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Transcription Factor Protein Interactomes Reveal Genetic Determinants in Heart Disease

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE159411 | GEO | 2022/02/18

REPOSITORIES: GEO

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Project description:Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD). The pathogenesis of HLHS is poorly understood and due to the likely oligogenic complexity of the disease, definitive HLHS-causing genes have not yet been identified. Postulating that impaired cardiomyocyte proliferation as a likely important contributing mechanism to HLHS pathogenesis, and we conducted a genome-wide siRNA screen to identify genes affecting proliferation of human iPSC-derived cardiomyocytes (hPSC-CMs). This yielded ribosomal protein (RP) genes as the most prominent class of effectors of CM proliferation. In parallel, whole genome sequencing and rare variant filtering of a cohort of 25 HLHS proband-parent trios with poor clinical outcome revealed enrichment of rare variants of RP genes. In addition, in another familial CHD case of HLHS proband we identified a rare, predicted-damaging promoter variant affecting RPS15A that was shared between the proband and a distant relative with CHD. Functional testing with an integrated multi-model system approach reinforced the idea that RP genes are major regulators of cardiac growth and proliferation, thus potentially contributing to hypoplastic phenotype observed in HLHS patients. Cardiac knockdown (KD) of RP genes with promoter or coding variants (RPS15A, RPS17, RPL26L1, RPL39, RPS15) reduced proliferation in generic hPSC-CMs and caused malformed hearts, heart-loss or even lethality in Drosophila. In zebrafish, diminished rps15a function caused reduced CM numbers, heart looping defects, or weakened contractility, while reduced rps17 or rpl39 function caused reduced ventricular size or systolic atrial dysfunction, respectively. Importantly, genetic interactions between RPS15A and core cardiac transcription factors TBX5 in CMs, Drosocross, pannier and tinman in flies, and tbx5 and nkx2-7 (nkx2-5 paralog) in fish, support a specific role for RP genes in heart development. Furthermore, RPS15A KD-induced heart/CM proliferation defects were significantly attenuated by p53 KD in both hPSC-CMs and zebrafish, and by Hippo activation (YAP/yorkie overexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play critical roles in cardiogenesis and constitute an emerging novel class of gene candidates likely involved in HLHS and other CHDs.

Project description:Background: Cardiac transcription factors are master regulators during heart development. Recently, some were shown to transdifferentiate noncardiac mesoderm cells and cardiac fibroblasts into cardiomyocytes. However, the individual roles of each transcription factors in activating cardiac gene program have not been elucidated. We examined cardiac-specific and genome-wide gene expression in fibroblasts induced with cardiac transcription factors Nkx2.5 (N), Tbx5 (T), Gata4 (G), Myocardin (M) alone or different combinations. Methodology/Principal Findings: We applied different combinations of human Nkx2.5 (N), Tbx5 (T), Gata4 (G) and Myocardin (M) lentiviruses into 10T1/2 fibroblasts. Immunostaining and quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that N, T, G or M alone did not induce expression of cardiac marker genes M-NM-1-myosin heavy chain (M-NM-1MHC) and cardiac troponin T (cTnT). Only T+M and T+G+M combinations induced M-NM-1MHC and cTnT expression. Microarray-based gene ontology analysis revealed that T alone inhibited most genes involved in cardiac-related processes and activated genes involved in Wnt receptor signaling pathway and in aberrant processes. M alone inhibited genes involved in Wnt receptor signaling pathway and activated genes involved in cardiac-related processes and in aberrant processes. G alone inhibited genes involved in ectoderm development. T+G+M combination was the most effective activator of genes associated with cardiac-related processes including muscle cell differentiation, sarcomere, striated muscle contraction, regulation of heart contraction, and glucose metabolism and fatty acid oxidation (two significant forms of cardiomyocyte energy metabolism). And unlike T, M, G alone or T+M, T+G+M did not activate genes associated with aberrant processes. Conclusions: Tbx5, Gata4 and Myocardin play different roles in activating cardiac gene program and in avoiding aberrant gene program activation. The combination of T+G+M activated cardiac gene program and avoided aberrant gene program activation. Two weeks after doxycline induction, total RNA was isolated from 10T1/2-tTA cells infected with different combinations of Tbx5, Gata4, and Myocardin lentiviruses. Biological triplicated.

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Transcription Factor Protein Interactomes Reveal Genetic Determinants in Heart Disease

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