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.

PU.1 (encoded by Spi1), an ETS-family transcription factor with many hematopoietic roles, is highly expressed in the earliest intrathymic T cell progenitors but must be down-regulated during T lineage commitment. The transcription factors Runx1 and GATA3 have been implicated in this Spi1 repression, but the basis of the timing was unknown. We show that increasing Runx1 and/or GATA3 down-regulates Spi1 expression in pro–T cells, while deletion of these factors after Spi1 down-regulation reactivates its expression. Leveraging the stage specificities of repression and transcription factor binding revealed an unconventional but functional site in Spi1 intron 2. Acute Cas9-mediated deletion or disruption of the Runx and GATA motifs in this element reactivates silenced Spi1 expression in a pro–T cell line, substantially more than disruption of other candidate elements, and counteracts the repression of Spi1 in primary pro–T cells during commitment. Thus, Runx1 and GATA3 work stage specifically through an intronic silencing element in mouse Spi1 to control strength and maintenance of Spi1 repression during T lineage commitment.

Establishment of cell lineage identity from multipotent progenitors is controlled by cooperative actions of lineage-specific and stably expressed transcription factors, combined with input from environmental signals. Lineage-specific master transcription factors activate and repress gene expression by recruiting consistently expressed transcription factors and chromatin modifiers to their target loci. Recent technical advances in genome-wide and multi-omics analysis have shed light on unexpected mechanisms that underlie more complicated actions of transcription factors in cell fate decisions. In this review, we discuss functional dynamics of stably expressed and continuously required factors, Notch and Runx family members, throughout developmental stages of early T cell development in the thymus. Pre-and post-commitment stagespecific transcription factors induce dynamic redeployment of Notch and Runx binding genomic regions. Thus, together with stage-specific transcription factors, shared transcription factors across distinct developmental stages regulate acquisition of T lineage identity.

No abstract

AMBRA1 is a member of the BECN1 (BECLIN1) complex protein, and it plays a role in autophagy, cell death, tumorigenesis, and proliferation. We recently reported that on TCR stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T cell activation, clonal expansion, and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability towards specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naïve T cell hyperactivity. The T cell hyperactivity observed in this study were similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naïve T cells were reduced in Ambra1-cKO mice, as same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.