Notch interaction with RUNX factors regulates initiation of the T-lineage program [RNA-seq]

RUNX factors play an essential role in the development of T cell. Dynamic interacting partner switching of RUNX1 induces the redeployment of RUNX1 at the T-lineage commitment checkpoint. Here, we attempted to reveal functional differences in RUNX factors between lymphoid progenitor (LP) and Noth-stimulated the earliest T progenitor stage, phase1. We identified CTCF as a LP-specific RUNX1-interacting partner. Indeed, LP-specific RUNX1 binding genomic sites had significantly enriched CTCF consensus motif and co-occupied with CTCF. After Notch stimulation, Notch1-IC directly interacted with RUNX1 and recruited it to the Notch-regulated T-signature gene loci with the Mediator/p300 transcriptional activation complex. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and its binding partners revealed that the RUNX1/CTCF complex in LP and the RUNX1/Mediator/p300 complex in phase1 negatively and positively regulate T-signature genes expression, respectively. Our results indicate that the Notch-mediated functional conversion of RUNX factors; re-organization of protein complexes and redeployment of genomic binding sites, has a crucial role in the initiation of T-lineage program. Overall design: Cas9-LPs with or without Notch stimulation were transduced with various sgRNAs. Total RNA was isolated from samples of 3 × 105 cultured cells using a RNeasy Micro Kit (Qiagen). 500 ng of total RNA was subjected to the 3'mRNA library preparation with QuantSeq 3' mRNA-Seq Library Prep Kit FWD (LEXOGEN) according to the manufacturer's instructions. After the PCR step, size distribution and yield of the library was determined by the D1000 high sensitivity tape station (Agilent) or Agilent High Sensitivity DNA kit on the bioanalyzer (Agilent). The pooled libraries were loaded on the Illumina Nextseq500 platform and analyzed by 75bp single read.