Inhibition of Dot1L Histone Methyltransferase Expands Bone Injury-Responsive CXCL12⁺ Stromal Progenitors
Adult bone marrow contains a heterogeneous network of skeletal stromal and progenitor cells (SSPC) that maintain bone homeostasis, support the hematopoietic niche, and drive the regenerative responses to injury. Despite their central role in bone regeneration, the mechanisms that regulate injury-induced SSPC activation and fate transitions remain incompletely understood. Here we identify Disruptor of telomeric silencing 1 like (DOT1L), the sole histone methyltransferase responsible for H3K79 methylation, as a critical regulator that restrains stromal activation. Using complementary genetic, pharmacologic, single-cell transcriptomic and injury models, we show that either Prrx1 lineage Dot1L haploinsufficiency or acute pharmacological Dot1L inhibition promote SSPC expansion, but through distinct programs: haploinsufficiency drives the expansion of Cxcl12+ CAR cells, whereas acute pharmacologic inhibition enriches fibroblastic-like stromal states. Single Cell Regulatory Network Inference and Clustering (SCENIC) analysis identifies DOT1L as a stabilizer of homeostatic marrow-supportive transcriptional programs, whose disruption facilitates transition to injury responsive stromal states. Partial loss of Dot1L in the Prrx1 lineage enhances injury-induced intramedullary mineralization in vivo. Collectively, these findings establish DOT1L as a gatekeeper of the stromal progenitor state that restrains lineage commitment and restrains the magnitude of the SSPC response following injury.