Biology subjects

Schaepe, J. M.

Publications and source records attributed to Schaepe, J. M..

4 recordsLinked to original sources

bioRxiv2025

The human transcription factor occupancy landscape viewed using high-resolution in situ base-conversion strand-specific single-molecule chromatin accessibility mapping

Chromatin accessibility profiling is a key tool for mapping the location of cis-regulatory elements (cREs) in the genome and tracking chromatin state dynamics during development, in response to various external and internal stimuli, and in disease contexts. Single-molecule footprinting (SMF) methods that rely on the labeling of individual accessible DNA bases have emerged in recent years as a powerful chromatin accessibility mapping approach, as they provide not just an average readout of accessibility over a given genomic position but also the distribution of accessibility states within a population at the level of individual original DNA molecules. However, methylation-based SMF approaches have often been limited either in their resolution or in labeling readout accuracy. We developed a deaminase-based high-resolution strand-specific single molecule footprinting approach that uses highly active sequence context-independent endogenous methylation-insensitive double-strand DNA (dsDNA) deaminases (CseDa01 and LbDa02 C[->]U/T-ssSMF), which convert accessible cytosines into uracils (which are converted to thymine after PCR amplification). We demonstrate the application of the method to mapping single-molecule accessibility states in human cell lines in both a short and a long-read format, and quantifying the occupancy states of individual transcription factors (TFs) as well as TF co-accessibility and strand-specific accessibility patterns.

genomics↗

bioRxiv2025

Thermodynamic principles link in vitro transcription factor affinities to single-molecule chromatin states in cells

The molecular details governing transcription factor (TF) binding and the formation of accessible chromatin are not yet quantitatively understood - including how sequence context modulates affinity, how TFs search DNA, the kinetics of TF occupancy, and how motif grammars coordinate binding. To resolve these questions for a human TF, erythroid Kruppel-like factor (eKLF/KLF1), we quantitatively compare, in high throughput, in vitro TF binding rates and affinities with in vivo single molecule TF and nucleosome occupancies across engineered DNA sequences. We find that 40-fold flanking sequence effects on affinity are consistent with distal flanks tuning TF search parameters and captured by a linear energy model. Motif recognition probability, rather than time in the bound state, drives affinity changes, and in vitro and in nuclei measurements exhibit consistent, minutes-long TF residence times. Finally, pairing in vitro biophysical parameters with thermodynamic models accurately predicts in vivo single-molecule chromatin states for unseen motif grammars. HighlightsO_LIKLF1:DNA binding is consistent with a three state binding model wherein the probability of recognizing the motif from a nonspecifically-bound state drives affinity. C_LIO_LISubstantial effects from proximal and distal motif-flanking sequence on KLF1 binding affinity are captured by an extended PWM model. C_LIO_LIKLF minutes-long residence time inferred from single-molecule footprinting in nuclei is consistent with in vitro measurement. C_LIO_LIIn vitro binding energies combined with thermodynamic models predict in vivo, single-molecule chromatin configurations across motif grammars and sequence contexts. C_LI

biophysics↗

bioRxiv2024

Single-molecule chromatin configurations link transcription factor binding to expression in human cells

The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TFs capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.

molecular biology↗

bioRxiv2022

Short tandem repeats bind transcription factors to tune eukaryotic gene expression

Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and their polymorphisms alter gene expression, yet how they regulate transcription remains unknown. We find that STRs can modulate transcription factor (TF)-DNA affinities and on rates by up to 70-fold by directly binding TF DNA-binding domains, with energetic impacts approaching or exceeding mutations to consensus sites. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density near motifs to speed target search. Confirming that STRs also impact TF binding in cells, neural networks trained only on in vivo occupancies predict identical effects to those observed in vitro. Approximately 90% of TFs preferentially bind STRs that need not resemble known motifs, providing a novel cis-regulatory mechanism to target TFs to cognate sites.

biophysics↗