Transcription Factor Protein Interactomes Reveal Genetic Determinants in Heart Disease. Transcription Factor Protein Interactomes Reveal Genetic Determinants in Heart Disease

Congenital Heart Disease (CHD) is present in 1% of live births, and while large-scale genetic studies have uncovered genes associated with CHDs, identifying causal mutations remains a challenge. We hypothesized that genetic determinants for CHDs could be found in the protein interactomes of GATA4 and TBX5, two cardiac transcription factors (TFs) that cause CHDs. Defining their interactomes in human cardiac progenitors via affinity purification-mass spectrometry and integrating the results with genetic data from the Pediatric Cardiac Genomic Consortium (PCGC) revealed an enrichment of de novo variants among proteins that interact with GATA4 or TBX5. A consolidative score designed to prioritize TF interactome members based on distinctive variant, gene, and proband features identified numerous likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied cardiac developmental genes, resulting in co-activation, and the GLYR1 variant associated with CHD disrupted interaction with GATA4. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating the contribution of genetic variants in CHD and can be extended to other genetic diseases. Overall design: 3 replicates for each condition were ran for Bulk RNAseq after siRNA knockdown of GATA4 or siControl in hiPSC-derived cardiac progenitors (3x siRNA Control and 3 x siRNA GATA4). 2 replicates for each sample were ran for Bulk RNAseq after siRNA knockdown of GLYR1 or siControl in hiPSC-derived cardiac progenitors (2x siRNA Control and 2x siRNA GLYR1 ). 4-5 replicates for each sample were ran for Bulk RNAseq of day6 hiPSC derived cardiac progenitors (CPs) (5x WT CPs, 4x GATA4 knockout CPs and 5x TBX5 knockout CPs). 3-1 replicates for each sample were ran for ChIPseq on day6 WT hiPSC derived cardiac progenitors (CPs), (3x GLYR1 WT CPs, 2x H3K36me3 WT CPs, 2x GATA4 WT CPs, 2x TBX5 WT CPs, 2x NKX2-5 WT CPs, 1x MEIS1 WT CPs, 1x ISL1 WT CPs). 2 replicates for each sample were ran for Bulk RNAseq of day0 WT hiPSC (2x GLYR1 WT hiPSC and 2x H3K36me3 WT hiPSCs, ). 1-3 replicate single-cell RNA-seq samples at 3 time-points (hiPSC, Day 6 and Day 18 into cardiac differentiation) under two genotypes (WT and P496L GLYR1 mutant) were processed across 3 batches (see column Y). For the analyses to infer differential binding between the two genotypes (WT and P496L GLYR1 mutant), 3 replicates for each genotype were used for GLYR1 ChIPseq in hiPSC-derived cardiac progenitors processed in batch 2 (see column Y). 5 replicates under each of the hiPSC and cardiac progenitor stages of GLYR1 ChIPseq were used to infer differential GLYR1 binding between the two stages.