The impact of genetic variation on genome biology and function

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。该基金的具体目标包括：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异的影响，我们开发了一个实验可处理的系统，使用转录因子（tf）和荧光素酶报告结构。使用一组100个高度保守的TF，我们将分别向每个TF cdna中引入3-5个预计具有破坏性的snv，并使用报告基因评估其影响。目标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助因子如何通过两种方式影响tf的活性。首先，我们将重新测试我们收集的含有tf的dSNV的一个子集和一个修饰的报告结构，该报告结构与已知的野生型相互作用蛋白或通用增强子蛋白（SP1）结合。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各不相同。这将使我们能够将tf和辅助因子的活性联系起来，并确定dsnv的影响是否可以通过辅助因子来调节。目的3 -基于细胞的损伤变异验证。为了避免因tf过表达引起的非生理反应，我们将选择少量经过验证的含有tf的dSNV，并将其稳定导入THP-1细胞系。由于该细胞系具有明确定义的调节分化的转录网络，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。这笔拨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，而且还将使我们能够生成一个独特而有价值的数据集，以便在更全球范围内研究这些方面。

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.