A Regulatory network of microRNAs confers lineage commitment during early developmental trajectories of B and T lymphocytes.

The commitment of hematopoietic multipotent progenitors (MPPs) towards a particular lineage involves activation of cell-type-specific genes and silencing of genes that promote alternate cell fates. Although the gene expression programs of early-B and early-T lymphocyte development are mutually exclusive, we show these cell types exhibit significantly correlated microRNA profiles. However, their corresponding miRNA-targetomes are distinct and predominated by transcripts associated with NK, DC, and myeloid lineages, suggesting miRNAs function in a cell-autonomous manner. Combinatorial expression of microRNAs: miR-186-5p, miR-128-3p, and miR-330-5p in MPPs significantly attenuate their myeloid differentiation potential due to the repression of myeloid-associated transcripts. Depletion of these miRNAs caused a pronounced de-repression of myeloid lineage targets in differentiating early-B and early-T cells, resulting in a mixed-lineage gene expression pattern. De novo motif analysis combined with an assay of promoter activities indicate that B, as well as T lineage determinants, drive the expression of these miRNAs in lymphoid lineages. Collectively, we present a paradigm that miRNAs are conserved between developing B and T lymphocytes, yet they target distinct sets of promiscuously-expressed lineage-inappropriate genes to suppress alternate cell-fate options. Thus, our studies provide a comprehensive compendium of miRNAs with functional implications for B and T lymphocyte development. Overall design: This study includes the miRNA-seq and Ago2-RIPseq profiles of Ebf1-/- progenitors, Early-B and Early-T cells; and H3K4me3 profiles of Ebf1-/- progenitors and Early-B cells. The Early-B cells were obtained by in vitro differentiation of Ebf1-/- progenitors by expressing transcription factor EBF1 (via MigR1-EBF1.ER vector), followed by culturing on OP9 stromal cells in presence of cytokines SCF, FLT3-L, IL-7; and 4-O-Hydroxy-tamoxifen for 2 days. The Early-T cells were obtained by culturing Ebf1-/- progenitors on OP9-DL1 stroma for 5 days in presence of cytokines SCF, FLT3-L and IL-7, in vitro. For miRNA-seq, miRNA libraries were prepared from total RNA samples using TrueSeq small RNA library preparation kit (Illumina). For Ago2-RIPseq, the INPUT (total RNA) or Ago2-immunoprecipitated (Ago2-IP) RNA was used for preparation of mRNA libraries using NEBNext Ultra II Directional RNA Library Prep kit (NEB). For ChIPseq, the INPUT (genomic DNA) or immunoprecipitated DNA (IP-DNA) was used for preparation of libraries using NEBNext Ultra II DNA Library prep kit (NEB) as per manufacturer?s instructions. All samples were processed in replicates and high-throughput sequencing was carried out on Illumina's HiSeq 2500.