Single-cell deletion analyses show control of pro–T cell developmental speed and pathways by Tcf7, Spi1, Gata3, Bcl11a, Erg, and Bcl11b

Abstract

As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment.

TrAGEDy: Trajectory Alignment of Gene Expression Dynamics

“…Gzmb is a key enzyme expressed in cytotoxic immune cells such as Natural killer (NK) cells while Srgn plays a role in storage of Gzmb in T cells (19). Expression of such genes (and Cd244 which both TradeSeq and TrAGEDy detected) is in keeping with Zhou and colleagues’ (10) observation that the Bcl11b KO T cells appear to develop a NK cell-like phenotype.…”

“…We thus decided to apply TrAGEDy to real scRNA-seq datasets where the underlying process has been characterised. We first applied TrAGEDy to a scRNA-seq dataset of in vitro T cell development in WT and Bcl11b KO condition mice over two timepoints (10). The data contain cells undergoing the early stages of T cell development in the thymus from thymus-seeding precursors (TSP) to the double negative three stage (DN3), where the cells undergo beta selection (11).…”

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

“…These genes are normally transiently activated in DN1-DN2a cells but repressed during later T cell development by Bcl11b and E-proteins (Fig. 4e, Table S5) 32, 54, 55 .…”

“…Blue; Runx-activated genes, orange; Runx-inhibited genes. f, Area-proportional Venn diagrams display overlap between functionally responsive Runx target genes with previously determined functional target genes of PU.1 13 , GATA3 55 , TCF1 55 , and Bcl11b 32 .…”

“…The raw reads from cDNA and hashtag oligo libraries were processed as previously described 55 . Briefly, cDNA libraries were aligned to the mouse reference genome GRCm38/mm10 using CellRanger3 and the hashtag oligo libraries were quantified and demultiplexed using in-house tools (hashtag_tool) 55 . Seurat v4 85 was utilized for QC and downstream analysis.…”