Evolutionarily divergent notch regulation underpins the initiation of human T-cell development

Abstract Hematopoietic stem and progenitor cells (HSPCs) differentiate into diverse blood cell lineages through intricate genetic and epigenetic decision processes. How these decision points are regulated has not been fully elucidated. We aimed to identify the factors influencing human HSPC differentiation into T lymphocytes using a single-cell RNA sequencing-based lineage tracing approach. Our approach revealed that a bifurcation between the mast cell and lympho-myeloid lineage trajectories occurs early, with lymphoid and myeloid clones remaining interconnected until later time points, when T cell lineage commitment is established. We identified a new mechanism that primes certain HSPCs to become T cells, even though all cells express NOTCH receptors and are exposed to Notch ligands. GXYLT2, a xylosyltransferase that regulates NOTCH activation post-translationally, was differentially expressed in HSPCs with a stronger T cell clonal bias. Considering this cell-intrinsic, heritable priming state, we investigated the factors influencing lineage priming before Notch ligand exposure. We discovered that class 1 histone deacetylases (HDACs) favor lymphoid outcomes, with HDAC inhibition promoting mast cell outcomes and activation enhancing T-lymphoid differentiation. Additionally, we identified binding regions for IRF1 and GATA3, which are early predictors of human lymphoid outcomes within the GXYLT2 promoter region. Further analysis of the GXYLT2 loci revealed a retrotransposon element that acts as a regulator of the loci, appearing to be evolutionarily divergent but conserved among primates. Removing this element increased T cell bias in our cultures. Our findings revealed a previously unrecognized role for GXYLT2 and its loci in influencing early decisions toward human T cell development outcomes.