Cumulative binding of multiple transcription factors and p300 regulates enhancer activity frequency
Background
Enhancers are DNA regions that regulate gene transcription, but they are not always open in all cells, even within the same cell population. Transcription factors compete with nucleosomes to access their binding sites, and this process leads to cell-to-cell variation in chromatin accessibility. While bulk assays like ATAC-seq are useful for identifying open DNA regions, they do not directly reveal the fraction of cells in which a specific regulatory region is open. In this study, researchers used single-molecule footprinting in mouse embryonic stem cells to measure the fraction of molecules at enhancers and promoters that are actually open, and to investigate how individual transcription factors and chromatin context contribute to accessibility frequency.
Key Findings
Most individual transcription factors showed a modest effect on chromatin opening in only a small fraction of cells. However, the frequency and extent of open chromatin increased as the number of transcription factors bound to a regulatory region increased. Experiments in which individual binding motifs were weakened or SOX2 was rapidly removed showed that accessibility was reduced, but not completely abolished, supporting a model of cumulative action of multiple transcription factors. Active enhancers were open in less than half of the cells, while promoters tended to be more accessible. Furthermore, testing hundreds of enhancers at the same genomic location revealed that full activity required the activity of the histone acetyltransferase p300. Inhibition of p300 reduced enhancer accessibility frequency across the genome.
Significance and Implications
This study supports a model in which enhancer activity frequency is determined by the combined contributions of multiple transcription factors and the chromatin environment created by p300, rather than being switched on by a single 'master regulator'. Changes in chromatin accessibility were associated with changes in recruitment of RNA polymerase II and enhancer activity. Measuring accessibility frequency provides a quantitative framework for understanding how the same enhancer can function differently in different cells. While the results are based on mechanistic studies in mouse embryonic stem cells and the researchers' experimental system, they do not immediately translate into therapeutic applications. However, they provide a more quantitative framework for interpreting cell-to-cell variation in gene expression and for predicting the function of regulatory regions.
Nature Genetics, Published online: 31 July 2026; doi:10.1038/s41588-026-02703-xThis study uses single-molecule footprinting to quantify chromatin accessibility at enhancers and promoters in mouse embryonic stem cells and to dissect the contributions of transcription factor binding and chromatin context.
Regulatory regions of the genome are not simply 'on' or 'off'. This study measures how often enhancers are open at the single-molecule level in a cell population and provides evidence that this frequency is regulated by the cumulative binding of multiple transcription factors and the activity of p300. This framework helps to understand why mutations that weaken the binding of a specific transcription factor often have only a partial effect. Future studies should investigate whether the same principles apply in other cell types and during differentiation.