Understanding how Dot1L activity in the growth plate regulates skeletal growth

了解生长板中的 Dot1L 活性如何调节骨骼生长

基本信息

批准号：
9979919
负责人：
Rosaria M. Guzzo
金额：
$ 8.2万
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：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979919
关键词：
Address Adolescent Architecture Biochemical Biological Biological Assay Biology Bone Growth Bone Morphogenetic Proteins Cancer Patient Cartilage Cells Cessation of life Child Health Childhood Childhood Cancer Treatment Childhood Leukemia Chondrocytes Chondrogenesis Chromatin Chromatin Remodeling Factor Chromatin Structure Cre driver Data Defect Deposition Development Diagnostic radiologic examination Embryo Enzymes Epigenetic Process Epiphysial cartilage Equilibrium Exhibits Funding Future Gene Expression Gene Expression Profiling Genes Genetic Genetic Polymorphism Genetic Transcription Goals Growth Growth Disorders Growth and Development function Height Histologic Histones Human Human Genetics Idiopathic scoliosis Impairment In Vitro Investigation Investigational Therapies Knowledge Laboratories Lead Lesion Limb Bud Link Literature Lysine Maintenance Malignant Childhood Neoplasm Malignant Neoplasms Measurement Mediating Methylation Modeling Modification Molecular Mus Pathway Analysis Pathway interactions Phase Phenotype Physiologic Ossification Play Proliferating Regulation Rest Role Signal Pathway Signal Transduction Signaling Protein Skeletal Development Stains Structural defect Structure Tamoxifen Technology Testing Thick Time Variant aggrecan base bone bone morphogenetic protein 2 cartilaginous chromatin immunoprecipitation conditional knockout differential expression driving force epigenetic regulation epigenetic therapy experimental study genetic variant genome wide association study in vitro Model in vivo inhibitor/antagonist insight interest laser capture microdissection leukemia treatment long bone loss of function mouse model novel postnatal postnatal development programs repaired skeletal skeletal dysplasia small molecule inhibitor telomere transcriptome sequencing

项目摘要

Abstract Genes that regulate chromatin organization and function have been implicated in the development of skeletal growth disorders. A major gap in our current knowledge is how epigenetic regulators control longitudinal bone growth at the growth plate. From a child's health perspective, a deeper understanding of the role of specific chromatin modifiers in the postnatal growth plate cartilage may lead to more informed use of targeted epigenetic therapies that are under investigation for the treatment of pediatric cancers. The goal of our proposal is to establish the requirement of a chromatin modifier Dot1L (Disruptor of telomere silencing-like 1) in growth plate cartilage and to uncover transcriptional mechanisms through which it regulates the progression of chondrocyte differentiation. Dot1L is a unique chromatin modifier because it is the only known enzyme that catalyzes the methylation of lysine residue 79 in histone 3 (H3K79), an established epigenetic mark for gene transcription. A human genetic variant of Dot1L is associated with taller pubertal stature and accelerated growth rate in adolescent idiopathic scoliosis. Recently, inactivation of the Dot1L gene in embryonic cartilage was shown to cause runting, growth plate disorganization, and early death in mice (1, 2). While inhibition of Dot1L is considered a promising therapy for treatment of leukemia in children(3-6), we know little about the influence of Dot1L on postnatal growth plate function. Thus, we developed an inducible strategy to genetically disrupt Dot1L in postnatal growth plate chondrocytes in mice. Postnatal inactivation of Dot1L in growth plate cartilage using the tamoxifen-inducible Aggrecan-Cre driver (AgcCreERT2) resulted in an intriguing skeletal phenotype characterized by impaired longitudinal bone growth, structural changes within growth plate cartilage, and focal closure of the growth plate. Our preliminary in vitro studies further suggest that Dot1L modulates signaling through the bone morphogenetic protein (Bmp) pathway, a key pathway controlling chondrocyte differentiation and endochondral ossification. Together, our data reveal a critical and understudied role for Dot1L in maintaining postnatal GP cartilage. We hypothesize that the chromatin modifier Dot1L maintains the proper balance of chondrocyte proliferation and maturation to support postnatal bone elongation at the growth plate. In this study, we will use cutting-edge genetic and molecular approaches to advance our understanding of Dot1L-mediated regulation of growth plate chondrocyte differentiation and postnatal bone growth by the following Specific Aims: (1) determine whether postnatal function of Dot1L in the GP restricts chondrocyte maturation; and (2) identify Dot1L-regulated genes and networks controlling chondrocyte differentiation in the postnatal growth plate. This contribution is significant since it will advance our understanding of the epigenetic regulation of cartilage genes linked to human skeletal dysplasia and growth disorders. The workflow and data generated by funding this R03 proposal will lay the groundwork for future projects focused on establishing the epigenetic circuits necessary for skeletal growth, development and repair. !

抽象的调节染色质组织和功能的基因与骨骼生长的发育有关失调。我们目前知识的一个主要差距是表观遗传调节剂如何控制纵向骨生长生长板。从孩子的健康角度，更深入地了解特定染色质的作用出生后生长板软骨中的修饰剂可能会导致更明智地使用靶向表观遗传正在研究治疗儿童癌症的疗法。我们提案的目标是确定生长板中染色质修饰剂 Dot1L（端粒沉默样破坏者 1）的要求软骨并揭示其调节软骨细胞进展的转录机制差异化。 Dot1L 是一种独特的染色质修饰剂，因为它是唯一已知的催化染色质的酶。组蛋白 3 (H3K79) 中赖氨酸残基 79 的甲基化是基因转录的既定表观遗传标记。一个人 Dot1L 的遗传变异与青少年特发性青春期身材较高和生长速度加快有关脊柱侧凸。最近，胚胎软骨中 Dot1L 基因的失活被证明会导致矮小、生长板小鼠的组织混乱和早期死亡 (1, 2)。虽然抑制 Dot1L 被认为是一种有前途的治疗方法对于儿童白血病（3-6岁）的研究，我们对Dot1L对出生后生长板功能的影响知之甚少。因此，我们开发了一种诱导策略来基因破坏小鼠出生后生长板软骨细胞中的 Dot1L。产后使用他莫昔芬诱导的 Aggrecan-Cre 驱动程序 (AgcCreERT2) 使生长板软骨中的 Dot1L 失活在一种有趣的骨骼表型中，其特征是纵向骨生长受损，内部结构发生变化生长板软骨和生长板的局灶性闭合。我们的初步体外研究进一步表明 Dot1L 通过骨形态发生蛋白 (Bmp) 途径调节信号传导，骨形态发生蛋白 (Bmp) 途径是控制软骨细胞的关键途径分化和软骨内骨化。总之，我们的数据揭示了 Dot1L 在以下领域中的关键且未被充分研究的作用：维持产后 GP 软骨。我们假设染色质修饰剂 Dot1L 保持正确的软骨细胞增殖和成熟的平衡，支持出生后生长时的骨伸长盘子。在这项研究中，我们将使用尖端的遗传和分子方法来加深我们对 Dot1L介导的生长板软骨细胞分化和出生后骨生长的调节通过以下因素具体目的：（1）确定GP中Dot1L的出生后功能是否限制软骨细胞成熟；和（2）识别控制出生后生长板中软骨细胞分化的 Dot1L 调控基因和网络。这贡献是重要的，因为它将增进我们对与软骨相关的基因的表观遗传调控的理解人类骨骼发育不良和生长障碍。通过资助此 R03 提案而生成的工作流程和数据将为未来的项目奠定基础，重点是建立骨骼生长所需的表观遗传回路，开发和修复。！