notch resistance.

While murine B- and T-lymphopoiesis require overlapping molecules, they occur in separate organs: the bone marrow (BM) and the thymus, respectively. The BM microenvironment is incapable of supporting T-lymphopoiesis because of insufficient interactions of Notch1 with delta-like ligand (Dll). Notch1/Dll interactions also play a role in the suppression of B-lymphopoiesis in the thymus. However, it is still unclear whether the Notch1/Dll interaction alone explains why the thymus does not support B-lymphopoiesis. In this study, we compared the precursor population colonizing the thymus with that in the BM by culturing them on stromal cells expressing abundant Dll1. We demonstrated that Flt3(+) Il7r(+) B220(+) Cd19(+) BM cells gave rise to B cells under this condition. We defined them as resistant to Dll1. In the thymus, Dll1-resistant cells were undetectable. This suggested that the absence of Dll1-resistant cells might explain the absence of B-lymphopoiesis in the thymus.

Stochastic differentiation into an osteoclast lineage from cloned macrophage-like cells.

Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor κB ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6 days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3 days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on 'autopilot' rather than requiring specific signals to drive this process.

A Notch ligand, Delta-like 1 functions as an adhesion molecule for mast cells.

Mast cells (MCs) accumulate in chronic inflammatory sites; however, it is not clear which adhesion molecules are involved in this process. Recently, the expression of Notch ligands was reported to be upregulated in inflammatory sites. Although Notch receptors are known as signaling molecules that can activate integrins, their contributions to the adhesion of MCs have not been studied. In this study, we demonstrated that mouse MCs efficiently adhered to stromal cells forced to express a Notch ligand, Delta-like 1 (Dll1). Surprisingly, the adhesion was a consequence of direct cell-cell interaction between MCs and Dll1-expressing stromal cells rather than activation of downstream effectors of Notch receptor(s)-Dll1. The adhesion of MCs to Dll1-expressing stromal cells remained even when the cell metabolism was arrested. The recognition was blocked only by inhibition of Notch receptor(s)-Dll1 interaction by addition of soluble DLL1, or mAbs against Dll1 or Notch2. Taken together, these results indicate that Notch receptor(s) and Dll1 directly promote the adhesion of MCs to stromal cells by acting as adhesion molecules. This appreciation that Notch receptor-ligand interactions have an adhesion function will provide an important clue to molecular basis of accumulation of MCs to inflammatory sites.