Bcl11b defines pro-T identity by site-specific cofactor recruitment and by repressing Id2 and Zbtb16 [RNA-seq]

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: 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.