The impact of genetic variation on genome biology and function

遗传变异对基因组生物学和功能的影响

• 批准号: RGPIN-2020-04947
• 负责人: Wilhelm, Brian
• 金额: $ 2.33万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717275
• 关键词: impact; genetic; variation; genome; biology

Our lab has an interest in transcriptional regulation and have previously shown how DNA methylation regulates the expression of alternative alleles of the GATA2 gene, a master regulator of blood cell differentiation. This work highlighted the phenomenon of loss of protein function through the expression of a single allele containing a damaging single nucleotide variant (dSNV). Our observations regarding the impact of dSNVs raises a number of questions with respect to the plasticity of genomes and the extent to which human genomes can tolerate damaging genetic variation. Despite the known potential for genetic variants to influence protein function, very little large-scale data has been generated to study this. The renewal of our NSERC grant is focused on better understanding the significance of this phenomenon and its impact on genome biology and function at a global level. To do this, we will focus on the activity of transcription factors as a model class of proteins since their activity is critical for cellular phenotypes and their function can easily be assayed. The specific aims of the grant include: Aim 1 - The global significance of damaging SNVs in the human genome. To generate high-throughput data to assess the impact of missense variants, we have developed an experimentally tractable system using transcription factors (TFs) and luciferase reporter constructs. Using a set of 100 highly conserved TFs we will individually introduce 3-5 SNVs predicted to be damaging into each of the TF cDNAs and assess their impact using reporter genes. Aim 2 Defining a potential role for cofactors in dSNV impact. We will test how protein co-factors can influence the activity of TFs in two ways. Firstly, we will retest a subset of our collection of dSNV containing TFs and a modified reporter construct in combination with either wild-type known interacting proteins or a generic enhancer protein (SP1). Secondly, we will test our collection of reporter system TFs and variants in a range of different cell lines, where the expression level of known interacting proteins of the TFs varies. This will allow us to correlate the activity of TFs and cofactors and determine whether the impact of dSNVs can be modulated by co-factors. Aim 3 Cell-based validation of damaging variants. To avoid non-physiological responses caused by the overexpression of TFs, we will select a small number of validated dSNV containing TFs and stably introduce these into the THP-1 cell line. Because this line has a well-defined transcriptional network regulating differentiation, we will use this system as a model to validate the impact of our dSNVs in a cellular context when cells are induced to differentiate. Rationale and significance. This grant will not only build upon our previous studies looking at transcriptional regulation, genetic variation, and protein function, but will also allow us to generate a unique and valuable dataset to examine these aspects on a more global scale.

我们的实验室对转录调控很感兴趣，并且之前已经展示了DNA甲基化如何调节GATA2基因的替代等位基因的表达，GATA2基因是血细胞分化的主要调节因子。这项工作强调了通过表达含有破坏性单核苷酸变异（dSNV）的单个等位基因而导致蛋白质功能丧失的现象。我们关于dsnv影响的观察提出了许多关于基因组可塑性和人类基因组能够容忍破坏性遗传变异的程度的问题。尽管已知遗传变异可能影响蛋白质功能，但很少有大规模的数据来研究这一点。我们NSERC拨款的更新集中在更好地理解这一现象的重要性及其在全球水平上对基因组生物学和功能的影响。为了做到这一点，我们将把重点放在转录因子的活性上，因为它们的活性对细胞表型至关重要，而且它们的功能很容易被测定。补助金的具体目的包括：