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Results for “single-molecule footprinting”

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6 recordsLinked to original sources
PubMed2026

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 transcriptional activation. Our data uncover dependencies between CREs genome-wide and suggest that coordinated enhancer activation is a widespread mechanism regulating gene expression.

Animals↗
PubMed2004

IHF and HU: flexible architects of bent DNA.

The energetic cost of bending short segments of DNA is very high. This bending is critical for the packaging of DNA and is exploited to regulate many cellular processes. In prokaryotes, IHF and HU are key architectural proteins present at high concentrations. New protein-DNA co-crystal structures, and the adaptation of advanced biophysical and biochemical techniques have led to an improved understanding of how these proteins interact with DNA. These techniques include time-resolved synchrotron X-ray footprinting, differential scanning calorimetry, isothermal titration calorimetry and single-molecule experiments.

Amino Acid Sequence↗
PubMedDate not supplied

Site-specific binding of the 9.5 kilodalton DNA-binding protein ORF80 visualized by atomic force microscopy.

Atomic force microscopy (AFM) has been used to examine the binding properties of the DNA-binding protein ORF80 to DNA. ORF80 is a 9.5 kDa protein that binds site-specifically to double-stranded DNA of the sequence TTAA-N(7)-TTAA. Direct sizing of the protein complexes on DNA fragments from the plasmid pRN1 with AFM shows that the protein ORF80 binds preferentially to two positions. These positions agree well with the ORF80 binding sites determined by footprinting analysis. The measurements allow an estimate of the stoichiometry of the DNA-protein complexes. In contrast to previous results, the single-molecule experiments suggest that only a low number of ORF80 molecules bind to a DNA-binding site.

Archaeal Proteins↗
PubMed2005

Single-molecule spectroscopic determination of lac repressor-DNA loop conformation.

The Escherichia coli lactose repressor protein (LacI) provides a classic model for understanding protein-induced DNA looping. LacI has a C-terminal four-helix bundle tetramerization domain that may act as a flexible hinge. In previous work, several DNA constructs, each containing two lac operators bracketing a sequence-induced bend, were designed to stabilize different possible looping geometries. The resulting hyperstable LacI-DNA loops exist as both a compact "closed" form with a V-shaped repressor and also a more "open" form with an extended hinge. The "9C14" construct was of particular interest because footprinting, electrophoretic mobility shift, and ring closure experiments suggested that it forms both geometries. Previous fluorescence resonance energy transfer (FRET) measurements gave an efficiency of energy transfer (ET) of 70%, confirming the existence of a closed form. These measurements could not determine whether open form or intermediate geometries are populated or the timescale of interconversion. We have now applied single-molecule FRET to Cy3, Cy5 double-labeled LacI-DNA loops diffusing freely in solution. By using multiple excitation wavelengths and by carefully examining the behavior of the zero-ET peak during titration with LacI, we show that the LacI-9C14 loop exists exclusively in a single closed form exhibiting essentially 100% ET.

Bacterial Proteins↗
PubMed2005

Detection of high-affinity and sliding clamp modes for MSH2-MSH6 by single-molecule unzipping force analysis.

Mismatch repair (MMR) is initiated by MutS family proteins (MSH) that recognize DNA mismatches and recruit downstream repair factors. We used a single-molecule DNA-unzipping assay to probe interactions between S. cerevisiae MSH2-MSH6 and a variety of DNA mismatch substrates. This work revealed a high-specificity binding state of MSH proteins for mismatch DNA that was not observed in bulk assays and allowed us to measure the affinity of MSH2-MSH6 for mismatch DNA as well as its footprint on DNA surrounding the mismatch site. Unzipping analysis with mismatch substrates containing an end blocked by lac repressor allowed us to identify MSH proteins present on DNA between the mismatch and the block, presumably in an ATP-dependent sliding clamp mode. These studies provide a high-resolution approach to study MSH interactions with DNA mismatches and supply evidence to support and refute different models proposed for initiation steps in MMR.

Adenosine Triphosphate↗
PubMed2026

HoT auto-blinking probes enable real-time, super-resolution chromatin imaging in live cells and tissues.

Single-molecule localization microscopy (SMLM) enables visualization of chromatin architecture at nanoscale resolution. However, high-performance DNA probes suitable for SMLM in both live cells and tissues remain limited. We developed Hoechst-6-Carboxytetramethylrhodamine (6-TAMRA) derivative (HoT) probes-rhodamine-based derivatives conjugated to a Hoechst moiety-through structural fine-tuning of rhodamine spirocyclization. HoTs are self-assembling, auto-blinking probes with excellent photostability and high temporal resolution. They permeate live cells, enabling long-term, real-time nanoscopic chromatin imaging in live and fixed cells and in tissue sections. In live cells, we identified nanoscale features in the 3D organization of chromatin and quantified DNA fiber kinetics at high resolution. We quantified DNA compaction in single cells within retinal and colon cancer sections. OligoSTORM (stochastic optical reconstruction microscopy)-labeled gene loci can be visualized and measured within their HoT-labeled chromatin footprints. Our work provides powerful tools for investigating chromatin structure and functions in living cells and tissues, with applications ranging from cancer diagnosis to retinal regeneration.

Chromatin↗