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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.

Mammalian genomes contain millions of transposable element sequences, but the overall impact they have on host biological processes remains poorly characterized. Retrotransposon insertions are known to actively shape embryonic development of model organisms, mainly by influencing transcription of host genes or by contributing functional protein or RNA products. Retrotransposons are also transcribed in human preimplantation embryos, and characterization of exapted retrotransposon families will help explain how human embryogenesis differs from other organisms. The second chapter of this dissertation presents studies on Human Endogenous Retrovirus-H (HERVH), which is a primate specific retrotransposon family functional in human embryonic stem cells (hESCs). The human genome contains over 2000 HERVH insertions and is associated with four distinct promoters, complicating efforts to understand host transcriptional regulation. Also, while HERVH RNA is clearly essential to maintain stemness, the mechanism of its action is unknown. To investigate the regulation of HERVH, we utilize bioinformatic prediction and reporter assays to identify transcription factor binding sites unique to the LTR7Y subfamily. A major finding of this dissertation is that HERVH regulation is recapitulated in naive and primed hESC culture, and the LTR7Y can be used to mark the naive state. We also find evidence that the primary mechanism of the HERVH RNA is in trans. We use genome engineering to delete an individual HERVH insertion and find it does not phenocopy family-wide HERVH knockdown or effect adjacent genes. Furthermore, ectopic overexpression of HERVH RNA effects the dynamics of BMP4/LY294002 induced mesendoderm differentiation, establishing a trans role for HERVH RNA in the postimplantation primitive streak. HERVH is the most active endogenous retrovirus (ERV) in preimplantation embryos, but we find other retrotransposons classes are more likely to modify adjacent cellular genes. In the third chapter of this dissertation, we identify a short interspersed element (SINE) insertion within ZBTB16, a developmentally important zinc-finger transcription factor. This SINE element serves as an alternative promoter, generating a transcript isoform found in the human oocyte that is capable of producing a truncated protein product. Using in vitro cell culture assays, we find the truncated ZBTB16 protein retains its function as a cell cycle regulator but shows altered subcellular localization and is resistant to post translational degradation, implying it may act to slow the cell cycle of early human embryos. The studies in this dissertation serve to clarify the complex regulation and mechanisms of function for retrotransposon families that are essential for human development. These findings advance the field of hESC biology by establishing clear markers of different pluripotent cell types, and ultimately add substantially to our understanding of how retrotransposons have shaped human pluripotency.

Since its early discovery as a messenger, RNA has been shown to play a diverse set of regulatory, structural and even catalytic roles. The more recent understanding that the genome is pervasively transcribed stimulated the discovery of a new prevalent class of long non coding RNAs (lncRNAs). While these are lower abundant and relatively less conserved than other class of functional RNAs, lncRNAs are emerging as key players in different cellular processes in development and disease.

Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. Given that most lincRNAs have no known function, the use and development of high-throughput strategies for identifying gene function will be an important component of efforts understand the biological roles played by noncoding RNAs. Here, we begin to develop a genetic interaction mapping platform for the rapid elucidation of gene function in death receptor signaling pathways.

The first steps in vision begin with signaling by photoreceptor cells of the retina. These post-mitotic neurons are reliant on the neighboring retinal pigmented epithelium (RPE) for daily homeostatic functions for life-long maintenance. Pathology affecting these integral cell layers can give rise to various retinal pathologies, with varying genetic patterns ranging from rare Mendelian diseases to more common complex multi-genic diseases. The goal of this work was to understand the phenotypic and genotypic factors that contribute to normal physiological function of the retina and the changes that manifest in disease. Human patients and mouse models of the Mendelian disease enhanced S-cone syndrome were subjected to RNA-Sequencing (RNA-Seq) and high-resolution hybrid microscopy methods to understand the genomic features that result in the progressive degenerative phenotype. It was revealed that a defect in proper photoreceptor shedding and phagocytosis, stemming for the aberrant development of these photoreceptors, gave rise to the progressive degeneration. Expanding the work to a more common, yet complex retinal degeneration, age-related retinal degeneration (ARD), the importance of genetic epistasis in disease was revealed. The use of inbred mouse lines with differing genetic backgrounds revealed that subtle differences in levels of inflammatory and RPE homeostatic factors can have profound effects on the predisposition and pathogenesis of ARD. The RNA-Seq from these studies revealed that non-coding RNAs may also mediate phenotypic diversity in disease. An evolutionary analysis of long intergenic non-coding RNA (lincRNA) across species with varying retinal environments elucidated those lincRNAs that were conserved in sequence. Further biochemical and bioinformatic experiments revealed that these lincRNAs may be critical for retinal physiology in adults and may represent another level of transcriptional regulation in the genetic architecture of the eye in health and disease.