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

• 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
• 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

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. !

摘要 调控染色质组织和功能的基因与骨骼生长发育有关 紊乱我们目前知识的一个主要空白是表观遗传调节剂如何控制骨的纵向生长。 生长板从儿童健康的角度，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物可能导致更明智地使用靶向表观遗传 正在研究的治疗儿科癌症的疗法。我们提案的目的是 在生长板中建立染色质修饰剂Dot 1 L（端粒沉默样1的破坏剂）的要求 软骨和揭示转录机制，通过它调节软骨细胞的进展 分化Dot 1 L是一种独特的染色质修饰剂，因为它是唯一已知的催化 组蛋白3（H3 K79）中赖氨酸残基79的甲基化，H3 K79是基因转录的表观遗传标记。人类 Dot 1 L的遗传变异与青少年特发性高血压患者青春期身高和生长速度加快相关 脊柱侧凸最近，Dot 1 L基因在胚胎软骨中的失活被证明会导致生长板萎缩， 组织解体和小鼠早期死亡（1，2）。虽然Dot 1 L的抑制被认为是一种有前途的治疗方法， 在儿童白血病（3-6岁）中，我们对Dot 1 L对出生后生长板功能的影响知之甚少。因此我们 开发了一种可诱导的策略，以遗传破坏小鼠出生后生长板软骨细胞中的Dot 1 L。产后 使用他莫昔芬诱导的聚集蛋白聚糖-Cre驱动剂（AgcCreERT 2）灭活生长板软骨中的Dot 1 L， 在以纵向骨生长受损为特征的有趣的骨骼表型中， 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，Dot 1 L 通过骨形态发生蛋白（Bmp）途径调节信号传导，Bmp途径是控制软骨细胞增殖的关键途径。 分化和软骨内骨化。总之，我们的数据揭示了Dot 1 L在以下方面的关键和未充分研究的作用： 保持出生后GP软骨。我们假设染色质修饰剂Dot 1 L维持适当的 软骨细胞增殖和成熟的平衡，以支持出生后骨骼在生长过程中的伸长 板在这项研究中，我们将使用尖端的遗传和分子方法来推进我们对 Dot 1 L通过以下途径介导生长板软骨细胞分化和出生后骨生长的调节 具体目的：（1）确定GP中Dot 1 L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Dot 1 L调节基因和网络。这 这一贡献是重要的，因为它将促进我们对软骨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R 03提案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， 发展和修复。 !