Biology subjects

Kleinendorst, R.

Publications and source records attributed to Kleinendorst, R..

6 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

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↗

bioRxiv2024

Mammalian promoters are characterised by low occupancy and high turnover of RNA polymerase II

The general transcription machinery and its occupancy at promoters are highly conserved across metazoans. This contrasts with the kinetics of mRNA production that considerably differ between model species such as Drosophila and mouse. The molecular basis for these kinetic differences is currently unknown. Here, we used Single Molecule Footprinting to measure RNA Polymerase II (Pol II) occupancy, the fraction of DNA molecules bound, at promoters in mouse and Drosophila cell lines. Single molecule data reveals that Pol II occupancy is on average 3-5 times more frequent at transcriptionally active Drosophila promoters than active mouse promoters. Kinetic modelling of the occupancy states suggests that these differences in Pol II occupancy are determined by the ratio between the transcription initiation and Pol II turnover rates. We used chemical perturbation of transcription initiation to determine Pol II turnover rate in both species. Integration of these data into the model shows that infrequent Pol II occupancy in mammals is explained by the combination of high Pol II turnover and low transcription initiation rates.

genomics↗

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↗

bioRxiv2017

The small non-coding vault RNA1-1 acts as a riboregulator of autophagy

Vault RNAs (vtRNA) are small, 88-100nt non-coding RNAs found in many eukaryotes. Although they have been linked to drug resistance, apoptosis and nuclear transport, their function remains unclear. Here we show that a human vtRNA, RNA1-1, specifically binds to the autophagy receptor sequestosome-1/p62. Antisense-mediated depletion of vault RNA1-1 augments, whereas increased vault RNA1-1 expression restricts, autophagic flux in a p62-dependent manner. Bulk autophagy induced by starvation reduces the levels of vault RNA1-1 and the fraction of RNA-bound p62. These findings show that RNAs can act as riboregulators of biological processes by interacting with proteins, and assign a function to a vault RNA.

molecular biology↗