Affinity-optimizing variants within the ZRS enhancer disrupt limb development

Abstract

An 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.

Unique functions of two overlappingPAX6retinal enhancers

“…Several issues challenge our understanding of the precise role of enhancers in developmental and disease processes, including relating the sequence-specific grammar of these elements to their function, defining their exact domains of action, and disentangling the additive, redundant or distinct functions of apparently similar enhancers. A few enhancers, such as the famous ZRS limb enhancer of SHH , have been so thoroughly characterised that they can now be manipulated at the single base pair level to achieve specific phenotypes (Lettice et al, 2003; Kvon et al, 2020; Lim et al, 2022). However, our understanding of the other hundreds of thousands of predicted enhancers in the human genome lags far behind this, and for the majority it remains challenging to identify even the target gene with confidence.…”

Ciona, an ideal research organism to study the role of enhancers

“…We have found signatures of enhancer grammar conserved across chordates—in Ciona , mice, and humans (Song et al, 2023). We've also expanded to look at how violations in regulatory principles governing enhancers can drive organismal‐level phenotypes in Ciona and other species, such as mice and humans (Jindal et al, 2023; Lim et al, 2022). We've found that single nucleotide variants (SNVs) can increase binding site affinity driving ectopic expression and organismal phenotypes as severe as a second heart in Ciona and extra fingers in mice and humans (Jindal et al, 2023; Lim et al, 2022).…”

“…We have found signatures of enhancer grammar conserved across chordates-in Ciona, mice, and humans (Song et al, 2023). We've also expanded to look at how violations in regulatory principles governing enhancers can drive organismal-level phenotypes in Ciona and other species, such as mice and humans (Jindal et al, 2023;Lim et al, 2022).…”

“…We've found that single nucleotide variants (SNVs) can increase binding site affinity driving ectopic expression and organismal phenotypes as severe as a second heart in Ciona and extra fingers in mice and humans (Jindal et al, 2023;Lim et al, 2022). Thus, the principle of suboptimization of developmental enhancers to encode tissue-specific expression, which we initially discovered in Ciona, applies to other organisms too.…”

Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters

“…This tight control underlies the robust emergence of form and function from a one-cell zygote. Fine regulatory changes of target genes, caused by even single nucleotide changes to individual enhancers, can both give rise to disease ( 1 – 3 ) as well as drive novelty across evolution ( 1, 4 ). Genetic methods have identified an extensive list of developmentally important genes in model systems ( 5, 6 ), yet how the transcription of these genes is regulated by enhancers, and specifically how DNA sequence encodes the requisite functional information, remains incompletely understood even for the best-studied examples ( 7 – 10 ).…”