Bcl11b defines pro-T identity by site-specific cofactor recruitment and by repressing Id2 and Zbtb16.

Multipotent progenitors confirm their T-lineage identity at the DN2a to DN2b pro-T cell transition, when expression of the essential transcription factor Bcl11b begins. This study defines the molecular basis of direct and indirect actions of Bcl11b in acquisition of T cell identity. In vivo and in vitro stage-specific deletions identify functional regulation targets genome-wide for mechanistic analysis. Bcl11b can associate with multiple co-factors and its direct action is needed to recruit these to selective target sites. Sites of Bcl11b-dependent cofactor recruitment are enriched at functionally regulated targets, and deletion of individual cofactors can relieve repression of many Bcl11b-repressed genes. In parallel, Bcl11b indirectly regulates a subset of its target genes by a gene network circuit via Id2 and Zbtb16, which are directly repressed by Bcl11b and control distinct alternative programs. Overall design: ChIP-seq; Ten million of BM-derived DN3 cells or Schd.adh.2c2 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. Six µg per 107 cells of anti-Bcl11b Abs (a mixture of A300-383A (Bethyl), A300-385A (Bethyl), ab18465 (Abcam) and 12120 (CST)), anti-Chd4 (A301-081A), anti-Mta2 (sc-9447), anti-Hdac2 (ab12169), anti-Rest (12C11-1B11), anti-Ring1b (A302-869A), anti-LSD1 (ab17721) or anti-Runx1 (ab23980) were hybridized to Dynabeads anti-Rabbit, Dynabeads anti-Mouse or Dynabeads Protein A/G (Invitrogen) and added to the diluted chromatin complex. They were incubated over night at 4°C, then washed and eluted for 6hr at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 µg/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 300,000 cells using an RNAeasy MicroKit (Qiagen). Libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB) from ~1 µg of total RNA following manufacturer's instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt. Please note that each *peaks.csv file was generated from both rep1 & rep2 and is linked to the corresponding rep1 sample records.