The impact of genetic variation on genome biology and function

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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.