Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Droplet formation assay for investigating phase-separation mechanisms of RNA Pol II transcription and CTCF functioning.

ABSTRACT: Clustering of RNA polymerase II carboxy-terminal domain (CTD) and CTCF DNA-binding domains (DBDs) have been viewed as emerging mechanisms of transcription regulation and three-dimensional chromatin organization. In this protocol, we address the need for a quantitative means of investigating phase-separation mechanisms of Pol II transcription and CTCF functioning. We describe steps for protein purification, droplet formation, and automeasuring droplet properties. We then detail quantification during Pol II CTD and CTCF DBD clustering and outline their limitations. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022)¹ and Zhou et al. (2022).².

SUBMITTER: Wang H

PROVIDER: S-EPMC10074247 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Droplet formation assay for investigating phase-separation mechanisms of RNA Pol II transcription and CTCF functioning.

Wang Hui H Zhou Rong R Ji Xiong X

STAR protocols 20230328 2

Clustering of RNA polymerase II carboxy-terminal domain (CTD) and CTCF DNA-binding domains (DBDs) have been viewed as emerging mechanisms of transcription regulation and three-dimensional chromatin organization. In this protocol, we address the need for a quantitative means of investigating phase-separation mechanisms of Pol II transcription and CTCF functioning. We describe steps for protein purification, droplet formation, and automeasuring droplet properties. We then detail quantification dur ...[more]

PMID: 36995933

Similar Datasets

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Project description:Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.

| S-EPMC5606208 | biostudies-literature

Chromatin remodeling by Pol II primes efficient Pol III transcription.

Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is coupled with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here a mechanism where RNA Polymerase II (Pol II) transcription is required to prime and maintain nucleosome depletion at Pol III loci and contributes to efficient Pol III recruitment upon re-initiation of growth from stationary phase in Fission yeast. The Pcr1 transcription factor participates in the recruitment of Pol II, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.

| S-EPMC10276017 | biostudies-literature

Investigating Biofilm Formation Mechanisms in Salmonella on Fresh Produce Using TraDIS-Xpress

Project description:This study investigates the mechanisms employed by Salmonella to colonise and establish itself on fresh produce at critical timepoints following infection. We established an alfalfa infection model and compared the findings to those obtained from glass surfaces. Our research revealed dynamic changes in the pathways associated with biofilm formation over time, with distinct plant-specific and glass-specific mechanisms for biofilm formation, alongside the identification of shared genes playing pivotal roles in both contexts.

2023-11-24 | E-MTAB-13495 | biostudies-arrayexpress

Enhancer SINEs Link Pol III to Pol II Transcription in Neurons.

Project description:Spatiotemporal regulation of gene expression depends on the cooperation of multiple mechanisms, including the functional interaction of promoters with distally located enhancers. Here, we show that, in cortical neurons, a subset of short interspersed nuclear elements (SINEs) located in the proximity of activity-regulated genes bears features of enhancers. Enhancer SINEs (eSINEs) recruit the Pol III cofactor complex TFIIIC in a stimulus-dependent manner and are transcribed by Pol III in response to neuronal depolarization. Characterization of an eSINE located in proximity to the Fos gene (FosRSINE1) indicated that the FosRSINE1-encoded transcript interacts with Pol II at the Fos promoter and mediates Fos relocation to Pol II factories, providing an unprecedented molecular link between Pol III and Pol II transcription. Strikingly, knockdown of the FosRSINE1 transcript induces defects of both cortical radial migration in vivo and activity-dependent dendritogenesis in vitro, demonstrating that FosRSINE1 acts as a strong enhancer of Fos expression in diverse physiological contexts.

| S-EPMC5732322 | biostudies-literature

Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation.

Project description:Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

| S-EPMC5928213 | biostudies-literature

Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble.

Project description:Liquid-liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.

| S-EPMC7939360 | biostudies-literature

Monodisperse droplet formation by spontaneous and interaction based mechanisms in partitioned EDGE microfluidic device.

Project description:The partitioned EDGE droplet generation device is known for its' high monodisperse droplet formation frequencies in two distinct pressure ranges, and an interesting candidate for scale up of microfluidic emulsification devices. In the current study, we test various continuous and dispersed phase properties and device geometries to unravel how the device spontaneously forms small monodisperse droplets (6-18 μm) at low pressures, and larger monodisperse droplets (>28 μm) at elevated pressures. For the small droplets, we show that the continuous phase inflow in the droplet formation unit largely determines droplet formation behaviour and the resulting droplet size and blow-up pressure. This effect was not considered as a factor of significance for spontaneous droplet formation devices that are mostly characterised by capillary numbers in literature. We then show for the first time that the formation of larger droplets is caused by physical interaction between neighbouring droplets, and highly dependent on device geometry. The insights obtained here are an essential step toward industrial emulsification based on microfluidic devices.

| S-EPMC6534564 | biostudies-literature

Investigating the role of Hmo1 in transcription by Pol I in yeast.

Project description:The purpose of this study was to investigate the role of the yeast protein, high mobility group protein (Hmo1), in rRNA synthesis. For over twenty years, Hmo1 has been proposed to be an activator of transcription by Pol I, but its regulatory mechanism remains largely undefined. Therefore, we used native elongating transcript sequencing (NET-seq) to explore the role of this factor in transcription by Pol I. Our NET-seq results demonstrated that in cells lacking Hmo1 (hmo1D), Pol I occupancy was significantly increased across the rDNA template, indicating an increased pause propensity in these cells. These data suggest that Hmo1 is an important transcription elongation factor for Pol I.

2023-11-20 | GSE247981 | GEO

CTCF organizes inter-A compartment interactions through RYBP-dependent phase separation.

Project description:Chromatin is spatially organized into three-dimensional structures at different levels including A/B compartments, topologically associating domains and loops. The canonical CTCF-mediated loop extrusion model can explain the formation of loops. However, the organization mechanisms underlying long-range chromatin interactions such as interactions between A-A compartments are still poorly understood. Here we show that different from the canonical loop extrusion model, RYBP-mediated phase separation of CTCF organizes inter-A compartment interactions. Based on this model, we designed and verified an induced CTCF phase separation system in embryonic stem cells (ESCs), which facilitated inter-A compartment interactions, improved self-renewal of ESCs and inhibited their differentiation toward neural progenitor cells. These findings support a novel and non-canonical role of CTCF in organizing long-range chromatin interactions via phase separation.

| S-EPMC9343660 | biostudies-literature

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

Project description:This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

| S-EPMC4381353 | 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