Biology subjects

Le, S. H.

Publications and source records attributed to Le, S. H..

3 recordsLinked to original sources

bioRxiv2022

Diverse logics and grammar encode notochord enhancers

The notochord is a key structure during chordate development. We have previously identified several enhancers regulated by Zic and ETS that encode notochord activity within the marine chordate Ciona robusta (Ciona). To better understand the role of Zic and ETS within notochord enhancers, we tested 90 genomic elements containing Zic and ETS sites for expression in developing Ciona embryos using a whole-embryo, massively parallel reporter assay. We discovered that 39/90 of the elements were active in developing embryos; however only 10% were active within the notochord, indicating that more than just Zic and ETS sites are required for notochord expression. Further analysis revealed notochord enhancers were regulated by three groups of factors: (1) Zic and ETS, (2) Zic, ETS and Brachyury (Bra), and (3) Zic, ETS, Bra and FoxA. One of these notochord enhancers, regulated by Zic and ETS, is located upstream of laminin alpha, a gene critical for notochord development in both Ciona and vertebrates. Reversing the ETS sites in this enhancer greatly diminish expression, indicating that enhancer grammar is critical for enhancer activity. Strikingly, we find clusters of Zic and ETS binding sites within the introns of mouse and human laminin alpha 1 with conserved enhancer grammar. Our analysis also identified two notochord enhancers regulated by Zic, ETS, FoxA and Bra binding sites: the Bra Shadow (BraS) enhancer located in close proximity to Bra, and an enhancer located near the gene Lrig. Randomizing the BraS enhancer demonstrates that although the Zic and ETS sites are necessary for enhancer activity, they are not sufficient. We find that FoxA and Bra sites contribute to BraS enhancer activity. Zic, ETS, FoxA and Bra binding sites occur within the Ciona Bra434 enhancer and vertebrate notochord Brachyury enhancers, suggesting a conserved regulatory logic. Collectively, this study deepens our understanding of how enhancers encode notochord expression, illustrates the importance of enhancer grammar, and hints at the conservation of enhancer logic and grammar across chordates.

developmental biology↗

bioRxiv2022

Affinity-optimizing variants within cardiac enhancers disrupt heart development and contribute to cardiac traits

Enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with disease. We find that suboptimal affinity ETS transcription factor binding sites are prevalent within Ciona and human developmental heart enhancers. Here we demonstrate in two diverse systems, Ciona intestinalis and human iPSC-derived cardiomyocytes (iPSC-CMs), that single nucleotide changes can optimize the affinity of ETS binding sites, leading to gain-of-function gene expression associated with heart phenotypes. In Ciona, ETS affinity-optimizing SNVs lead to ectopic expression and phenotypic changes including two beating hearts. In human iPSC-CMs, an affinity-optimizing SNV associated with QRS duration occurs within an SCN5A enhancer and leads to increased enhancer activity. Our mechanistic approach provides a much-needed systematic framework that works across different enhancers, cell types and species to pinpoint causal enhancer variants contributing to enhanceropathies, phenotypic diversity and evolutionary changes. In BriefThe prevalent use of low-affinity ETS sites within developmental heart enhancers creates vulnerability within genomes whereby single nucleotide changes can dramatically increase binding affinity, causing gain-of-function enhancer activity that impacts heart development. HighlightsETS affinity-optimizing SNVs can lead to migration defects and a multi-chambered heart. An ETS affinity-optimizing human SNV within an SCN5A enhancer increases expression and is associated with QRS duration. Searching for ETS affinity-optimizing variants is a systematic and generalizable approach to pinpoint causal enhancer variants.

genomics↗

bioRxiv2022

Affinity-optimizing variants within the ZRS enhancer disrupt limb development

SummaryAn emerging regulatory principle governing enhancers is the use of suboptimal affinity binding sites to encode tissue-specific gene expression. Here we investigate if optimizing single-nucleotide variants that violate this principle can disrupt tissue-specific gene expression and development. The ZRS enhancer mediates expression of Shh in the posterior of the developing limb buds and is critical for limb and digit development. We find that the ZRS contains suboptimal-affinity ETS binding sites. Two human mutations and a synthetic mutation that optimize the affinity of the ETS-A site from 0.15 to 0.25 relative binding affinity cause polydactyly with the same penetrance and severity. Further increasing the affinity of the ETS-A site results in more penetrant and severe phenotypes. The prevalent use of suboptimal affinity binding sites within enhancers to encode tissue-specificity creates a vulnerability within genomes whereby variants that optimize affinity, even subtly, can be pathogenic. This provides a generalizable approach to identify causal variants that underlie enhanceropathies. In BriefSubtle increases in low-affinity sites underlie human limb defects, while greater increases in affinity lead to more severe and penetrant phenotypes. HighlightsPrediction and validation of pathogenic enhancer variants Very subtle increases in affinity of low-affinity sites are pathogenic Penetrance and severity of phenotype scales with increase in affinity

genetics↗