The role of Dot1L activity in chondrogenic differentiation

Dot1L 活性在软骨分化中的作用

基本信息

批准号：
10669804
负责人：
Rosaria M. Guzzo
金额：
$ 47.23万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-21 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10669804
关键词：
Address Biological Assay Biology Bone Development Bone Growth Bone Morphogenetic Proteins CRISPR/Cas technology Cartilage Catalytic Domain Cell Differentiation process Cells ChIP-seq Chemicals Child Chondrocytes Chondrogenesis Chromatin Chromatin Remodeling Factor Complex Data Defect Development Disease Engineering Enzymes Epigenetic Process Epiphysial cartilage Female Functional disorder Future Gatekeeping Gene Expression Gene Expression Regulation Genes Genetic Transcription Goals Growth Growth and Development function Hindlimb Histones Hypertrophy Impairment In Vitro Knock-out Knowledge Limb Bud Limb structure Lysine Malignant Childhood Neoplasm Mesenchyme Methylation Methyltransferase Molecular Molecular Conformation Morphology Mus Mutant Strains Mice Outcome Study Pathologic Pathway interactions Pattern Phenotype Physiologic Ossification Physiological Play Post-Translational Protein Processing Process Proliferating Proteins Publishing Regulation Reporting Research Personnel Role Signal Pathway Signal Transduction Skeletal Development Technology Testing Therapeutic adverse outcome bone cartilage cell conditional knockout epigenetic regulation experimental study gene network genome wide methylation genome-wide analysis histone methyltransferase histone modification in vivo innovation insight long bone male methylation pattern mutant novel pharmacologic premature programs protein function single-cell RNA sequencing skeletal small molecule targeted treatment therapeutic target transcriptome sequencing transcriptomics

项目摘要

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.

抽象的该新应用的目的是定义染色质修饰剂点的作用（端粒的破坏器在正常的骨骼生长和发育中，沉默1。 dot1l是唯一催化催化的酶赖氨酸79在组蛋白3（H3K79）中的甲基化，在基因的表观遗传调节中起重要作用表达。用于DOT1L的唯一分子函数属于其催化或甲基转移酶（MT）结构域，但是新的研究表明，DOT1L的非催化功能也有助于调节基因表达和细胞分化。目前，关于如何 DOT1L调节骨骼生长和发育。这代表了我们知识上的关键差距，因为正在研究DOT1L靶向方法作为儿科癌症的疗法。我们最近报道了肢体间充质中DOT1L表达的有条件丧失诱导的异常骨骼表型表征通过长骨缩短和生长板（GP）软骨细胞增殖的缺陷。有趣的是，小分子 DOT1L催化活性的抑制不会损害体外软骨细胞的增殖，这表明是潜在的但 DOT1L在这些复杂过程中非催化功能的关键作用。化学抑制dot1l 软骨肢体芽微瘤测定导致过早的软骨细胞肥大通过错误调节骨形态发生蛋白（BMP）信号通路的途径。我们实验室的新体内数据提供了引人注目的证据表明催化非活性DOT1L突变蛋白可以恢复软骨GP功能障碍和长骨小鼠的生长不足，肢体间充质中的DOT1L功能有条件丧失。这些数据在一起，支持我们的新型中心假设，即DOT1L通过以下方式调节GP的长骨生长：i）非催化活性支持软骨细胞增殖； ii）限制软骨细胞成熟的MT依赖性活动。我们已经组建了一个强大的研究人员，具有骨骼生物学，表观遗传学和DOT1L生物学方面的专业知识解决这个新颖的假设。在AIM 1中，我们将建立DOT1L催化活性的功能要求在体内的软骨内骨生长中，使用新型的Dot1l MT突变小鼠。我们的研究将确定一个是否催化死亡的DOT1L突变体可以在DOT1L CKOPRRX1小鼠中挽救骨骼缺陷。我们将应用单个单元格转录组分析以鉴定新型DOT1L调节的基因和途径。在AIM 2中，我们将定义 DOT1L的调节函数可提供软骨分化的特定阶段控制。使用dot1l 敲除与MT突变细胞，机械研究将评估DOT1L催化与软骨细胞增殖与肥大的非催化功能。最后，Chipseq实验将识别全基因组甲基化模式（H3K79ME2）与软骨细胞增殖和成熟有关。这些研究的结果有望在正常骨骼生长期间产生有关DOT1L的新知识和开发，对靶向dot1l治疗具有影响。