Notch-dependent functional conversion of RUNX factors regulates the initiation of T-lineage program

Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)- and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1 binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain (Notch1-IC) directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex re-organization and genomic redeployment in initiating T-lineage program. The cell suspension obtained from the resected tumor specimens was processed according to 10×Genomics specifications, using the 10×Chromium Next GEM single-cell 3ʹ reagent kit and library construction kit. The cell suspension was processed in the Chromium Controller to create gel beads in emulsion (GEMs), incubated at a specific temperature to generate cDNA containing barcodes and Unique Molecular Identifiers (UMIs). Subsequently, the cDNA was washed, amplified, and its quality assessed, then enzymatically fragmented and size-selected, followed by adapter ligation and indexed PCR amplification. Finally, after quantification and quality assessment of the library, sequencing was performed on the NovaSeq 6000.