Notch signaling is the dominant intercellular signaling input during the earliest stages of T cell development in the thymus. Although Notch1 is known to be indispensable, we show that it does not mediate all Notch signaling in precommitment stages: Notch2 initially works in parallel to promote early murine T cell development and antagonize other fates. Notch-regulated target genes before and after T lineage commitment change dynamically, and we show that this partially reflects shifts in genome-wide DNA binding by RBPJ, the transcription factor activated by complex formation with the Notch intracellular domain. Although Notch signaling and transcription factor PU.1 can activate some common targets in precommitment T progenitors, Notch signaling and PU.1 activity have functionally antagonistic effects on multiple targets, delineating separation of pro-T cells from alternative PU.1-dependent fates. These results define a distinct mechanism of Notch signal response that distinguishes the initial stages of murine T cell development.
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.
Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two murine Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.
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.
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