The role of Dot1L activity in chondrogenic differentiation

摘要 这一新应用的目标是定义染色质修饰物DOT1L(端粒干扰因子)的作用 沉默-1)在正常的骨骼生长和发育中。DOT1L是唯一一种催化 组蛋白3(H3K79)赖氨酸79甲基化在基因表观遗传调控中的重要作用 表情。已经证明的DOT1L的唯一分子功能在于它的催化或 甲基转移酶(MT)结构域，然而新的研究表明DOT1L的非催化功能也 有助于调节基因表达和细胞分化。目前，人们对此知之甚少。 DOT1L调节骨骼的生长和发育。这代表着我们知识上的一个严重差距，因为 DOT1L靶向治疗方法正在研究中，作为儿童癌症的治疗方法。我们最近报道说， 肢体间充质中DOT1L表达的条件性丢失导致一种异常的骨骼表型 由长骨缩短、缺损区生长板(GP)软骨细胞增殖。有趣的是，小分子 抑制DOT1L催化活性并不损害软骨细胞的体外增殖，这表明潜在的但 DOT1L的非催化功能在这些复杂过程中的关键作用。DOT1L在体内的化学抑制作用 软骨性肢芽微团分析通过错误调节导致软骨细胞过早肥大 骨形态发生蛋白(BMP)信号通路的研究。我们实验室的新活体数据提供了令人信服的 催化失活的DOT1L突变蛋白可以修复软骨GP功能障碍和长骨的证据 肢体间充质中DOT1L功能条件性丧失的小鼠的生长缺陷。这些数据加在一起支持 我们新的中心假设是DOT1L通过：i)非催化活性来调节骨质疏松症的长骨生长 支持软骨细胞增殖；以及ii)限制软骨细胞成熟的MT依赖的活动。我们 组建了一支强大的研究团队，他们拥有骨骼生物学、表观遗传学和DOT1L生物学方面的专业知识 来解释这一新的假设。在目标1中，我们将确定DOT1L催化活性的功能要求 在体内软骨内骨生长中，使用新型DOT1L MT突变小鼠。我们的研究将确定一个 催化死亡的DOT1L突变体可以修复DOT1L cKOPrrx1小鼠的骨缺陷。我们将应用单细胞 转录分析以确定DOT1L调节的新基因和途径。在目标2中，我们将定义 DOT1L的调节功能，提供对软骨细胞分化的阶段特异性控制。使用DOT1L 基因敲除与MT突变细胞相比，机制研究将评估DOT1L催化与 非催化作用对软骨细胞增殖与肥大的影响。最后，ChIPseq实验将确定 全基因组甲基化模式(H3K79me2)与软骨细胞的增殖和成熟相关。 这些研究的结果有望在正常骨骼生长过程中产生关于DOT1L的新知识 和发展，对靶向DOT1L治疗的影响。

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.