Abstract The goal of this new application is to define the role of the chromatin modifier Dot1L (Disruptor of telomeric silencing-1 like) in normal skeletal growth and development. Dot1L is the only enzyme that catalyzes the methylation of lysine 79 in histone 3 (H3K79), which plays an important role in the epigenetic regulation of gene expression. The only molecular function that has been demonstrated for Dot1L resides in its catalytic or methyltransferase (MT) domain, however new studies indicate that non-catalytic functions of Dot1L also contributes to the regulation of gene expression and cell differentiation. Currently, very little is known about how Dot1L regulates skeletal growth and development. This represents a critical gap in our knowledge because Dot1L targeted approaches are being studied as therapies for pediatric cancers. We recently reported that the conditional loss of Dot1L expression in limb mesenchyme induced an aberrant skeletal phenotype characterized by long bone shortening, and defects in growth plate (GP) chondrocyte proliferation. Interestingly, small molecule inhibition of Dot1L catalytic activity did not impair chondrocyte proliferation in vitro, suggesting an underlying but critical role for non-catalytic functions of Dot1L in these complex processes. Chemical inhibition of Dot1L in chondrogenic limb bud micromass assays resulted in premature chondrocyte hypertrophy through mis-regulation of the Bone morphogenetic protein (Bmp) signaling pathway. New in vivo data from our lab provides compelling evidence that a catalytic inactive Dot1L mutant protein can restore the cartilage GP dysfunction and long bone growth deficits in mice with conditional loss of Dot1L function in limb mesenchyme. Together, these data, support our novel central hypothesis that Dot1L regulates long bone growth at the GP through: i) non-catalytic activities that support chondrocyte proliferation; and ii) MT-dependent activities which restrict chondrocyte maturation. We have assembled a strong team of investigators with expertise in skeletal biology, epigenetics, and Dot1L biology to address this novel hypothesis. In Aim 1, we will establish the functional requirement for Dot1L catalytic activity in endochondral bone growth in vivo, using novel Dot1L MT mutant mice. Our studies will determine whether a catalytic-dead Dot1L mutant can rescue skeletal defects in Dot1L cKOPrrx1 mice. We will apply single cell transcriptomic analyses to identify novel Dot1L-regulated genes and pathways. In Aim 2, we will define the regulatory functions of Dot1L that provide stage-specific control of chondrogenic differentiation. Using Dot1L knockout versus MT mutant cells, mechanistic studies will assess the direct contribution of Dot1L catalytic versus non-catalytic functions to chondrocyte proliferation versus hypertrophy. Lastly, ChIPseq experiments will identify genome-wide methylation patterns (H3K79me2) associated with chondrocyte proliferation and maturation. Ou...