Transcriptional control of early T and B cell developmental choices.

T and B cells share a common somatic gene rearrangement mechanism for assembling the genes that code for their antigen receptors; they also have developmental pathways with many parallels. Shared usage of basic helix-loop-helix E proteins as transcriptional drivers underlies these common features. However, the transcription factor networks in which these E proteins are embedded are different both in membership and in architecture for T and B cell gene regulatory programs. These differences permit lineage commitment decisions to be made in different hierarchical orders. Furthermore, in contrast to B cell gene networks, the T cell gene network architecture for effector differentiation is sufficiently modular so that E protein inputs can be removed. Complete T cell-like effector differentiation can proceed without T cell receptor rearrangement or selection when E proteins are neutralized, yielding natural killer and other innate lymphoid cells.

Runx factors launch T cell and innate lymphoid programs via direct and gene network-based mechanisms.

Runx factors are essential for lineage specification of various hematopoietic cells, including T lymphocytes. However, they regulate context-specific genes and occupy distinct genomic regions in different cell types. Here, we show that dynamic Runx binding shifts in mouse early T cell development are mostly not restricted by local chromatin state but regulated by Runx dosage and functional partners. Runx cofactors compete to recruit a limited pool of Runx factors in early T progenitor cells, and a modest increase in Runx protein availability at pre-commitment stages causes premature Runx occupancy at post-commitment binding sites. This increased Runx factor availability results in striking T cell lineage developmental acceleration by selectively activating T cell-identity and innate lymphoid cell programs. These programs are collectively regulated by Runx together with other, Runx-induced transcription factors that co-occupy Runx-target genes and propagate gene network changes.

Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16.

Multipotent progenitor cells confirm their T cell-lineage identity in the CD4-CD8- double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.

Transcription factors specifically control change.

Transcription factors are defined by their sequence-specific binding to DNA and by their selective impacts on gene expression, depending on specific binding sites. The factor binding motifs in the DNA should thus represent a blueprint of regulatory logic, suggesting that transcription factor binding patterns on the genome (e.g., measured by ChIP-seq) should indicate which target genes the factors are directly controlling. However, although genetic data confirm high impacts of transcription factor perturbation in embryology, transcription factors bind to far more sites than the number of genes they dynamically regulate, when measured by direct perturbation in a given cell type. Also, deletion of carefully chosen transcription factor binding sites often gives disappointingly weak results. In a new study in the previous issue of Genes & Development, Lo and colleagues (pp. 1079-1095) reconcile these contradictions by using an elegant experimental system to directly compare the roles of transcription factor-binding site interaction in gene regulation maintenance with roles of the same factor-site interactions in gene regulation through developmental change. They examine Oct4:Sox2 shared target genes under maintained versus reinduced pluripotency conditions within the same cell clone. The results show that the same factor-site interaction impacts can appear modest in assays in developmental steady-state but are far more important as regulatory catalysts of developmental change.

A two-amino-acid substitution in the transcription factor RORγt disrupts its function in TH17 differentiation but not in thymocyte development.

The transcription factor RORγt regulates differentiation of the TH17 subset of helper T cells, thymic T cell development and lymph-node genesis. Although elimination of RORγt prevents TH17 cell-mediated experimental autoimmune encephalomyelitis (EAE), it also disrupts thymocyte development, which could lead to lethal thymic lymphoma. Here we identified a two-amino-acid substitution in RORγt (RORγtM) that 'preferentially' disrupted TH17 differentiation but not thymocyte development. Mice expressing RORγtM were resistant to EAE associated with defective TH17 differentiation but maintained normal thymocyte development and normal lymph-node genesis, except for Peyer's patches. RORγtM showed less ubiquitination at Lys69 that was selectively required for TH17 differentiation but not T cell development. This study will inform the development of treatments that selectively target TH17 cell-mediated autoimmunity but do not affect thymocyte development or induce lymphoma.

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment.

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.

Building a Human Thymus: A Pointillist View.

In this issue of Immunity, Zeng et al. use single-cell RNA sequencing analyses of rare samples to shed light on the emergence of thymic stromal cell types, the first developing T lymphocytes, and their possible pre-thymic precursors in the early human fetus.

Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding.

Transcription factors normally regulate gene expression through their action at sites where they bind to DNA. However, the balance of activating and repressive functions that a transcription factor can mediate is not completely understood. Here, we showed that the transcription factor PU.1 regulated gene expression in early T cell development both by recruiting partner transcription factors to its own binding sites and by depleting them from the binding sites that they preferred when PU.1 was absent. The removal of partner factors Satb1 and Runx1 occurred primarily from sites where PU.1 itself did not bind. Genes linked to sites of partner factor "theft" were enriched for genes that PU.1 represses despite lack of binding, both in a model cell line system and in normal T cell development. Thus, system-level competitive recruitment dynamics permit PU.1 to affect gene expression both through its own target sites and through action at a distance.

Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells.

How chromatin reorganization coordinates differentiation and lineage commitment from hematopoietic stem and progenitor cells (HSPCs) to mature immune cells has not been well understood. Here, we carried out an integrative analysis of chromatin accessibility, topologically associating domains, AB compartments, and gene expression from HSPCs to CD4+CD8+ T cells. We found that abrupt genome-wide changes at all three levels of chromatin organization occur during the transition from double-negative stage 2 (DN2) to DN3, accompanying the T lineage commitment. The transcription factor BCL11B, a critical regulator of T cell commitment, is associated with increased chromatin interaction, and Bcl11b deletion compromised chromatin interaction at its target genes. We propose that these large-scale and concerted changes in chromatin organization present an energy barrier to prevent the cell from reversing its fate to earlier stages or redirecting to alternatives and thus lock the cell fate into the T lineages.

Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity.

T cell development comprises a stepwise process of commitment from a multipotent precursor. To define molecular mechanisms controlling this progression, we probed five stages spanning the commitment process using RNA-seq and ChIP-seq to track genome-wide shifts in transcription, cohorts of active transcription factor genes, histone modifications at diverse classes of cis-regulatory elements, and binding repertoire of GATA-3 and PU.1, transcription factors with complementary roles in T cell development. The results highlight potential promoter-distal cis-regulatory elements in play and reveal both activation sites and diverse mechanisms of repression that silence genes used in alternative lineages. Histone marking is dynamic and reversible, and though permissive marks anticipate, repressive marks often lag behind changes in transcription. In vivo binding of PU.1 and GATA-3 relative to epigenetic marking reveals distinctive factor-specific rules for recruitment of these crucial transcription factors to different subsets of their potential sites, dependent on dose and developmental context.

Programming for T-lymphocyte fates: modularity and mechanisms.

T-cell development in mammals is a model for lineage choice and differentiation from multipotent stem cells. Although T-cell fate choice is promoted by signaling in the thymus through one dominant pathway, the Notch pathway, it entails a complex set of gene regulatory network and chromatin state changes even before the cells begin to express their signature feature, the clonal-specific T-cell receptors (TCRs) for antigen. This review distinguishes three developmental modules for T-cell development, which correspond to cell type specification, TCR expression and selection, and the assignment of cells to different effector types. The first is based on transcriptional regulatory network events, the second is dominated by somatic gene rearrangement and mutation and cell selection, and the third corresponds to establishing a poised state of latent regulator priming through an unknown mechanism. Interestingly, in different lineages, the third module can be deployed at variable times relative to the completion of the first two modules. This review focuses on the gene regulatory network and chromatin-based kinetic constraints that determine activities of transcription factors TCF1, GATA3, PU.1, Bcl11b, Runx1, and E proteins in the primary establishment of T-cell identity.

Speed and navigation control of thymocyte development by the fetal T-cell gene regulatory network.

T-cell differentiation is a tightly regulated developmental program governed by interactions between transcription factors (TFs) and chromatin landscapes and affected by signals received from the thymic stroma. This process is marked by a series of checkpoints: T-lineage commitment, T-cell receptor (TCR)ß selection, and positive and negative selection. Dynamically changing combinations of TFs drive differentiation along the T-lineage trajectory, through mechanisms that have been most extensively dissected in adult mouse T-lineage cells. However, fetal T-cell development differs from adult in ways that suggest that these TF mechanisms are not fully deterministic. The first wave of fetal T-cell differentiation occurs during a unique developmental window during thymic morphogenesis, shows more rapid kinetics of differentiation with fewer rounds of cell division, and gives rise to unique populations of innate lymphoid cells (ILCs) and invariant γδT cells that are not generated in the adult thymus. As the characteristic kinetics and progeny biases are cell-intrinsic properties of thymic progenitors, the differences could be based on distinct TF network circuitry within the progenitors themselves. Here, we review recent single-cell transcriptome data that illuminate the TF networks involved in T-cell differentiation in the fetal and adult mouse thymus.

Cold Spring Harbor Laboratory 2022: emerging insights and viewpoints in immunology.

Some of the current and former organizers of the Cold Spring Harbor Laboratory (CSHL) 'Gene Expression and Signaling in the Immune System' (GESIS) meeting offer opinions on emerging questions in immunology, discussing the strong value of this recurring scientific meeting in the field.

Multiple Curricula for B Cell Developmental Programming.

B-1 B cells differ from conventional B-2 B cells functionally, but how these differences relate to the ontogeny of these lineages has been unclear. Two recent Immunity articles, Kristiansen et al. (2016) and Montecino-Rodriguez et al. (2016), now provide insight into the origins of B-1 and B-2 B cells, revealing a multi-layered developmental program and successive waves of B cell precursors.

Runx1 and Runx3 drive progenitor to T-lineage transcriptome conversion in mouse T cell commitment via dynamic genomic site switching.

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.

Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1.

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.