RNA-seq of CDKN2A and EZH2-deficient pro-B and DN3 lymphocyte progenitors

This goal of this study was to identify genes that are deregulated in the absence of EZH2 in early lymphocyte progenitors. Due to a requirement for EZH2 to repress Cdkn2a in early B and T cell development we generated Cdkn2a-/-Ezh2fl/fl Il7racre/+ mice. We examined gene expression by RNA-sequencing in sorted pro-B cells (B220+CD19+CD43+), DN3 cells (Lin- CD25+ CD117-), from Cdkn2a-/- Ezh2fl/fl Il7racre/+ and Cdkn2a-/- Il7racre/+ control mice. Reads were aligned to the mm10 reference genome by Tophat2.1.0. Reads were assigned to genes using the htseq-count tool from HTSeq v 0.6.1 and gene annotations from Ensembl release 78. Differential expression was calculated across 3 independent replicates by EdgeR. We found that pro-B and DN3 cells remain specified to their respective lineages despite loss of EZH2. In contrast, loss of EZH2 led to expression of alternate lineage determinants in pro-B but not DN3 cells indicating that EZH2 is required for lineage commitment in B, but not T lymphocyte progenitors. Overall design: Expression profiling analysis of pro-B and DN3 cells in Cdkn2a-/-Ezh2fl/fl Il7racre/+ mice and Cdkna2a-/- Il7racre/+ controls generated by RNA-sequencing. 3 independent replicates per sample.