Notch2 complements Notch1 to mediate inductive signaling that initiates early T cell development

Notch signaling is the dominant intercellular signaling input during the earliest stages of T cell development in the thymus. Although Notch1 is known to be indispensable, we show that it does not mediate all Notch signaling in pre-commitment stages: Notch2 initially works in parallel to promote early murine T cell development and antagonize other fates. Notch-regulated target genes before and after T-lineage commitment change dynamically, and we show that this partially reflects shifts in genome-wide DNA binding by RBPJ, the transcription factor activated by complex formation with the Notch intracellular domain. Although Notch signaling and transcription factor PU.1 can activate some common targets in pre-commitment T progenitors, Notch signaling and PU.1 activity have functionally antagonistic effects on multiple targets, delineating separation of pro-T cells from alternative PU.1-dependent fates. These results define a distinct mechanism of Notch signal response that distinguishes the initial stages of murine T-cell development. ChIP-seq; Five to ten million of Phase1 or Phase2 cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by additional 10 min with addition of formaldehyde up to 1%. The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco). Pelleted nucleus were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30sec sonication followed by 30sec rest, with max power. Five micro­g of anti-Rbpj mAbs (a mixture of 2.5 micro­g of 5313 (CST) and 2.5 micro­g of ab180588 (Abcam)) were pre-bound to Dynabeads anti-Rabbit Ig (Invitrogen) and then added to the diluted chromatin complexes in parallel aliquots. The samples were incubated overnight at 4 °C, then washed and eluted for 6 h at 65 °C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 micro­g/ml proteinase K). Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (E6240, NEB) and sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt. RNA-seq; Total RNA was isolated from 200,000 cells using an RNAeasy MicroKit (Qiagen). Libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB) from ~1 microg of total RNA following manufacturer's instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt.