MLL3 and MLL4 sustain hematopoietic stem cell multipotency by opposing a B-cell default state.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are sustained by networks of transcription factors and epigenetic regulators that prime lineage-specific programs yet maintain multipotency. Two epigenetic regulators, MLL3 and MLL4, play important but distinct roles in maintaining this balance. MLL3 promotes HSC differentiation, whereas MLL4 opposes differentiation. These activities are essential for both normal homeostasis and leukemia suppression, yet it is not clear how MLL3 and MLL4 regulate HSC and MPP gene expression to control HSC/MPP fate decisions. To resolve these mechanisms, we performed an extensive series of single-cell genomic studies after conditionally deleting Mll3, Mll4 or both genes together. Mll3 deletion had only limited effects on HSC/MPP enhancer networks at steady state, whereas Mll4 deletion led to precocious activation of myeloid enhancers. Surprisingly, compound Mll3/4 deletion eliminated all myeloid, erythroid, and megakaryocytic potential within the hematopoietic hierarchy and caused all progenitors to rapidly default to a B-cell-like identity. These changes were accompanied by widespread inactivation of HSC/MPP enhancers and superenhancers and ectopic activation of B-cell superenhancers. Disabling MLL3/4 histone methyltransferase activity did not recapitulate the pervasive changes in cell identity that were observed when MLL3 and MLL4 were fully inactivated, indicating that MLL3 and MLL4 activate HSC/MPP enhancers independently from their enzymatic activities. Our findings show that HSC/MPP multipotency requires sustained tension between MLL3/4-dependent enhancers that maintain myeloid, erythroid, and megakaryocyte potential and MLL3/4-independent enhancers that prime B-cell identity. MLL3 and MLL4 therefore serve as critical linchpins of multilineage hematopoiesis.