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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster V10 - Single base-pair changes within heart enhancers increase transcription factor binding affinity and disrupt embryonic heart development

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Enhancers are genomic elements that act as switches to control the location, level, and timing of gene expression to ensure the successful development and integrity of an organism. Enhancers harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease, but pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge.

Materials and

Methods:  We determine enhancer activity using enhancer reporter constructs in Ciona intestinalis type A (aka Ciona robusta) and in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Standard electroporation techniques are used to introduce the modified enhancer reporter constructs into Ciona fertilized eggs, and standard confocal imaging is used to acquire representative images to assess enhancer activity. iPSC-CMs were cultured using standard conditions and enhancer activity was measured using RNA sequencing.

Results, Conclusions, and Discussions: Here we find that low-affinity transcription factor binding sites are necessary for precise gene expression during heart development. Strikingly, in Ciona robusta embryos, we find that single base-pair changes can optimize the binding affinity of ETS binding sites, causing excess gene expression, cell migration defects, and phenotypes as severe as an extra beating heart. Mouse and human developmental heart enhancers also contain low-affinity transcription factor binding sites that are likely important for encoding precise gene expression patterns. Indeed, in human induced pluripotent stem cell-derived cardiomyocytes, a single base-pair change within a GATA4 enhancer increases ETS transcription factor binding affinity and causes excess enhancer activity. Our work illustrates a potential vulnerability in genomes created by the prevalence of low-affinity transcription factor binding sites, namely that single base-pair affinity-optimizing variants within enhancers can lead to excess gene expression and changes in cellular identity and organismal-level phenotypes. Understanding how tissue-specific enhancers in genomes encode specificity could inform the design of synthetic tissue-specific enhancers.

Acknowledgements (Optional):