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

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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.