
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.06.611737
preprint
1
Article
Mutagenesis Sensitivity Mapping of Human Enhancers In Vivo
Kosicki Michael http://orcid.org/0000-0001-7173-8852

Zhang Boyang
Pampari Anusri
Akiyama Jennifer A
Playzer-Frick Ingrid
Novak Catherine S
Tran Stella
Zhu Yiwen
Kato Momoe
Hunter Riana D
von Maydell Kianna
Barton Sarah
Beckman Erik
Kundaje Anshul http://orcid.org/0000-0003-3084-2287

Dickel Diane E http://orcid.org/0000-0001-5497-6824

Visel Axel http://orcid.org/0000-0002-4130-7784

Pennacchio Len A http://orcid.org/0000-0002-8748-3732

08 9 2024
2024.09.06.611737https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.06.611737
nihpp-2024.09.06.611737.pdf
Distant-acting enhancers are central to human development. However, our limited understanding of their functional sequence features prevents the interpretation of enhancer mutations in disease. Here, we determined the functional sensitivity to mutagenesis of human developmental enhancers in vivo. Focusing on seven enhancers active in the developing brain, heart, limb and face, we created over 1700 transgenic mice for over 260 mutagenized enhancer alleles. Systematic mutation of 12-basepair blocks collectively altered each sequence feature in each enhancer at least once. We show that 69% of all blocks are required for normal in vivo activity, with mutations more commonly resulting in loss (60%) than in gain (9%) of function. Using predictive modeling, we annotated critical nucleotides at base-pair resolution. The vast majority of motifs predicted by these machine learning models (88%) coincided with changes to in vivo function, and the models showed considerable sensitivity, identifying 59% of all functional blocks. Taken together, our results reveal that human enhancers contain a high density of sequence features required for their normal in vivo function and provide a rich resource for further exploration of human enhancer logic.
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