Decoding the role of non-coding mutations in gene regulation by cardiac transcription factors

About This Grant

Project Summary/Abstract Congenital heart diseases (CHDs) are the most common birth defect, occurring approximately in 1% of live births. Over 97% of mutations associated with CHD occur within the non-coding genome, potentially disrupting transcription factor (TF) binding to regulatory DNA and dysregulating gene regulation essential for heart development. However, due to their overwhelming numbers, it has remained challenging to identify causal mechanisms between non-coding mutations and human diseases. The long-term goal of the proposed work is to describe the molecular mechanisms by which disease-associated non-coding mutations dysregulate gene expression, leading to human diseases. Herein, we propose a high-throughput biochemical strategy to study the role of CHD-associated non-coding mutations in cardiac TF binding and gene regulation. Our hypothesis is that CHD-associated non-coding mutations will impact cardiac TF- DNA binding and disrupt gene regulation necessary for proper heart development. We have leveraged genome-wide association studies (GWAS) to identify 121 CHD-associated mutations in the non-coding genome. To account for the resolution limitations of GWAS, we performed a linkage disequilibrium expansion on the CHD-associated mutations to include single-nucleotide variants from diverse ancestries, resulting in 3,232 mutations. Additionally, we included all possible alleles for each genomic variant, resulting in 12,928 permutations. In Specific Aim 1, we will measure the impact of ~12,000 single- nucleotide non-coding variants on the binding of three master cardiac developmental TFs GATA4, NKX2-5, and TBX5. We will measure binding aYinities through Single Nucleotide Polymorphism-Systematic Evolution of Ligands by EXponential enrichment (SNP-SELEX) and identify non-coding mutations that decrease or increase cardiac TF binding aYinity. Our approach will allow us to quantitatively measure TF binding aYinities for ~12,000 genomic loci in a single experiment. In Specific Aim 2, we will determine the impact of the ~12,000 CHD-associated non-coding mutations on regulatory element activity during cardiomyocyte diYerentiation. The gene regulatory activity of ~12,000 promoter and enhancer variants will be measured in human embryonic stem cells, cardiac progenitors, and cardiomyocytes by massively parallel reporter assay (MPRA). This integrated approach will train undergraduate and graduate students in high-throughput biochemistry, functional genomics, and large-scale data analysis. Completion of the proposed project will contribute to our understanding of the mechanisms of non-coding mutations in CHDs and can be extended to study gene dysregulation in multiple human diseases.