The impact of genetic variation on genome biology and function

我们的实验室对转录调控感兴趣，以前已经展示了DNA甲基化如何调控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体(DSNV)的单个等位基因来丧失蛋白质功能的现象。我们对dSNvs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。尽管已知基因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。我们的NSERC资助的续期是为了更好地了解这一现象的意义及其对全球基因组生物学和功能的影响。为此，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检测。这笔赠款的具体目标包括：目标1--破坏人类基因组中SNV的全球意义。为了产生高通量的数据来评估错义变体的影响，我们开发了一个使用转录因子(TF)和荧光素酶报告结构的实验上易于处理的系统。使用一组100个高度保守的转录因子，我们将分别在每个转录因子cDNA中引入3-5个预测具有破坏性的SNV，并使用报告基因评估它们的影响。目标2-确定协同因素在dSNV影响中的潜在作用。我们将从两个方面测试蛋白质辅助因子如何影响转录因子的活性。首先，我们将重新测试我们收集的dSNV的子集，其中包含TF和一个修改的报告结构，与野生型已知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及其变体，其中已知相互作用蛋白的表达水平各不相同。这将使我们能够将转录因子和辅因子的活性联系起来，并确定dSNV的影响是否可以被辅因子调节。AIM 3-基于细胞的破坏性变异验证。为了避免TFS过表达引起的非生理反应，我们将选择少量有效的含有TFS的dSNV并将其稳定地导入THP-1细胞系。因为这条线有一个明确的转录网络调节分化，我们将使用这个系统作为一个模型，以验证我们的dSNV在细胞环境中的影响，当细胞被诱导分化时。理论基础和意义。这笔拨款不仅将建立在我们之前研究转录调控、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的、有价值的数据集，以在更全球的范围内检查这些方面。

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.