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 while 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.
Multipotent blood progenitor cells enter the thymus and begin a protracted differentiation process in which they gradually acquire T-cell characteristics while shedding their legacy of developmental plasticity. Notch signalling and basic helix-loop-helix E-protein transcription factors collaborate repeatedly to trigger and sustain this process throughout the period leading up to T-cell lineage commitment. Nevertheless, the process is discontinuous with separately regulated steps that demand roles for additional collaborating factors. This Review discusses new evidence on the coordination of specification and commitment in the early T-cell pathway; effects of microenvironmental signals; the inheritance of stem-cell regulatory factors; and the ensemble of transcription factors that modulate the effects of Notch and E proteins, to distinguish individual stages and to polarize T-cell-lineage fate determination.
The identities of the regulators that mediate commitment of hematopoietic precursors to the T lymphocyte lineage have been unknown. The last stage of T lineage commitment in vivo involves mechanisms to suppress natural killer cell potential, to suppress myeloid and dendritic cell potential, and to silence the stem cell or progenitor cell regulatory functions that initially provide T cell receptor–independent self-renewal capability. The zinc finger transcription factor Bcl11b is T cell–specific in expression among hematopoietic cell types and is first expressed in precursors immediately before T lineage commitment. We found that Bcl11b is necessary for T lineage commitment in mice and is specifically required both to repress natural killer cell–associated genes and to down-regulate a battery of stem cell or progenitor cell genes at the pivotal stage of commitment.
The first checkpoint in T cell development, beta selection, has remained incompletely characterized for lack of specific surface markers. We show that CD27 is upregulated in DN3 thymocytes initiating beta selection, concomitant with intracellular TCR-beta expression. Clonal analysis determined that CD27high DN3 cells generate CD4+CD8+ progeny with more than 90% efficiency, faster and more efficiently than the CD27low majority. CD27 upregulation also occurs in gammadelta-selected DN3 thymocytes in TCR-beta-/- mice and in IL2-GFP transgenic reporter mice where GFP marks the earliest emerging TCR-gammadelta cells from DN3 thymocytes. With CD27 to distinguish pre- and postselection DN3 cells, a detailed gene expression analysis defined regulatory changes associated with checkpoint arrest, with beta selection, and with gammadelta selection. gammadelta selection induces higher CD5, Egr, and Runx3 expression as compared to beta selection, but it triggers less proliferation. Our results also reveal differences in Notch/Delta dependence at the earliest stages of divergence between developing alphabeta and gammadelta T-lineage cells.
Summary Cells acquire their ultimate identities by activating combinations of transcription factors that initiate and sustain expression of the appropriate cell-type specific genes. T-cell development depends on the progression of progenitor cells through three major phases associated with distinct transcription factor ensembles that control their recruitment to and proliferation in the thymus, their lineage commitment, and their responsiveness to T-cell receptor (TCR) signals, before the allocation of cells to particular effector programs. All three phases are essential for proper T cell development, as are the mechanisms that determine the boundaries between each phase: cells failing to shut off one set of regulators before the next gene network phase is activated are predisposed to leukemic transformation.
GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.
GATA-3 is essential for T cell development from the earliest stages. However, highly abundant GATA-3 can drive T-lineage precursors to a non-T fate, depending on Notch signaling and developmental stage. GATA-3 overexpression blocked pro-T cell survival when Notch-Delta signals were present, but enhanced viability in their absence. In double-negative (DN1) and DN2 but not DN3 fetal thymocytes, GATA-3 overexpression rapidly induced mast cell lineage respecification with high frequency by direct transcriptional reprogramming. Normal DN2 thymocytes also displayed mast cell potential, when interleukin 3 and stem cell factor were added in the absence of Notch signaling. Our results suggest a close relationship between the pro-T and mast cell programs and a new role for Notch in T-lineage fidelity.
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).
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