Aberrant extracellular matrix and cardiac development in models lacking the PR-DUB component ASXL3

Abstract: Background: Clinical and research based genetic testing has uncovered genes that encode chromatin modifying complex components required for organogenesis. Covalent histone modifications play a key role in establishing transcriptional plasticity during development, required for cell fate specification, and have been implicated as a developmental mechanism that accounts for autism spectrum disorder (ASD) and CHD co-occurrence. ASXL3 has been identified as a high confidence ASD gene. ASXL3 is a component of the Polycomb Repressive Deubiquitination (PR-DUB) complex, which deubiquitinates histone H2A. However, the role of ASXL3 in cardiac development remains unknown. Methods: We used CRISPR/Cas9 gene editing to generate clinically relevant Asxl3 frameshift alleles in a mouse model and human embryonic stem cells (hESCs). To evaluate ASXL3 function in developing hearts, we performed structural, molecular, immunostaining and histological analyses. Transcriptomic and cellular compositional changes were assessed with bulk RNA sequencing of mouse hearts and single-cell RNA sequencing (scRNA-seq) of human cardiac tissue differentiated from isogenic hESC lines. Results: Biallelic genetic inactivation of Asxl3 leads to perinatal lethality and increased levels of histone H2A mono-ubiquitination, which are regulated by PR-DUB. Asxl3+/fs and Asxl3fs/fs mice display cardiac abnormalities including ventricular hypoplasia, septal defects, and bifid cardiac apex with variable penetrance. The presence of underdeveloped ventricles is preceded by increased progenitor proliferation in the ventricles, as determined by EdU incorporation. Differential gene expression, assessed by bulk RNA sequencing implicates extracellular matrix dysfunction as a pathogenic mechanism. This correlates with a reduction in vimentin-positive cardiac fibroblasts. scRNA-seq of cardiac cultures differentiated from human ASXL3fs/fs ESC lines exhibit altered ratios of cardiac fibroblasts and cardiomyocytes. Similar to the mouse data, genes essential for extracellular matrix composition and signaling are differentially expressed between ASXL3+/+ and ASXL3fs/fs human in vitro differentiated cardiac tissue. The observed transcriptomic changes predict diminished cell-cell signaling interactions between cardiac fibroblasts and cardiomyocyte progenitors in ASXL3 cultures. Conclusions: Collectively, our data implicates species-specific roles for ASXL3 in both human and mouse cardiac development. These results highlight the role of extracellular matrix gene programs by cardiac fibroblast during cardiomyocyte development and provide insight into mechanisms of altered cardiogenesis by autism risk genes.