EBF1 and PAX5 exert dose dependent epigenetic repression of T-lineage potential [scMultiome]

B-lymphocyte development is dependent on the coordinated action of transcription factors controlling the expression of stage and lineage specific genes. To better understand gene regulation and changes in the epigenetic landscape in early B-cell development, we conducted combined SC-RNA/ATAC seq analysis of bone marrow progenitor populations, allowing us to build a developmental trajectory based on changes in epigenetic accessibility. This analysis identified a rapid shift in DNA accessibility, where T-lineage primed progenitors gained the epigenetic landscape of B-lymphocytes. The epigenetic switch correlated well with initiation of EBF1 and PAX5 transcription as well as their functional activity. As ectopic expression of EBF1 in Pax5-/- pro-B cells was not sufficient to mediate either the full activation of the B-lineage program of silencing of T-lineage associated genes, we conclude that PAX5 and EBF1 are independently essential for the establishment of the epigenetic landscape in normal B-cell development. This idea was supported by the finding that pro-B cells from mice with combined heterozygote deletion of the Ebf1 and Pax5 genes, carried a non-differentiated epigenetic landscape and expressed T-lineage associated genes. The importance of epigenetic silencing for the preservation of B-cell fate was supported by the finding that inhibition of the histone methylases EZH1 and EZH2 in pro-B cells, allowed for the activation of T-lineage genes and generation of T-lineage cells in response to Notch signaling. Hence, B-lymphoid commitment is associated with a transcription factor dose dependent epigenetic switch, silencing an inherent T-lineage potential in early lymphoid progenitors.