Project Summary During cardiac development, coordinate gene expression changes facilitate the progressive lineage restriction of multipotent progenitors into a terminal identity that is maintained over their lifespan. Compromised differentiation and/or cell state have been linked to multiple diseases, including aspects of congenital heart disease and heart failure. Thus, the mechanisms underlying cellular identity are of intense interest. Models underlying fate determination and the identity often focus on transcription factors and/or niche signals. Current paradigms fail to reconcile how the interplay between a finite number of morphogens and lineage specific transcription factors result in 200+ cell types with distinct and stable identities. I hypothesize that nuclear architecture represents a critical mechanism for achieving coordinated regulation of hundreds of genes underlying cellular identity by governing their accessibility or availability. Supporting our hypothesis, we have built a strong body of work demonstrating that nuclear architecture regulates cardiac cellular identity in development and disease. First, we discovered mechanisms by which critical transcription factors not only govern transcription, but also choreograph genome folding to regulate cardiac neural crest fate determination. Second, our work shows that spatial positioning of chromatin safeguards cardiac cellular identity and likely contributes to human cardiac disease (i.e. laminopathies). Decades of work have shown that gene expression programs are regulated by the recruitment and activity of activator and opposing repressor proteins. In addition to revolutionizing our understanding of transcription, this work has led to therapies directly targeting transcription factors. The mechanisms that similarly balance formation, maintenance and dissolution of nuclear architecture are poorly understood. In the EIA application I outline an interdisciplinary vision to uncover how these mechanisms control cardiac cellular identity. In Theme 1, I propose strategies to identify and decipher how molecular players guiding establishment, maintenance and disassembly of genome folding impact cardiac cell state. In Theme 2, I propose strategies to uncover how epigenetic, transcriptional, and mechanical inputs regulate spatial positioning of the genome in relation to the nuclear lamina in physiologic and pathologic conditions. We have established a multipronged program that will use high throughput 3D imaging, genetic manipulations with precise spatiotemporal resolution, tunable cardiac microtissues, epigenome engineering, super-resolution imaging and state-of-the-art genomics to tackle the propose studies, with a focus grounded in physiological relevance. The orthogonal approaches promote rigor, but require flexibility. My strong track record of building an impactful body of work support our pursuit of this paradigm shifting work. The proposed studies have the potential to reshape our understanding of how epigenetics, transcriptional, and mechano-related mechanisms direct 3D genome organization and orchestrate cardiac cellular identity. By viewing cardiac diseases through the prism of genome organization, we predict a wealth of unrealized therapeutic opportunities.

Project Narrative A continuing mystery in human biology is how the same genetic material can give to the more that 200 different types of cells found in the body. My proposal is focused on understanding how the genetic material in cardiac cells is organized in three-dimensions and revealing how that organization is maintained throughout the lifetime of a cell. These studies will help identify new therapeutic strategies for patients with disease that result from the failure to properly establish or maintain the correct organization in cardiac cells.