Happy to share our latest work on In Vivo Systematic Mutagenesis of a Human Enhancer @AxelVisel @diane_dickel @LenPennacchio @LBNLBioSci @BerkeleyLab
cell.com/cell/fulltext/…
Thread👇 (1/12)
cell.com/cell/fulltext/…
Thread👇 (1/12)
A major challenge in human genomics is how to systematically assess the phenotypic impact of enhancer variants identified in human genetics studies. Mouse transgenics is a powerful tool for this purpose but has limited throughput, high cost, and so-called position effects (2/12) 
enSERT avoids position effects from random integration and results in a much higher integration rate for large constructs (50%vs12%). Having come from #Drosophila where site-specific transgenics is a norm (but not as efficient), it was satisfying to get it to work in mice (4/12)
We then used enSERT to systematically mutagenize all nucleotides in the human ZRS enhancer of the Sonic Hedgehog gene (SHH), a powerful regulator of limb development. The ZRS activates SHH expression in the limbs over an extreme distance of nearly 1 million base pairs. (5/12)
Changes in the ZRS have been implicated in vertebrate evolution (see our previous work on limb loss in snakes: ) and also cause polydactyly in human and several other tetrapods (6/12)
We show that ~70% of all rare non-coding ZRS variants previously proposed as causal led to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knock-in mice (7/12)
In what was perhaps the most surprising result, ~30% of previously assumed pathogenic variants led to normal ZRS enhancer activity, suggesting that care should be taken when interpreting rare non-coding human variants without experimental validation (8/12)
Finally, we performed systematic mutagenesis of the ZRS enhancer and demonstrate that many nucleotides spread across the entire length of the enhancer are critical for its normal function suggesting widespread epistatic interactions within the enhancer (9/12)
Notably, random mutagenesis uncovered novel gain-of-function mutations that have not yet been reported in human patients with polydactyly (10/12)
We propose that preemptive in vivo saturation mutagenesis screens of disease-associated enhancers to establish supportive functional data will facilitate the interpretation of future human genetics findings (11/12)
And check out this awesome summary video by @AxelVisel
Although I'd prefer to be a Jedi than a Stormtrooper :)
Although I'd prefer to be a Jedi than a Stormtrooper :)
