Yike Huang1, Marjon J.A.M. Verstegen1, Sjoerd J.D. Tjalsma1, Peter H.L. Krijger1, Kavvya Gupta2, Minhee Park2,3, Alistair Boettiger2 and Wouter de Laat1 1Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Center Utrecht, Utrecht 3584 CT, the Netherlands; 2Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA Corresponding author: w.l.delaatumcutrecht.nl

↵3 Present address: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon 34141, Republic of Korea.

Abstract

Enhancers are tissue-specific regulatory DNA elements that can activate transcription of genes over distance. Their target genes most often are located in the same contact domain—chromosomal entities formed by cohesin DNA loop extrusion and typically flanked by CTCF-bound boundaries. Enhancers shared by multiple unrelated genes are underexplored but may be more common than anticipated. Here, we analyzed the interplay between an enhancer and two distal functionally unrelated genes residing at opposite domain boundaries. The enhancer strongly activated their expression and supported their frequent interactions. Cohesin structured the domain and supported their transcription, but the genes did not rely on each other's transcription or show gene competition. Deleting either domain boundary not only extended the contact domain but led to reduced contacts within the original domain and reduction in the expression of both genes. Conversely, by isolating either gene with the enhancer in shorter domains, through insertion of new CTCF boundaries, intradomain contact frequencies increased, and the gene isolated with the enhancer was upregulated. Collectively, this shows that an enhancer can independently activate unrelated distal genes and that long-range gene regulation benefits from operating in small contact domains.

Received August 19, 2024. Accepted January 6, 2025.