Long, H. K., Prescott, S. L. & Wysocka, J. Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167, 1170–1187 (2016).
Kvon, E. Z. et al. Comprehensive in vivo interrogation reveals phenotypic impact of human enhancer variants. Cell 180, 1262–1271 (2020).
Lambert, S. A. et al. The human transcription factors. Cell 172, 650–665 (2018).
Fickett, J. W. Quantitative discrimination of MEF2 sites. Mol. Cell. Biol. 16, 437–441 (1996).
Gotea, V. et al. Homotypic clusters of transcription factor binding sites are a key component of human promoters and enhancers. Genome Res. 20, 565–577 (2010).
Johnson, D. S., Mortazavi, A., Myers, R. M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).
Dickel, D. E. et al. Ultraconserved enhancers are required for normal development. Cell 172, 491–499 (2018).
Hong, J.-W., Hendrix, D. A. & Levine, M. S. Shadow enhancers as a source of evolutionary novelty. Science 321, 1314 (2008).
Osterwalder, M. et al. Characterization of mammalian in vivo enhancers using mouse transgenesis and CRISPR genome editing. Methods Mol. Biol. 2403, 147–186 (2022).
Snetkova, V. et al. Ultraconserved enhancer function does not require perfect sequence conservation. Nat. Genet. 53, 521–528 (2021).
Attanasio, C. et al. Fine tuning of craniofacial morphology by distant-acting enhancers. Science 342, 1241006 (2013).
Inoue, F. & Ahituv, N. Decoding enhancers using massively parallel reporter assays. Genomics 106, 159–164 (2015).
Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).
Visel, A. et al. A high-resolution enhancer atlas of the developing telencephalon. Cell 152, 895–908 (2013).
Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L. A. VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007).
Spurrell, C. H. et al. Genome-wide fetalization of enhancer architecture in heart disease. Cell Rep. 40, 111400 (2022).
May, D. et al. Large-scale discovery of enhancers from human heart tissue. Nat. Genet. 44, 89–93 (2011).
Dickel, D. E. et al. Genome-wide compendium and functional assessment of in vivo heart enhancers. Nat. Commun. 7, 12923 (2016).
Pampari, A. et al. ChromBPNet: bias factorized, base-resolution deep learning models of chromatin accessibility reveal cis-regulatory sequence syntax, transcription factor footprints and regulatory variants. Preprint at bioRxiv https://doi.org/10.1101/2024.12.25.630221 (2025).
Shrikumar, A., Greenside, P. & Kundaje, A. Learning important features through propagating activation differences. In Proc. 34th International Conference on Machine Learning (eds Precup, D. & Teh, Y. W.) Vol. 70, 3145–3153 (PMLR, 2017).
Firulli, B. A., Redick, B. A., Conway, S. J. & Firulli, A. B. Mutations within helix I of Twist1 result in distinct limb defects and variation of DNA binding affinities. J. Biol. Chem. 282, 27536–27546 (2007).
Selleri, L. & Rijli, F. M. Shaping faces: genetic and epigenetic control of craniofacial morphogenesis. Nat. Rev. Genet. 24, 610–626 (2023).
Prescott, S. L. et al. Enhancer divergence and cis-regulatory evolution in the human and chimp neural crest. Cell 163, 68–83 (2015).
Kim, S. et al. DNA-guided transcription factor cooperativity shapes face and limb mesenchyme. Cell 187, 692–711 (2024).
Olson, E. N. Gene regulatory networks in the evolution and development of the heart. Science 313, 1922–1927 (2006).
Smith, R. P. et al. Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat. Genet. 45, 1021–1028 (2013).
Lettice, L. A. et al. Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly. Dev. Cell 22, 459–467 (2012).
Lettice, L. A., Devenney, P., De Angelis, C. & Hill, R. E. The conserved Sonic Hedgehog limb enhancer consists of discrete functional elements that regulate precise spatial expression. Cell Rep. 20, 1396–1408 (2017).
Spitz, F. & Furlong, E. E. M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012).
Gorkin, D. U. et al. An atlas of dynamic chromatin landscapes in mouse fetal development. Nature 583, 744–751 (2020).
ENCODE Project Consortium et al. Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583, 699–710 (2020).
Roadmap Epigenomics Consortium et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).
Rebboah, E. et al. The ENCODE mouse postnatal developmental time course identifies regulatory programs of cell types and cell states. Preprint at bioRxiv https://doi.org/10.1101/2024.06.12.598567 (2024).
Ameen, M. et al. Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease. Cell 185, 4937–4953 (2022).
Taskiran, I. I. et al. Cell-type-directed design of synthetic enhancers. Nature 626, 212–220 (2024).
de Almeida, B. P., Reiter, F., Pagani, M. & Stark, A. DeepSTARR predicts enhancer activity from DNA sequence and enables the de novo design of synthetic enhancers. Nat. Genet. 54, 613–624 (2022).
Rafi, A. M. et al. A community effort to optimize sequence-based deep learning models of gene regulation. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02414-w (2024).
Karbalayghareh, A., Sahin, M. & Leslie, C. S. Chromatin interaction-aware gene regulatory modeling with graph attention networks. Genome Res. 32, 930–944 (2022).
Tan, J. et al. Cell-type-specific prediction of 3D chromatin organization enables high-throughput in silico genetic screening. Nat. Biotechnol. 41, 1140–1150 (2023).
Farley, E. K., Olson, K. M., Zhang, W., Rokhsar, D. S. & Levine, M. S. Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers. Proc. Natl Acad. Sci. USA 113, 6508–6513 (2016).
Farley, E. K. et al. Suboptimization of developmental enhancers. Science 350, 325–328 (2015).
Jindal, G. A. et al. Single-nucleotide variants within heart enhancers increase binding affinity and disrupt heart development. Dev. Cell 58, 2206–2216 (2023).
Crocker, J. et al. Low affinity binding site clusters confer hox specificity and regulatory robustness. Cell 160, 191–203 (2015).
Boisclair Lachance, J.-F., Webber, J. L., Hong, L., Dinner, A. R. & Rebay, I. Cooperative recruitment of Yan via a high-affinity ETS supersite organizes repression to confer specificity and robustness to cardiac cell fate specification. Genes Dev. 32, 389–401 (2018).
Borok, M. J., Tran, D. A., Ho, M. C. W. & Drewell, R. A. Dissecting the regulatory switches of development: lessons from enhancer evolution in Drosophila. Development 137, 5–13 (2010).
Galupa, R. et al. Enhancer architecture and chromatin accessibility constrain phenotypic space during Drosophila development. Dev. Cell 58, 51–62 (2023).
Fuqua, T. et al. Dense and pleiotropic regulatory information in a developmental enhancer. Nature 587, 235–239 (2020).
Le Poul, Y. et al. Regulatory encoding of quantitative variation in spatial activity of a enhancer. Sci. Adv. 6, eabe2955 (2020).
Museridze, M. et al. Entangled and non-modular enhancer sequences producing independent spatial activities. Sci. Adv. 10, eadr9856 (2024).
Swanson, C. I., Schwimmer, D. B. & Barolo, S. Rapid evolutionary rewiring of a structurally constrained eye enhancer. Curr. Biol. 21, 1186–1196 (2011).
Trevino, A. E. et al. Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution. Cell 184, 5053–5069 (2021).
Feng, J., Liu, T., Qin, B., Zhang, Y. & Liu, X. S. Identifying ChIP-seq enrichment using MACS. Nat. Protoc. 7, 1728–1740 (2012).
Lundberg, S. & Lee, S.-I. A unified approach to interpreting model predictions. Preprint at https://doi.org/10.48550/arXiv.1705.07874 (2017).
Gupta, S., Stamatoyannopoulos, J. A., Bailey, T. L. & Noble, W. S. Quantifying similarity between motifs. Genome Biol. 8, R24 (2007).
Kosicki, M. et al. Massively parallel reporter assays and mouse transgenic assays provide correlated and complementary information about neuronal enhancer activity. Nat. Commun. 16, 4786 (2025).
Ma, X. R. et al. Molecular convergence of risk variants for congenital heart defects leveraging a regulatory map of the human fetal heart. Preprint at medRxiv https://doi.org/10.1101/2024.11.20.24317557 (2024).
Tenney, A. P. et al. Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis. Nat. Genet. 55, 1149–1163 (2023).
Lee, A. S. et al. A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders. Nat. Commun. 15, 8268 (2024).