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

这项研究的目的是确定适应机制，以应对基因剂量的变化，如 拷贝数变异（CNVs），使用X染色体作为模型。基因在剂量敏感性方面有所不同， 其是人类CNV致病性的主要决定因素。更好地理解分子机制 在发育过程中补偿关键剂量敏感基因的异常将澄清结果 致病性CNV，如非整倍体，通常与出生缺陷和疾病有关， 特别是心脏和神经发育障碍。 哺乳动物的X染色体是研究基因剂量调节的极好模型，因为它代表了一种基因剂量调节。 一种自然形式的染色体非整倍性，由于男性（XY）中存在单个X染色体而导致 女性（XX）2例。剂量补偿机制已经发展，以避免由于X而导致的适应性下降 男性单倍不足。很明显，每个性连锁基因的剂量敏感性将是一个重要的因素。 这是所需补偿模式和水平的重要因素。然而，人们对 补偿机制和水平与剂量敏感性有关。 在这里，我们将应用一种新的单细胞/细胞核RNA-seq方法来测定绝对基因表达水平。 在来自啮齿动物/人（真兽目哺乳动物）、负鼠（有袋动物）和 鸡肉（目标1.1）。这种方法避免了相对表达式比较的需要（例如X：A 表达比率）和转录组标准化，将同时测量 X连锁基因及其祖先常染色体直向同源基因。我们将第一次能够将X染色体连锁的 基因一个接一个的适应单倍性和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重复综合征用于功能性测试剂量敏感性X连锁基因的调节和额外剂量的影响 在干细胞分化成与表型相关的细胞类型（例如神经元细胞和心脏细胞）期间， 肌细胞）（目的2）。这些使用全基因组和功能方法进行的逐基因研究的结果 将潜在地提供响应于原Y的可变剂量补偿机制的见解 染色体退化在进化过程中，从而提供了一个更好的理解的发病机制， 由剂量敏感基因的遗传或获得性CNV引起的疾病。

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.