Ancestral roles of histone-modifying genes in heart development and disease

University Of Maryland Baltimore, Baltimore MD

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Abstract

Project Summary Large numbers of gene variants were identified from genomic sequencing of Congenital Heart Disease patients, but lack of functional verification in heart development precludes assigning ?disease gene? status. Genetic control of heart development is conserved from Drosophila to humans, thus investigations in flies can illuminate gene functions in human heart development and disease. We developed a gene validation system in Drosophila to screen large numbers of genes for roles in heart development, and quantitative analysis tools to assess multiple phenotypic parameters. We also developed novel strategies to test patient-¬derived genetic mutations in flies for in vivo evidence linking specific gene variants to disease. We observed that many histone modifying genes mutated in disease patients have roles in fly heart development. We propose studies designed to elucidate ancestral roles of histone-¬modifying genes in heart development and disease, and to generate personalized fly heart disease models for specific gene variants. Using the high-throughput Drosophila gene validation screen for essential roles in heart development, we will test candidate disease genes identified from publicly available datasets and collaborators? unpublished datasets. We will also screen Drosophila genes encoding enzymes for histone methylation/demethylation and acetylation/deacetylation for roles in heart development. Histone modifying genes validated by screening will be phenotyped using multiple quantifiable morphological and functional readouts. We will identify histone modifications that are most important for heart development. Genes will be prioritized based on multiple criteria, and for highest priority genes we will examine the transcriptional profile of heart tissue from flies in which the gene of interest was silenced in cardiac cells. We will identify conserved targets of histone modification effects by comparing our results to data from murine models and patient tissue samples. We will generate transgenic and knock-in fly models to provide in vivo functional evidence for involvement of high priority gene variants in congenital heart disease. In pReplacement, we will express wild type or mutant transgene versions of a given human disease gene in the fly heart while simultaneously silencing the endogenous fly homolog. We will also generate ?knock-in? Drosophila models using CRISPR/Cas9-mediated gene editing. In this pCRISPR approach, the endogenous Drosophila homolog is precisely modified to encode a protein with amino acid changes identical to those encoded in the patient-¬derived mutant allele. We will also use Drosophila to model polygenic disease based on selected patients, each of whom carries multiple gene variants that include one mutant histone-modifying gene. Additional aims to incorporate this award as part of the INCLUDE project expanded scope in two ways. First, we will develop a novel model system to identify and study genes involved in congenital heart disease in people with Down syndrome (DS-CHD). Second, we will perform transcriptomic profiling studies using this new model organism for CHD in people with Down syndrome. Approximately half of people with Down syndrome have CHD, but the responsible genes are not known. This poses a challenge to understanding the molecular mechanism underlying DS-CHD. Using mouse Down syndrome models, our collaborator was able to map the DS-CHD causal genes within two small loci (or linkage groups) together containing 18 genes, and further demonstrated that CHD requires a combination of genes from both loci. However, systematic testing of all 72 possible two-gene combinations in the mouse model would be prohibitively expensive. Because early heart development is controlled by highly conserved genetic networks from flies to humans, a Drosophila- based model testing and analysis system is an ideal approach to identification of the specific gene combinations responsible for DS-CHD. We thus propose to generate and characterize Drosophila models of CHD in people with Down syndrome based on our collaborator?s discoveries and our pilot studies. We will also perform transcriptomic profiling on fly defective hearts resulting from co-expression of causal gene combination(s), and compare this data to results from mouse models and tissue from people with Down syndrome. Successful completion of this supplemental project will lead to the identification of the specific gene combination causing CHD in people with Down syndrome. This is essential for understanding the molecular mechanism underlying DS-CHD and future development of precision medicine-based therapeutic approaches to prevention and treatment.