Lineage reprogramming of T lymphoid cells by myeloid transcription factors

Abstract

Lineage decisions in hematopoiesis involve the balanced action of transcription factors that establish and maintain cell fates by modulating gene expression. T lineage specification from multipotent hematopoietic progenitors progresses through a series of defined uncommitted stages in which differentiation into other lineages, including those of natural killer, macrophage and dendritic cells, remains possible. T lineage commitment, the conversion of pro-T cells into pre-T cells, involves a restriction of differentiation potentials of these early progenitors. We predicted that T lineage commitment is also determined by the balance of lineage associated transcription factors and hypothesized that expression of myeloid transcription factors, PU.1 and C/EBPalpha, in pre-T cells would instruct myeloid differentiation thereby restoring the normal capacity of pro-T cells to differentiate into alternative lineages. First, we found that the endogenous expression pattern of myeloid transcription factors was correlated with loss of alternative lineage potentials: PU.1 was down-regulated at the pro-T cell (DN1/2) to pre-T cell boundary (DN3/4) while C/EBPalpha was sharply down-regulated after the DN1 stage. Next, to investigate further this restriction point, we expressed C/EBPalpha and PU.1 in fully committed pre-T cells and found that C/EBPalpha induced the formation of functional macrophages. In contrast, PU.1 converted them into myeloid dendritic cells under identical culture conditions. C/EBPalpha-induced reprogramming is complex because upregulation of same but not all myelomonocytic markers required endogenous PU.1. Notch signaling partially inhibited C/EBPalpha-induced macrophage formation and completely blocked PU.1-induced dendritic cell formation. Likewise, expression of Notch or DATA-3, both essential T lineage transcription factors, inhibited C/EBPalpha-induced reprogramming. Finally, we also investigated whether a boundary exists after which T lineage cells become resistant to reprogramming. We found that, unlike DN3 and DN4 thymocytes, DP thymocytes and CD4+ splenic T cells were resistant to complete reprogramming mediated by C/EBPalpha and PU.1. In summary, we have demonstrated that expression of myeloid transcription factors in T lineage cells induces stage-specific changes in their phenotype. Our data show that committed T cell progenitors remain susceptible to the lineage instructive effects of myeloid transcription factors and suggest that Notch signaling induces T lineage restriction by downregulating C/EBPalpha and PU.1 in multilineage progenitors,