Despite the vast majority of the human genome being non-coding, modern techniques have identified distal regions that regulate gene transcription, known as enhancers, which are enriched for such disease-associated variants.
Current and pressing challenges are to understand the formation and functional scope of enhancers, whether disease-associated or not; challenges which require higher throughput approaches within dynamic, multicellular systems. Due to its many strengths, the zebrafish as a genetic model system provides an excellent stage for testing enhancers in vivo and is highly relevant to the development and disease of many human tissues. We aim to identify and functionally characterize stage- and heart-specific transcriptional enhancers during zebrafish embryonic development.
General characteristics of the identified enhancers will provide insights into the spatio-temporal dynamics of regulatory potential. Sequences from the identified enhancers will be used to predict stage- and heart-specific transcription factor binding and may reveal modes of combinatorial transcription factor activity. Candidate enhancers, predicted transcription factors, and predicted binding sites will be subjected to genome editing in the zebrafish with the CRISPR/Cas9 system and assessed for functional roles in heart development. Orthologous pairs of human and zebrafish enhancers will be determined yielding insights into how such regulatory elements are functionally maintained throughout evolution.
Finally, previously identified human heart disease-associated variants occurring within enhancer regions will be delineated and functionally assessed within live zebrafish embryos, revealing the mechanistic scope of regulatory regions for disease formation. This proposal outlines a powerful systems biology approach, combining cutting edge sequencing methods with sophisticated computation and in vivo developmental genetics, which will be as insightful into basic biological mechanisms as it will be translational to human health.