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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760605
• 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感兴趣，并且先前已经展示了DNA甲基化如何调节GATA 2基因的替代等位基因的表达，GATA 2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体（dSNV）的单个等位基因而丧失蛋白质功能的现象。我们对dSNV影响的观察提出了一些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。尽管已知遗传变异影响蛋白质功能的潜力，但很少有大规模的数据来研究这一点。我们的NSERC资助的更新重点是更好地理解这一现象的重要性及其对全球基因组生物学和功能的影响。为了做到这一点，我们将集中在转录因子的活性作为蛋白质的模型类，因为它们的活性是至关重要的细胞表型和它们的功能可以很容易地进行测定。资助的具体目标包括：目标1 -人类基因组中破坏性SNV的全球意义。为了产生高通量数据来评估错义变体的影响，我们已经开发了一个实验上易于处理的系统，使用转录因子（TF）和荧光素酶报告构建体。使用一组100个高度保守的TF，我们将分别引入3-5个预测会破坏每个TF cDNA的SNV，并使用报告基因评估它们的影响。 目的2 -定义辅助因子在dSNV影响中的潜在作用。我们将测试蛋白质辅因子如何以两种方式影响TF的活性。首先，我们将重新测试我们收集的含有TF和修饰的报告构建体的dSNV的子集，其与野生型已知的相互作用蛋白或通用增强子蛋白（SP1）组合。其次，我们将在一系列不同的细胞系中测试我们收集的报告系统TF和变体，其中已知的TF相互作用蛋白的表达水平不同。这将使我们能够关联TF和辅因子的活性，并确定dSNV的影响是否可以通过辅因子调节。目标3 -基于细胞的破坏性变体验证。为了避免TF过表达引起的非生理反应，我们将选择少量经验证的含有TF的dSNV并将其稳定地引入THP-1细胞系中。由于该细胞系具有调节分化的明确定义的转录网络，因此我们将使用该系统作为模型来验证当细胞被诱导分化时我们的dSNV在细胞环境中的影响。理由和意义。这笔赠款不仅将建立在我们以前研究转录调控，遗传变异和蛋白质功能的基础上，还将使我们能够生成一个独特而有价值的数据集，以在更全球范围内研究这些方面。