Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single-molecule imaging of transcription dynamics in somatic stem cells.

ABSTRACT: Molecular noise is a natural phenomenon that is inherent to all biological systems^1,2. How stochastic processes give rise to the robust outcomes that support tissue homeostasis remains unclear. Here we use single-molecule RNA fluorescent in situ hybridization (smFISH) on mouse stem cells derived from haematopoietic tissue to measure the transcription dynamics of three key genes that encode transcription factors: PU.1 (also known as Spi1), Gata1 and Gata2. We find that infrequent, stochastic bursts of transcription result in the co-expression of these antagonistic transcription factors in the majority of haematopoietic stem and progenitor cells. Moreover, by pairing smFISH with time-lapse microscopy and the analysis of pedigrees, we find that although individual stem-cell clones produce descendants that are in transcriptionally related states-akin to a transcriptional priming phenomenon-the underlying transition dynamics between states are best captured by stochastic and reversible models. As such, a stochastic process can produce cellular behaviours that may be incorrectly inferred to have arisen from deterministic dynamics. We propose a model whereby the intrinsic stochasticity of gene expression facilitates, rather than impedes, the concomitant maintenance of transcriptional plasticity and stem cell robustness.

SUBMITTER: Wheat JC

PROVIDER: S-EPMC8577313 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

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

Single-Molecule Imaging Reveals Dynamics of CREB Transcription Factor Bound to Its Target Sequence.

Project description:Proper spatiotemporal gene expression is achieved by selective DNA binding of transcription factors in the genome. The most intriguing question is how dynamic interactions between transcription factors and their target sites contribute to gene regulation by recruiting the basal transcriptional machinery. Here we demonstrate individual binding and dissociation events of the transcription factor cAMP response element-binding protein (CREB), both in vitro and in living cells, using single-molecule imaging. Fluorescent-tagged CREB bound to its target sequence cAMP-response element (CRE) for a remarkably longer period (dissociation rate constant: 0.21 s(-1)) than to an unrelated sequence (2.74 s(-1)). Moreover, CREB resided at restricted positions in the living cell nucleus for a comparable period. These results suggest that CREB stimulates transcription by binding transiently to CRE in the time range of several seconds.

| S-EPMC4454023 | biostudies-literature

Nucleosomal arrangement affects single-molecule transcription dynamics.

Project description:In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, ?, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, ? can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes ? in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.

| S-EPMC5111697 | biostudies-literature

Single-molecule imaging of transcription at damaged chromatin.

Project description:How DNA double-strand breaks (DSBs) affect ongoing transcription remains elusive due to the lack of single-molecule resolution tools directly measuring transcription dynamics upon DNA damage. Here, we established new reporter systems that allow the visualization of individual nascent RNAs with high temporal and spatial resolution upon the controlled induction of a single DSB at two distinct chromatin locations: a promoter-proximal (PROP) region downstream the transcription start site and a region within an internal exon (EX2). Induction of a DSB resulted in a rapid suppression of preexisting transcription initiation regardless of the genomic location. However, while transcription was irreversibly suppressed upon a PROP DSB, damage at the EX2 region drove the formation of promoter-like nucleosome-depleted regions and transcription recovery. Two-color labeling of transcripts at sequences flanking the EX2 lesion revealed bidirectional break-induced transcription initiation. Transcriptome analysis further showed pervasive bidirectional transcription at endogenous intragenic DSBs. Our data provide a novel framework for interpreting the reciprocal interactions between transcription and DNA damage at distinct chromatin regions.

| S-EPMC6326756 | biostudies-literature

Single-molecule imaging of transcription factor binding to DNA in live mammalian cells.

Project description:Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-? (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.

| S-EPMC3664538 | biostudies-literature

Quantifying transcription factor binding dynamics at the single-molecule level in live cells.

Project description:Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

| S-EPMC5522764 | biostudies-literature

Single-molecule Dynamics of Enhanceosome Assembly In Embryonic Stem Cells

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

Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells.

Project description:The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.

| S-EPMC5078992 | biostudies-literature

BayFish: Bayesian inference of transcription dynamics from population snapshots of single-molecule RNA FISH in single cells.

Project description:Single-molecule RNA fluorescence in situ hybridization (smFISH) provides unparalleled resolution in the measurement of the abundance and localization of nascent and mature RNA transcripts in fixed, single cells. We developed a computational pipeline (BayFish) to infer the kinetic parameters of gene expression from smFISH data at multiple time points after gene induction. Given an underlying model of gene expression, BayFish uses a Monte Carlo method to estimate the Bayesian posterior probability of the model parameters and quantify the parameter uncertainty given the observed smFISH data. We tested BayFish on synthetic data and smFISH measurements of the neuronal activity-inducible gene Npas4 in primary neurons.

| S-EPMC5582403 | biostudies-other

Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells.

Project description:The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.

| S-EPMC7083905 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data