Single-molecule Dynamics of Enhanceosome Assembly In Embryonic Stem Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE54103 | GEO | 2014/03/12
SECONDARY ACCESSION(S): PRJNA235183
REPOSITORIES: GEO
Similar Datasets
Project description:These ChIP-exo data were used to validate the predictions from our live-cell single-molecule imaging experiment The ChIP-exo mapping of ultra-fine localization of endogenous Sox2, halo-Sox2, and two halo-Sox2 mutants (halo-Sox2M and halo-Sox2D) in embryonic stem cells.
2014-03-12 | E-GEOD-54103 | biostudies-arrayexpress
Project description:Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
| S-EPMC4040518 | biostudies-literature
Project description:Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and each of their binding partners based on which we inferred their most likely interaction sites. Our results indicate that H3K9me promotes specific complex formation between HP1 proteins and their interactors in a spatially restricted manner, while attenuating their ability to form off-chromatin complexes. As opposed to being an inert platform or scaffold to direct HP1 binding, our studies propose a novel function for H3K9me as an active participant in enhancing HP1-associated complex formation in living cells.
2024-05-01 | GSE263472 | GEO
Project description:Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity.
2023-05-31 | GSE179007 | GEO
Project description:Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer/promoter dynamics [ChIP-seq]
| PRJNA616370 | ENA
Project description:Enhancer-promoter dynamics are critical for the spatiotemporal control of gene expression, but it remains unclear how these dynamics are controlled by the nucleosome remodelling and deacetylase (NuRD) complex that is found at nearly all active enhancers and promoters. We show that MBD3-dependent assembly of the intact NuRD complex increases CTCF/Cohesin binding (using ChIP-seq) and the probability of the interaction of intermediate-range (~1Mb) genomic sequences (using population Hi-C). Using single-molecule imaging, we then revealed that these changes are related to how the chromatin moves, and that the intact NuRD complex increases how fast the nucleus is explored by NuRD-bound enhancers and promoters.
2021-04-01 | GSE147789 | GEO
Project description:Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed in vitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK-tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK-tag substantially altered ParB’s DNA compaction rates, its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.
2023-09-30 | GSE212751 | GEO
Project description:Targeting cellular RNA by small molecules has come to the forefront of biotechnology and holds great promise for therapeutic use. Strategies to identify, validate and optimize these molecules are essential, but are still lacking in some aspects. In particular, the site-specific covalent labeling and modification of RNA in living cells poses many challenges. Here, we describe a general structure-guided approach to engineer non-covalent RNA aptamer–ligand complexes into their covalent counterparts using a molecular tether. The key is to modify the native ligand with an electrophilic handle that allows it to react specifically with a guanine at the RNA ligand binding site. We show that site-specific cross-linking between ligand and RNA is achieved in mammalian cells upon transfection of a genetically encoded version of the preQ1-I riboswitch aptamer. Further, we showcase the versatility of the tether by engineering the first covalent fluorescent light-up aptamer (coFLAP) out of the non-covalent Pepper FLAP. The coPepper system maintains strong fluorescence in live-cell imaging even after repeated washing. Thus, any background signal arising from unspecific fluorophore accumulation in the cell can be eliminated. In addition, we generated a bifunctional Pepper ligand containing a second handle for bioorthogonal chemistry to allow for easily traceable and efficient pulldown of the covalently linked target RNA. Finally, we provide evidence for the suitability of this tethering strategy for specific drug targeting. Taken together, our results show that functionalized ligands generated by rational design can cross-link site-specifically with target RNAs in cells, and hence, open up a wide range of applications in RNA biology that require irreversible small molecule binding.
2024-09-30 | GSE271728 | GEO
Project description:Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the central nervous system. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 T helper (Th)17 cells can directly interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we conducted single cell RNA sequencing of human primary oligodendrocytes cultured alone, in contact or separated by an insert with primary Th17 polarized T cells and identified several cell adhesion molecules that may modulate T cell contact mediated damage of oligodendrocytes. Furthermore, we used flow cytometry and immunofluorescence studies on central nervous system tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule and activated leukocyte cell adhesion molecule in multiple sclerosis, EAE and controls although their regulation differ between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of melanoma cell adhesion molecule but not activated leukocyte cell adhesion molecule at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live-imaging, immunofluorescence, and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking activated leukocyte cell adhesion molecule but not melanoma cell adhesion molecule limited prolonged interactions between oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferred significant protection of oligodendrocytic processes.
2023-08-31 | GSE230828 | GEO
Project description:Here we describe and validate a protocol for small molecule-mediated induction of the neural crest state as a new tool for generating this important cell type and facilitating future studies of its development.
2023-10-13 | GSE230350 | GEO