UCSF

The molecular machinery underlying heart development out of primordial anterior mesoderm remains incompletely understood. This is in part due to a restricted research emphasis on canonical genomic elements and the inability to precisely model the timing of cardiac lineage commitment. The genome is pervasively transcribed far beyond the coding exome. Therefore, we hypothesized that the transcriptome as a whole, including all noncoding and splice isoforms would most precisely define the identity of the cell as it differentiated into a cardiomyocyte. Using an enhancer fragment of the heart-critical Smarcd3 Brg1/Brm associated factor (BAF) subunit, we developed an in vitro reporter differentiation system that precisely delineated the first heart specification from murine embryonic stem cells (mESCs). From this, de novo total RNA isoform coexpression networks were generated to reveal a staged progression by which hundreds of thousands of gene isoforms were organized into hundreds of subnetwork modules that dynamically programmed nascent mesoderm to became restricted to a cardiovascular fate.

Many transcripts within the nucleus bind and influence the genomic regulation of chromatin modifying complexes. Therefore, we next aimed to elucidate the BRG1/BAF RNA interactome during its key role at cardiac fate commitment. Using targeting immunoprecipitations coupled to molecular techniques to isolate and identify protein:RNA adducts, we found at least 7 subunits engaged RNA molecules via previously unrealized discrete domains. Furthermore, these subunits interfaced RNA through tens of thousands of binding events that frequently coincided the defining transcript isoform transitions of this developmental window.

These interrogations into the transcriptional basis for cardiac lineage differentiation also identified 6 annotated long intergenic noncoding RNAs (lincRNA) with discrete gene structure, epigenetic regulation, and cardiac progenitor specificity in vivo. We ablated these lincRNAs in the embryo via Cas9 editing, which revealed regulatory roles within their local genomic environments, including between Bmp4 and Rubie. While none of the 6 transcripts were required for proper heart morphogenesis, compound heterozygous Bmp4+/-; Rubie+/- offspring did reveal a genetic interaction between these genes in formation of the right ventricular outflow tract. These experiments provide new insight into the complex role RNA plays during cardiac lineage commitment and congenital heart disease.