Inhibition of Dot1L Histone Methyltransferase Expands Bone Injury-Responsive CXCL12⁺ Stromal Progenitors

The regenerative capacity of adult bone relies on the rapid activation and lineage engagement of skeletal stromal and progenitor cells (SSPCs). While signaling pathways that regulate these processes have been extensively studied, the epigenetic mechanisms that constrain progenitor activation and lineage permissiveness during adult bone repair remain poorly defined. Disruptor of telomeric silencing 1 like (Dot1L), the sole histone methyltransferase responsible for H3K79 methylation, is essential for skeletal development, yet its function in adult skeletal regeneration has not been established. Here, we identify Dot1L as a key epigenetic regulator that limits the early regenerative response to bone injury. Genetic reduction of Dot1L activity in the Prrx1⁺ mesenchymal lineage enhances stromal progenitor activation, proliferative engagement, and differentiation capacity, revealing a previously unrecognized role for Dot1L in restraining progenitor responsiveness in adult bone. Notably, acute pharmacologic inhibition of Dot1L using the selective H3K79 methyltransferase inhibitor EPZ-5676 similarly enhances early progenitor activation, indicating that reduced Dot1L enzymatic activity is sufficient to modulate regenerative engagement. At the cellular level, reduced Dot1L activity expands injury-responsive Cxcl12⁺ stromal populations and increases osteogenic progenitor abundance in vivo following injury. Consistent with these cellular changes, Dot1L reduction is associated with accelerated early bone formation in vivo. Collectively, these findings position Dot1L as an epigenetic gatekeeper that constrains early progenitor activation during the initial phase of adult skeletal repair.