Integrative analysis of 3D nucleome and chromatin accessibility reveals a chromatin barrier established for T-lineage commitment during early T cell development [Dnase-Seq, HiC-Seq, Mnase-Seq, RNA-Seq]. Mus musculus

Hematopoiesis consists of step-wise commitment of multiple distinct intermediate differentiation stages before mature blood cells are generated. Although extensive studies have been performed on regulatory pathways in mature blood cells, few have examined chromatin reorganization underlying the changes in transcription programs from early progenitors of hematopoiesis to mature immune cells. In particular, what roles chromatin organization plays in the cell fate commitment during the differentiation of early hematopoietic progenitor cells to committed T cells remains unclear. Here, we carried out an integrative analysis of 3D nucleome, chromatin accessibility and gene expression from hematopoietic stem cells (HSC) to CD4/CD8 double positive (DP) T cells. Analysis of these data sets revealed extensive reorganization of A/B compartments, topologically associating domains (TADs), and chromatin accessibility at transcriptional regulatory regions during the development process. Remarkably, all three levels of chromatin organization display a monotonic pattern of change. While gradual and progressive changes in chromatin reorganization was observed at most development stages, an abrupt genome-wide change in A/B compartment structure, TAD domain scores and chromatin accessibility occurred during the transition from double negative stage 2 (DN2) to DN3, which was accompanied with the loss of cell fate plasticity of DN3 to differentiate to alternative lineages, suggesting that a chromatin barrier is established at the DN3 stage to lock the cells in the T cell fate. This chromatin barrier may be reinforced by another genome-wide reorganization of chromatin at the DN4-to-DP transition. The binding of PU.1, a key factor for the fate choice of early progenitor cells, and BCL11B, critically required for T cell commitment at later stages, was associated with increased long-distance interaction and chromatin accessibility. Deletion of Bcl11b led to decreased chromatin interaction for BCL11B target genes, suggesting that it may contribute to the establishment of the 3D nucleome structure required for the lineage differentiation and commitment. Overall design: To understand the contribution of chromatin interaction landscape to early development stages of hematopoesis, we generated genome-wide profiles of chromatin interaction (with an improved HiC protocal), chromatin accessibility (with scDNase-Seq) and gene expression (with RNA-Seq) from hematopoietic stem cells (HSC) and its immediate progeny (MPP) to common lymphocyte progenitor cells (CLP), early T progenitor cells (ETP or DN1), double negative stage 2 (DN2), DN3, DN4 and double positive (DP) cells. To examine the impact of BCL11B on gene expression, we conditionally knocked out Bcl11b in DP cells and mearured the expression change by RNA-Seq. To examine the effect of BCL11B on chromatin interaction and accessibility, we conditional knocked out Bcl11b in Naive CD4+ T cells and measured change in chromatin interaction by Hi-C, chromatin accessibility by scDNase-Seq, and expression by RNA-Seq.