article epub_ahead yes Elia Mascolo author 776a6ed0-a053-11f0-8635-80b95e0e0d530000-0003-2977-7844 Reka E Körei author 50FDE43E-AA30-11E9-A72B-8A12E6697425 Noa O. Borst author Nicholas H Barton author 4880FE40-F248-11E8-B48F-1D18A9856A870000-0002-8548-5240 Justin Crocker author Gašper Tkačik author 3D494DCA-F248-11E8-B48F-1D18A9856A870000-0002-6699-1455 GaTk department NiBa department Understanding the evolution of continuous genomes project Promoters and enhancers are cis-regulatory elements (CREs), DNA sequences that bind transcription factor (TF) proteins to up- or down-regulate target genes. Decades-long efforts yielded TF-DNA interaction models that predict how strongly an individual TF binds arbitrary DNA sequences and how individual binding events on the CRE combine to affect gene expression. These insights can be synthesized into a global, biophysically realistic, and quantitative genotype-phenotype (GP) map for gene regulation, a ‘holy grail’ for the application of evolutionary theory. A global map provides a rare opportunity to simulate the long-term evolution of regulatory sequences and pose several fundamental questions: How long does it take to evolve CREs de novo? How many non-trivial regulatory functions exist in sequence space? How connected are they? For which regulatory architecture is CRE evolution most rapid and evolvable? In this article, the second of a two-part series, we review the application of evolutionary concepts — epistasis, robustness, evolvability, tunability, plasticity, and bet-hedging — to the evolution of gene regulatory sequences. We then evaluate the potential for a unifying theory for the evolution of regulatory sequences and identify key open challenges. Elsevier2026 eng 0959-437X 1879-038010.1016/j.gde.2026.102472 98 Mascolo, Elia, Reka E Körei, Noa O. Borst, Nicholas H Barton, Justin Crocker, and Gašper Tkačik. “Long-Term Evolution of Regulatory DNA Sequences. Part 2: Theory and Future Challenges.” <i>Current Opinion in Genetics and Development</i>. Elsevier, 2026. <a href="https://doi.org/10.1016/j.gde.2026.102472">https://doi.org/10.1016/j.gde.2026.102472</a>. Mascolo E, Körei RE, Borst NO, Barton NH, Crocker J, Tkačik G. Long-term evolution of regulatory DNA sequences. Part 2: Theory and future challenges. <i>Current Opinion in Genetics and Development</i>. 2026;98. doi:<a href="https://doi.org/10.1016/j.gde.2026.102472">10.1016/j.gde.2026.102472</a> Mascolo, E., Körei, R. E., Borst, N. O., Barton, N. H., Crocker, J., &#38; Tkačik, G. (2026). Long-term evolution of regulatory DNA sequences. Part 2: Theory and future challenges. <i>Current Opinion in Genetics and Development</i>. Elsevier. <a href="https://doi.org/10.1016/j.gde.2026.102472">https://doi.org/10.1016/j.gde.2026.102472</a> Mascolo, Elia, et al. “Long-Term Evolution of Regulatory DNA Sequences. Part 2: Theory and Future Challenges.” <i>Current Opinion in Genetics and Development</i>, vol. 98, 102472, Elsevier, 2026, doi:<a href="https://doi.org/10.1016/j.gde.2026.102472">10.1016/j.gde.2026.102472</a>. Mascolo E, Körei RE, Borst NO, Barton NH, Crocker J, Tkačik G. 2026. Long-term evolution of regulatory DNA sequences. Part 2: Theory and future challenges. Current Opinion in Genetics and Development. 98, 102472. E. Mascolo, R. E. Körei, N. O. Borst, N. H. Barton, J. Crocker, and G. Tkačik, “Long-term evolution of regulatory DNA sequences. Part 2: Theory and future challenges,” <i>Current Opinion in Genetics and Development</i>, vol. 98. Elsevier, 2026. E. Mascolo, R.E. Körei, N.O. Borst, N.H. Barton, J. Crocker, G. Tkačik, Current Opinion in Genetics and Development 98 (2026). 217592026-04-26T22:01:46Z2026-04-28T12:41:00Z