Gene-by-gene studies of dosage regulation pathways of the mammalian active X chromosome

哺乳动物活性 X 染色体剂量调节途径的逐基因研究

• 批准号: 9788500
• 负责人: Xinxian Deng
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-19 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788500
• 关键词: Aneuploidy; Attention; Biological Assay; Cardiac Myocytes; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Chickens; Clinical Research; Complex; Congenital Abnormality; Copy Number Polymorphism; Development; Didelphidae; Disease; Disease model; Dosage Compensation (Genetics); Down Syndrome; ES Cell Line; Epigenetic Process; Etiology; Event; Evolution; Female; Financial compensation; Gene Dosage; Gene Expression; Genes; Genetic; Genomics; Goals; Haploidy; Human; Individual; Inherited; Link; Mammals; Marsupialia; Measurement; Measures; Methods; Modeling; Molecular; Mouse Cell Line; Mus; Muscle Cells; Neurons; Orthologous Gene; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pattern; Persons; Phenotype; Process; Rattus; Regulation; Rodent; Testing; Time; Tissues; Transgenic Organisms; Up-Regulation; X Chromosome; X Inactivation; Y Chromosome; arm; cell type; chromosomal location; chromosome Xq duplication syndrome; combinatorial; developmental disease; digital; dosage; fitness; genome-wide; indexing; induced pluripotent stem cell; insight; male; novel; overexpression; research study; response; sex; stem cell differentiation; transcriptome; transcriptome sequencing

The goal of this study is to identify mechanisms of adaptation in response to gene dosage changes such as copy number variants (CNVs), using the X chromosome as a model. Genes vary in terms of dosage sensitivity, which is a major determinant of CNV pathogenicity in human. A better understanding of molecular mechanisms that compensate for anomalies in critical dosage-sensitive genes during development will clarify consequences of pathogenic CNVs such as aneuploidy which are often associated with birth defects and diseases, in particular cardio- and neuro-developmental disorders. The mammalian X chromosome is an excellent model to study gene dosage regulation since it represents a natural form of chromosomal aneuploidy resulting from the presence of a single X chromosome in males (XY) and two in females (XX). Dosage compensation mechanisms have evolved to avoid decreased fitness due to X haploinsufficiency in males. It has become clear that dosage sensitivity of each sex-linked gene would be an important factor in the patterns and levels of compensation needed. However, little is known about variability in compensation mechanisms and levels in relation to dosage sensitivity. Here we will apply a novel single-cell/nucleus RNA-seq method to assay absolute gene expression levels (expression per cell) in thousands of cells from rodent/human (eutherian mammal), opossum (marsupial), and chicken (Aim 1.1). This approach, which circumvents the need of relative expression comparison (e.g. X:A expression ratios) and transcriptome normalization, will concurrently measure expression differences between X-linked genes and their ancestral autosomal orthologs. We will, for the first time, be able to classify X-linked genes one-by-one in terms of their adaptation to haploidy and search for genetic and epigenetic features associated with X upregulation (Aim 1.2). We will also develop ex vivo models including a disease model for Xq duplication syndrome to functionally test regulation of dosage-sensitive X-linked genes and effects of extra dosage during stem cell differentiation into cell types related to the phenotype (e.g. neuronal cells and cardio- myocytes) (Aim 2). Results from these gene-by-gene studies using genome-wide and functional approaches will potentially provide insights of variable dosage compensation mechanisms in response to the proto-Y chromosome degeneration during evolution, and thus provide a better understanding of the pathogenesis of diseases that result from inherited or acquired CNVs of dosage-sensitive genes.

本研究的目的是确定响应基因剂量变化的适应机制，例如 拷贝数变异（CNV），使用 X 染色体作为模型。基因在剂量敏感性方面有所不同， 这是CNV对人类致病性的主要决定因素。更好地理解分子机制 补偿发育过程中关键剂量敏感基因的异常将阐明后果 致病性 CNV，例如通常与出生缺陷和疾病相关的非整倍体， 特别是心脏和神经发育障碍。 哺乳动物 X 染色体是研究基因剂量调节的绝佳模型，因为它代表了 男性中存在单个 X 染色体 (XY) 导致的染色体非整倍体的自然形式 两名女性 (XX)。剂量补偿机制已经发展，以避免由于 X 导致的适应性下降 男性单倍体不足。很明显，每个性连锁基因的剂量敏感性将是一个 所需补偿的模式和水平的重要因素。然而，人们对于其变异性知之甚少。 与剂量敏感性相关的补偿机制和水平。 在这里，我们将应用一种新型的单细胞/核 RNA-seq 方法来测定绝对基因表达水平 （每个细胞的表达）来自啮齿动物/人类（真兽类哺乳动物）、负鼠（有袋动物）和 鸡（目标 1.1）。这种方法避免了相对表达比较的需要（例如 X:A 表达比率）和转录组标准化，将同时测量之间的表达差异 X连锁基因及其祖先常染色体直系同源物。我们将首次能够对 X 连锁进行分类 基因一一适应单倍体并寻找遗传和表观遗传特征 与 X 上调相关（目标 1.2）。我们还将开发离体模型，包括疾病模型 Xq 重复综合征功能测试剂量敏感 X 连锁基因的调节和额外的影响 干细胞分化为与表型相关的细胞类型（例如神经元细胞和心脏细胞）期间的剂量 肌细胞）（目标 2）。使用全基因组和功能方法进行逐个基因研究的结果 将有可能提供针对原型 Y 的可变剂量补偿机制的见解 进化过程中染色体变性，从而更好地了解疾病的发病机制 由剂量敏感基因的遗传性或获得性 CNV 引起的疾病。