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

Abstract

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.

Transcription factors regulate early T cell development via redeployment of other factors

“…In pro-T cell stages, Runx1 is particularly interesting, among consistently expressed TFs. [23,35] Bifunctional transcription factor, Runx1, is a functionally important interacting molecule for PU.1 and contributes to PU.1-mediated gene regulation in Phase 1, [27] while collaborating with Bcl11b to establish T lineage identity through activating and repressing Bcl11b-regulated target genes in T lineage committed Phase 2 stages. [33] Another representative of constitutively expressed factors with context dependent roles for early T cell development is Notch family members.…”

“…[67] The similarity of in vitro-generated Phase 1 and Phase 2 subsets and their in vivo counterparts are validated by transcriptome analysis. [32,35] The T . They stay at Phase 1 for 5 days, followed by transition through commitment by around 6-9 days, and most of cells progress into Phase 2 after 10 days total.…”

“…Specific loss of Runx proteins or cell surface Notch proteins was observed at 3 days post-sgRNA introduction (day 5 or 13 of overall culture for Phase 1 or Phase 2, respectively). [34,35] Thus, we succeeded in establishing the acute and stage-specific deletion system before and after T lineage commitment, which allows us to analyze effects of targeted genes disruption within 3 days.…”

“…[27] We found that Runx1 and Runx3 are substantially co-expressed in individual Phase 1 cells, whereas Runx3 expression declines and Runx1 expression slightly increases in Phase 2. [32,35] Therefore, we tested collaborative effects of Runx3 with Runx1 in early T cell development using a CRISPR/Cas9-mediated acute and stage-specific deletion system (Figure 3). [35] As we expected, Runx1 or Runx3 single KO caused very few gene expression changes in Phase 1.…”

“…[12] In this review, we focus on an unexpected new layer of regulatory network, mediated by stage-specific TFs, PU.1 and Bcl11b, with stage-specific actions of stably expressed and continuously required factors, Runx and Notch family members, in the T lineage commitment. [26,27,[33][34][35]…”