The human genome contains more than 20,000 genes, yet most cells use only a fraction of them at any given moment. How cells decide which genes to activate—and how strongly to activate them—remains one of biology’s most difficult questions. Researchers at the European Molecular Biology Laboratory (EMBL) Heidelberg have now developed a synthetic biology method that allows them to investigate this process with unprecedented control. Their approach, called mCHIRA, lets scientists place hundreds or even thousands of regulatory DNA sequences at a defined location in the genome and observe how those sequences influence gene regulation inside living cells.
The work, led by Arnaud Krebs’s group, addresses a major challenge in molecular genetics. Regulatory DNA sequences do not operate in isolation. Their activity depends not only on their own nucleotide sequence, but also on the surrounding genomic environment, the chemical marks attached to DNA-associated proteins, and the availability of regulatory proteins. Because these factors vary from one genomic region to another, it can be difficult to determine whether a regulatory sequence is intrinsically powerful or simply benefits from its natural location. mCHIRA gives researchers a controlled experimental setting in which sequence and genomic context can be examined separately.
The team focused on enhancers, stretches of DNA that help control when and where genes are transcribed. Enhancers are recognized by transcription factors, proteins that bind specific DNA motifs and influence the molecular machinery responsible for copying DNA information into messenger RNA. Once produced, messenger RNA carries genetic instructions to ribosomes, which use them to assemble proteins. Although transcription factors are often studied individually, cells typically regulate genes through combinations of many such proteins. The EMBL researchers found that this cumulative binding can have a much greater effect than the activity of any single transcription factor alone.
Their results indicate that multiple transcription factors increase the probability that an enhancer becomes accessible to the transcriptional machinery. In practical terms, each binding event can contribute to a regulatory state in which the DNA is more likely to be exposed and active. This effect is not merely additive: combinations of transcription factors can substantially increase enhancer activation frequency. The finding helps explain how relatively small changes in regulatory DNA may produce large differences in gene activity, particularly when those changes affect binding sites used by several transcription factors.
The researchers also examined H3K27 acetylation, an epigenetic mark associated with active enhancers. Epigenetic marks do not change the DNA sequence itself; instead, they influence how DNA is packaged and read by the cell. H3K27 acetylation is linked to a more permissive chromatin environment, helping regulatory regions remain accessible. According to the study, cumulative transcription factor binding and H3K27 acetylation work together to drive the frequency with which enhancers become active. This provides a more detailed view of enhancer regulation, in which DNA sequence, protein binding, chromatin accessibility, and chemical modification operate as interconnected parts of the same system.
mCHIRA was designed to make these relationships experimentally measurable. The method uses a synthetically constructed genomic region that serves as a standardized landing site for regulatory elements. Scientists can insert different enhancer or promoter sequences into this locus and compare their behavior under the same broader genomic conditions. By testing many sequence variants in parallel, researchers can identify the effects of individual mutations or combinations of mutations without the results being dominated by unrelated differences in chromosomal location.
“Basically, this method enables us to insert, in a specific locus in the genome, hundreds or thousands of regulatory elements, such as enhancers or promoters, so that we could study how the genomic environment is shaping transcription factor binding, enhancer accessibility, and finally transcription,” said Valentina Baderna, one of the study’s first authors and a former PhD student in the Krebs Group. The approach could be especially valuable for studying disease-associated variants found in noncoding DNA, much of which lies outside protein-coding genes but contains important regulatory information.
To capture the molecular events taking place on individual DNA molecules, the team used single-molecule footprinting. This technique records patterns left by transcription factors and other molecular components as they bind to DNA, allowing researchers to distinguish regulatory states within individual cells rather than relying only on population averages. The analysis was supported by FootprintCharter, a computational framework developed by co-first author Guido Barzaghi in collaboration with Judith Zaugg’s computational biology group at EMBL Heidelberg and the University of Basel. The software organizes the complex patterns generated by single-molecule footprinting without requiring researchers to define all possible molecular states in advance.
“Measuring multiple aspects of DNA regulation at once, single-molecule footprinting makes for an excitingly data-rich technology,” Barzaghi said. “FootprintCharter has been our first attempt at distilling its information in an unsupervised way.” Combining the experimental and computational tools allowed the researchers to quantify transcription factor binding, chromatin accessibility, and epigenetic signatures at high resolution. Instead of observing only whether a gene is on or off, they could examine the intermediate molecular states that determine how frequently an enhancer becomes active.
The findings point toward a general framework for understanding gene regulation: regulatory sequences provide binding instructions, transcription factors accumulate on those sequences, and epigenetic modifications help determine whether the resulting molecular complex can engage the transcriptional machinery. Krebs said that synthetic biology and quantitative genomics can break the complexity of the genome into manageable experimental units, making it possible to uncover rules that connect DNA sequence to cellular behavior. In the future, the researchers expect that integrating these methods with artificial intelligence could enable the systematic testing and prediction of vast numbers of regulatory sequences, accelerating the study of development, disease, and personalized genomic variation.
Subject of Research:
Synthetic biology, gene regulation, enhancer activity, transcription factor binding, chromatin accessibility, and epigenetics.
Article Title:
Cumulative TF binding and H3K27 Acetylation drive enhancer activation frequency.
Web References:
https://doi.org/10.1038/s41588-026-02703-x
https://academic.oup.com/bioinformatics/article/41/10/btaf502/8256680
https://biomedizin.unibas.ch/en/research/research-groups/zaugg-lab/
References:
Nature Genetics, DOI: 10.1038/s41588-026-02703-x.
Image Credits:
Daniela Velasco/EMBL.
Keywords:
Synthetic biology, gene regulation, enhancers, transcription factors, H3K27 acetylation, epigenetics, chromatin accessibility, single-molecule footprinting, mCHIRA, FootprintCharter, genomics, Nature Genetics.
