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The thymus generates cells of the T cell lineage that seed the lymphatic and blood systems. Transcription factor regulatory networks control the lineage programming and maturation of thymic precursor cells. Whether extrathymic antigenic events, such as the microbial colonization of the mucosal tract also shape the thymic T cell repertoire is unclear. We show here that intestinal microbes influence the thymic homeostasis of PLZF-expressing cells in early life. Impaired thymic development of PLZF⁺ innate lymphocytes in germ-free (GF) neonatal mice is restored by colonization with a human commensal, Bacteroides fragilis, but not with a polysaccharide A (PSA) deficient isogenic strain. Plasmacytoid dendritic cells influenced by microbes migrate from the colon to the thymus in early life to regulate PLZF⁺ cell homeostasis. Importantly, perturbations in thymic PLZF⁺ cells brought about by alterations in early gut microbiota persist into adulthood and are associated with increased susceptibility to experimental colitis. Our studies identify a pathway of communication between intestinal microbes and thymic lymphocytes in the neonatal period that can modulate host susceptibility to immune-mediated diseases later in life.

PU.1 is an ETS-family transcription factor that plays a broad range of roles in hematopoiesis. A direct regulator of myeloid, dendritic-cell, and B cell functional programs, and a well-known antagonist of terminal erythroid cell differentiation, it is also expressed in the earliest stages of T-cell development of each cohort of intrathymic pro-T cells. Its expression in this context appears to give T-cell precursors initial, transient access to myeloid and dendritic cell developmental competence and therefore to represent a source of antagonism or delay of T-cell lineage commitment. However, it has remained uncertain until recently why T-cell development is also intensely dependent upon PU.1. Here, we review recent work that sheds light on the molecular biology of PU.1 action across the genome in pro-T cells and identifies the genes that depend on PU.1 for their correct regulation. This work indicates modes of chromatin engagement, pioneering, and cofactor recruitment (\"coregulator theft\") by PU.1 as well as gene network interactions that not only affect specific target genes but also have system-wide regulatory consequences, amplifying the impact of PU.1 beyond its own direct binding targets. The genes directly regulated by PU.1 also suggest a far-reaching transformation of cell biology and signaling potential between the early stages of T-cell development when PU.1 is expressed and when it is silenced. These cell-biological functions can be important to distinguish fetal from adult T-cell development and have the potential to illuminate aspects of thymic function that have so far remained the most mysterious.

Runt domain-related (Runx) transcription factors are essential for early T cell development in mice from uncommitted to committed stages. Single and double Runx knockouts via Cas9 show that target genes responding to Runx activity are not solely controlled by the dominant factor, Runx1. Instead, Runx1 and Runx3 are coexpressed in single cells; bind to highly overlapping genomic sites; and have redundant, collaborative functions regulating genes pivotal for T cell development. Despite stable combined expression levels across pro-T cell development, Runx1 and Runx3 preferentially activate and repress genes that change expression dynamically during lineage commitment, mostly activating T-lineage genes and repressing multipotent progenitor genes. Furthermore, most Runx target genes are sensitive to Runx perturbation only at one stage and often respond to Runx more for expression transitions than for maintenance. Contributing to this highly stage-dependent gene regulation function, Runx1 and Runx3 extensively shift their binding sites during commitment. Functionally distinct Runx occupancy sites associated with stage-specific activation or repression are also distinguished by different patterns of partner factor cobinding. Finally, Runx occupancies change coordinately at numerous clustered sites around positively or negatively regulated targets during commitment. This multisite binding behavior may contribute to a developmental “ratchet” mechanism making commitment irreversible.

DNA damage, especially DNA double strand breaks (DSBs) and replication stress, activates a complex post-translational network termed DNA damage response (DDR). Our review focuses on three PI3-kinase related protein kinases—ATM, ATR and DNA-PKcs, which situate at the apex of the mammalian DDR. They are recruited to and activated at the DNA damage sites by their respective sensor protein complexes—MRE11/RAD50/NBS1 for ATM, RPA/ATRIP for ATR and KU70–KU80/86 (XRCC6/XRCC5) for DNA-PKcs. Upon activation, ATM, ATR and DNA-PKcs phosphorylate a large number of partially overlapping substrates to promote efficient and accurate DNA repair and to coordinate DNA repair with other DNA metabolic events ( e.g. , transcription, replication and mitosis). At the organism level, robust DDR is critical for normal development, aging, stem cell maintenance and regeneration, and physiological genomic rearrangements in lymphocytes and germ cells. In addition to endogenous damage, oncogene-induced replication stresses and genotoxic chemotherapies also activate DDR. On one hand, DDR factors suppress genomic instability to prevent malignant transformation. On the other hand, targeting DDR enhances the therapeutic effects of anti-cancer chemotherapy, which led to the development of specific kinase inhibitors for ATM, ATR and DNA-PKcs. Using mouse models expressing kinase dead ATM, ATR and DNA-PKcs, an unexpected structural function of these kinases was revealed, where the expression of catalytically inactive kinases causes more genomic instability than the loss of the proteins themselves. The spectrum of genomic instabilities and physiological consequences are unique for each kinase and depends on their activating complexes, suggesting a model in which the catalysis is coupled with DNA/chromatin release and catalytic inhibition leads to the persistence of the kinases at the DNA lesion, which in turn affects repair pathway choice and outcomes. Here we discuss the experimental evidences supporting this mode of action and their implications in the design and use of specific kinase inhibitors for ATM, ATR and DNA-PKcs for cancer therapy.

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77 ᴳFᴾ transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim ⁻/⁻ mice had an increased number of GFP ʰⁱ cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.

Among the variety of tissue-resident NK-like populations recently distinguished from recirculating classical NK (cNK) cells, liver innate lymphoid cells (ILC) type 1 (ILC1s) have been shown to represent a distinct lineage that originates from a novel promyelocytic leukaemia zinc finger (PLZF)-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2, and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre–pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP), and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny. Although a limited set of ILC1 genes depended on PLZF for expression, characteristically includingIl7r,most of these genes were also differentially expressed between ILC1s and cNKs, indicating that PLZF together with other, yet to be defined, factors contribute to the divergence between these lineages.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

Immunotherapeutic targeting of surface regulatory proteins and pharmacologic inhibition of critical signaling pathways has dramatically shifted our approach to the care of individuals with B cell malignancies. This evolution in therapy reflects the central role of the B cell receptor (BCR) signaling complex and its co-receptors in the pathogenesis of B lineage leukemias and lymphomas. Members of the Fc receptor-like gene family ( FCRL1-6 ) encode cell surface receptors with complex tyrosine-based regulation that are preferentially expressed by B cells. Among them, FCRL1 expression peaks on naïve and memory B cells and is unique in terms of its intracellular co-activation potential. Recent studies in human and mouse models indicate that FCRL1 contributes to the formation of the BCR signalosome, modulates B cell signaling, and promotes humoral responses. Progress in understanding its regulatory properties, along with evidence for its over-expression by mature B cell leukemias and lymphomas, collectively imply important yet unmet opportunities for FCRL1 in B cell development and transformation. Here we review recent advances in FCRL1 biology and highlight its emerging significance as a promising biomarker and therapeutic target in B cell lymphoproliferative disorders.

The generation and differentiation of B lymphocytes (B cells) is a flexible process with many critical regulatory factors. Previous studies indicated that non-coding RNAs play multiple roles in the development of lymphocytes. However, little has been known about the circular RNA (circRNA) profiles and their competing endogenous RNA (ceRNA) networks in B-cell development and differentiation. Here, four B-cell subsets were purified from single-cell suspensions of mouse bone marrow. Then RNA sequencing (RNA-Seq) was used to display expression profiles of circRNAs, miRNAs and mRNAs during B-cell differentiation. 175, 203, 219 and 207 circRNAs were specifically expressed in pro-B cells, pre-B cells, immature B cells and mature B cells, respectively. The circRNA-associated ceRNA networks constructed in two sequential stages of B-cell differentiation revealed the potential mechanism of circRNAs in these processes. This study is the first to explore circRNA profiles and circRNA-miRNA-mRNA networks in different B-cell developmental stages of mouse bone marrow, which contribute to further research on their mechanism in B-cell development and differentiation.

Cell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.