Cell-to-Cell Transcription Variability as Measured by Single-Molecule RNA FISH to Detect Epigenetic State Switching

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

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Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling

PLoS Pathogens ◽

10.1371/journal.ppat.1008164 ◽

2019 ◽

Vol 15 (11) ◽

pp. e1008164 ◽

Cited By ~ 6

Author(s):

Michi Miura ◽

Supravat Dey ◽

Saumya Ramanayake ◽

Abhyudai Singh ◽

David S. Rueda ◽

...

Keyword(s):

Single Molecule ◽

Stochastic Modelling ◽

Rna Fish ◽

Kinetics Of

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A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

10.1101/159004 ◽

2017 ◽

Cited By ~ 1

Author(s):

Rajarshi P Ghosh ◽

J Matthew Franklin ◽

Will E. Draper ◽

Quanming Shi ◽

Jan T. Liphardt

Keyword(s):

Single Molecule ◽

Precision Measurement ◽

Single Molecules ◽

Living Cells ◽

Background Suppression ◽

Molecular Heterogeneity ◽

Single Molecule Tracking ◽

High Brightness ◽

Cellular Processes ◽

State Switching

AbstractCellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

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Axonal mRNA in human embryonic stem cell derived neurons

10.1101/066142 ◽

2016 ◽

Author(s):

Rebecca L. Bigler ◽

Joyce W. Kamande ◽

Raluca Dumitru ◽

Mark Niedringhaus ◽

Anne Marion Taylor

Keyword(s):

Single Molecule ◽

Cortical Neurons ◽

Embryonic Stem ◽

Mrna Translation ◽

Model Organisms ◽

Functional Roles ◽

Rna Fish ◽

Rat Cortical Neurons ◽

Neuronal Types ◽

Microfluidic Chambers

The identification of axonal mRNAs in model organisms has led to the discovery of multiple proteins synthesized within axons for functional roles such as axon guidance and injury response. The extent to which protein synthesis within the axon is conserved in humans is unknown. Here we used axon-isolating microfluidic chambers to characterize the axonal transcriptome of human embryonic stem cells (hESC-neurons) differentiated using a protocol for glutamatergic neurons. Using gene expression analysis, we identified mRNAs proportionally enriched in axons, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata and dendrites. Further, we found that the most abundant mRNAs within hESC-neuron axons were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, β-actin and GAP43, within hESC-neuron axons using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human axons and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human axons provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique axonal mechanisms across species and neuronal types.

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Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae

eLife ◽

10.7554/elife.05007 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 47

Author(s):

Anne E Dodson ◽

Jasper Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Single Molecule ◽

Genome Stability ◽

Gene Repression ◽

Cell Divisions ◽

Rna Fish ◽

Eukaryotic Gene ◽

The Stability ◽

Lines Of Evidence

Heterochromatin exerts a heritable form of eukaryotic gene repression and contributes to chromosome segregation fidelity and genome stability. However, to date there has been no quantitative evaluation of the stability of heterochromatic gene repression. We designed a genetic strategy to capture transient losses of gene silencing in Saccharomyces as permanent, heritable changes in genotype and phenotype. This approach revealed rare transcription within heterochromatin that occurred in approximately 1/1000 cell divisions. In concordance with multiple lines of evidence suggesting these events were rare and transient, single-molecule RNA FISH showed that transcription was limited. The ability to monitor fluctuations in heterochromatic repression uncovered previously unappreciated roles for Sir1, a silencing establishment factor, in the maintenance and/or inheritance of silencing. In addition, we identified the sirtuin Hst3 and its histone target as contributors to the stability of the silenced state. These approaches revealed dynamics of a heterochromatin function that have been heretofore inaccessible.

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Room-Temperature Switching of Spin-State in Single-Molecule Junctions

10.21203/rs.3.rs-78276/v1 ◽

2020 ◽

Author(s):

Jing Li ◽

QingQing Wu ◽

Wei Xu ◽

Hai-Chuan Wang ◽

Hewei Zhang ◽

...

Keyword(s):

Spin State ◽

Single Molecule ◽

Room Temperature ◽

High Spin State ◽

Scanning Tunneling ◽

Molecular Spintronics ◽

State Switching ◽

Square Planar ◽

Single Molecule Junctions ◽

Conductance Changes

Abstract The emerging of molecular spintronics offers a unique chance for the design of molecular devices with different spin-state, and the control of spin-state becomes essential for molecular spin switches. However, the intrinsic spin switching from low-spin to high-spin state is a temperature-dependent process with a small energy barrier that low temperature is required to maintain the low-spin state, and thus the room-temperature operation of single-molecule devices have not yet been achieved. Here, we investigated the single-molecule charge transport through a diamagnetic square planar nickel(II) porphyrin using the scanning tunneling microscope break-junction (STM-BJ) technique. The reversible single-molecule conductance switches are demonstrated by utilizing a coordination-induced spin-state switching to manipulate the spin state between S = 0 and S = 1 at room temperature. Furthermore, the different coordinated complexes could be distinguished from the conductance traces, which cannot be realized by the ensemble investigations such as NMR and UV-vis spectrums. The combined DFT calculations revealed that the conductance changes come from the different spin-states of the molecules varying the number of coordination ligands, suggesting coordination-induced spin-state switching provides a new way towards room-temperature molecular spintronics.

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