Biology subjects

Krebs, A. R.

Publications and source records attributed to Krebs, A. R..

7 recordsLinked to original sources

bioRxiv2026

SMFnet reveals principles of combinatorial transcription factor assembly

Combinatorial assembly of transcription factors (TFs) opens chromatin at cis-regulatory elements (cREs) to establish cell-type-specific gene expression. Dissecting the role of individual TFs and their collective function has been limited by the diversity of TF binding-site arrangements across cREs. A single regulatory sequence can support multiple configurations of TF and nucleosome occupancy, raising the possibility that distinct chromatin states reflect different TF assemblies. Predictive models linking regulatory sequence to chromatin accessibility collapse these distinct molecular states into population averages, obscuring how TF assemblies shape accessible chromatin. Here, we introduce SMFNet, a generative diffusion model that predicts distributions of single-molecule chromatin accessibility profiles from DNA sequence, and PRISM, which identifies sequence features associated with individual molecular states. Together, these approaches enable us to systematically associate single molecule chromatin states at cREs with specific TF binding motifs. Experimental perturbation of these motifs redistributes the corresponding states as predicted by SMFNet, supporting their interpretation as TF-dependent configurations underlying chromatin opening. We then use controlled in silico motif screens to systematically dissect individual and combinatorial TF contributions to chromatin opening. Individual TFs show distinct chromatin-opening signatures, differing in accessible-region width and nucleosome-positioning precision even when associated with similar levels of chromatin accessibility. Combining motifs reveals partner- and spacing-dependent effects on chromatin accessibility, TF occupancy and nucleosome positioning. Genetic perturbation of TF binding sites using natural variation at endogenous cRE confirms that TF-specific properties shape chromatin accessibility in vivo. Together, our findings reveal sequence-encoded principles of combinatorial TF assembly during chromatin opening.

genetics↗

bioRxiv2025

Molecular co-accessibility identifies coordinated regulation between distant cis-regulatory elements

In metazoans, gene expression is typically regulated by a cis-regulatory landscape (CRL) composed of a promoter and multiple enhancers. How these cis-regulatory elements (CREs) coordinate their function across large genomic distances remains unclear. For example, is the simultaneous activation of multiple enhancers required to promote transcription? Here, we combined single-molecule footprinting with long-read sequencing to quantify how often chromatin accessibility and transcription factor binding co-occur across entire CRLs in the Drosophila genome. Analysis of thousands of individual DNA molecules at each locus revealed that CREs form a specific network with shared single-molecule chromatin accessibility profiles. Co-accessibility is not limited to adjacent CREs, and is frequently observed between CREs brought into proximity by chromatin looping. Co-accessible CREs exhibit strong coordination in their cell type-specific accessibility, linking enhancer activity with transcription activation. Our data uncover dependencies between CREs genome-wide, and suggest that coordinated enhancer activation is a widespread mechanism regulating gene expression.

genomics↗

bioRxiv2025

FootprintCharter: unsupervised detection and quantification of footprints in single molecule footprinting data

SummarySingle molecule footprinting profiles the heterogeneity of TF occupancy at cis-regulatory elements across cell populations at unprecedented resolution. The single molecule nature of the data in principle allows for observing the footprint of individual transcription factors and nucleosomes. However, we currently lack algorithms to quantify these occupancy patterns of chromatin binding factors in an automated way and without prior assumptions on their genomic location. Here we present FootprintCharter, an unsupervised tool to detect and quantify footprints for transcription factors (TFs) and nucleosomes from single molecule footprinting data. FootprintCharter allows for the quantification of complex molecular states such as positioning of unphased nucleosomes and combinatorial co-binding of multiple TFs. Availability and implementationFootprintCharter is freely available on Bioconductor with version 2.1.6 of https://bioconductor.org/packages/SingleMoleculeFootprinting with the functions FootprintCharter, PlotFootprints and Plot_FootprintCharter_SM.

bioinformatics↗

bioRxiv2025

Cumulative TF binding and H3K27 Acetylation drive enhancer activation frequency

In eukaryotes, transcription factors (TFs) must continuously compete with nucleosomes to access their binding sites, leading to cell-to-cell variability in chromatin accessibility at regulatory regions. Although critical to understand enhancer function in transcription, the mechanisms that define how frequently an enhancer is active in a cell population remain unclear. Here, we used single molecule footprinting to quantify the frequency at which chromatin is accessible at enhancers in response to TF perturbations, and changes of their chromatin environment. We find that individually most TFs open chromatin in a small fraction of the cells, and that cumulative TF binding controls enhancers activation frequency. Moreover, testing the functionality of hundreds of enhancers when inserted at an ectopic genomic location identifies H3K27Ac as a chromatin mark required for their full activation. Our data support a model where enhancer activation frequency depends on the cumulative function of multiple TFs, enhanced by activating chromatin marks.

molecular biology↗

bioRxiv2023

Identification of transcription factor co-binding patterns with non-negative matrix factorization

Transcription factor (TF) binding to DNA is critical to transcription regulation. Although the binding properties of numerous individual TFs are well-documented, a more detailed comprehension of how TFs interact cooperatively with DNA, forming either complex or co-binding to the same region, is required. Indeed, the combinatorial binding of TFs is essential to cell differentiation, development, and response to external stimuli. We present COBIND, a novel method based on non-negative matrix factorization (NMF) to identify TF co-binding patterns automatically. COBIND applies NMF to one-hot encoded regions flanking known TF binding sites (TFBSs) to pinpoint enriched DNA patterns at fixed distances. We applied COBIND to 8,293 TFBS datasets from UniBind for 404 TFs in seven species. The method uncovered already established co-binding patterns (e.g., between POU5F1 and SOX2 or SOX17) and new co-binding configurations not yet reported in the literature and inferred through motif similarity and protein-protein interaction knowledge. Our extensive analyses across species revealed that 84% of the studied TFs share a co-binding motif with other TFs from the same structural family. The co-binding patterns captured by COBIND are likely functionally relevant as they harbor higher evolutionarily conservation than isolated TFBSs. Open chromatin data from matching human cell lines further supported the co-binding predictions. Finally, we used single-molecule footprinting data from mouse embryonic stem cells to confirm that the co-binding events captured by COBIND were likely occurring on the same DNA molecules.

bioinformatics↗

bioRxiv2022

Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation

Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with local DNA demethylation. Yet, whether this epigenetic remodelling is necessary for enhancer activation is unknown. Here, we developed a single molecule multi-omics approach to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their activity in multiple cell lineages. While reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where presence of DNA methylation antagonises the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding are a direct function of active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at cell-type-specific active enhancers.

genomics↗

bioRxiv2020

Single molecule occupancy patterns of transcription factors reveal determinants of cooperative binding in vivo

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements. Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we advance Single Molecule Footprinting to detect individual molecular interactions of transcription factors and nucleosomes with DNA at mouse cis-regulatory elements. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that for most types of transcription factors high DNA co-occupancy can occur in absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy for binding cooperativity. These findings elucidate the binding cooperativity mechanism used by transcription factors in absence of strict organisation of their binding motifs, a characteristic feature of most of enhancers.

genomics↗