Notch interaction with RUNX factors regulates initiation of the T-lineage program [ChIP-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: Ten million of Cas9-LPs with or without Notch stimulation were fixed with 1 mg/ml disuccinimidyl glutarate (Thermo Scientific) in PBS for 30 min at room temperature followed by an additional 10 min with addition of formaldehyde up to 1 %. The reaction was quenched by addition of 1/10 volume of 0.125 M glycine and the cells were washed with cold PBS. Pelleted nuclei were dissolved in lysis buffer (0.5 % SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl [pH 8.0], and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30 sec sonication followed by 30 sec rest, with high power. Five micrograms of anti-RUNX1 (abcam; ab23980), anti-CTCF (CST; 3418), anti-Med12 (Bethyl; A300-774A), or anti-p300 Abs (CST; 57625) were prebound to Dynabeads anti-rabbit Ig and then added to the diluted chromatin complexes. The samples were incubated overnight at 4 ?, then washed and eluted overnight at 65 ? in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1 % SDS, and 50 mg/ml proteinase K). Eluted chromatin fragments were cleaned up using ChIP DNA Clean & Concentrator (Zymo). ChIP-seq libraries were constructed using NEBNext Ultra ? DNA Library Prep with Sample Purification Beads (E7103S, NEB) and NEBNext Multiplex Oligos for Illumina (E7500S, NEB) and sequenced on Illumina NextSeq500 in single read mode with the read length of 75 nt.