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

这项研究的目标是确定适应机制对基因剂量变化的反应，如 拷贝数变异(CNV)，以X染色体为模型。基因在剂量敏感性方面有所不同， 这是人类CNV致病性的一个主要决定因素。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后果 致病的CNV，如非整倍体，通常与出生缺陷和疾病有关，在 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一个很好的模型，因为它代表一个 由男性单个X染色体引起的自然形式的染色体非整倍体(XY) 女性2例(XX)。剂量补偿机制已经进化，以避免由于X 男性单倍体功能不全。很明显，每个与性别相关的基因的剂量敏感性将是一个 这是所需补偿方式和水平的重要因素。然而，人们对 与剂量敏感性有关的补偿机制和水平。 在这里，我们将应用一种新的单细胞/核rna-seq方法来检测基因的绝对表达水平。 (每个细胞的表达)来自啮齿动物/人类(真兽类)、负鼠(有袋类)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同源基因。我们将第一次能够将X连锁 从基因对单倍体的适应性和寻找遗传和表观遗传学特征的角度逐一分析 与X上调有关(目标1.2)。我们还将开发包括疾病模型在内的体外模型 XQ重复综合征功能测试剂量敏感X连锁基因的调节及Extra的作用 干细胞分化为与表型相关的细胞类型(如神经细胞和心脏细胞)的剂量 心肌细胞)(目标2)。使用全基因组和功能方法进行的这些逐个基因研究的结果 将潜在地提供对Proto-Y反应的可变剂量补偿机制的见解 染色体在进化过程中的退化，从而提供了更好的理解 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.