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.
Outcomes from these studies are expected to generate new knowledge on Dot1L during normal skeletal growth
and development, with implications for targeting Dot1L therapeutically.