scholarly journals On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

2021 ◽  
Author(s):  
Danuta M Jeziorska ◽  
Edward A J Tunnacliffe ◽  
Jill M Brown ◽  
Helena Ayyub ◽  
Jacqueline A Sloane-Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Dynamic Behaviour ◽  
Live Cells ◽  
Development And Differentiation ◽  
The Stability ◽  
Transcriptional Bursting ◽  
Behaviour Changes ◽  
Transcriptional State

Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as α-globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

scholarly journals Mammalian gene expression variability is explained by underlying cell state

2019 ◽  
Author(s):  
Robert Foreman ◽  
Roy Wollman
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Qualitative Difference ◽  
Live Cells ◽  
Expression Variability ◽  
Cell State ◽  
Relative Contribution ◽  
Allele Specific ◽  
Poisson Limit ◽  
Transcriptional Bursting

AbstractGene expression variability in mammalian systems plays an important role in physiological and pathophysiological conditions. This variability can come from differential regulation related to cell state (extrinsic) and allele-specific transcriptional bursting (intrinsic). Yet, the relative contribution of these two distinct sources is unknown. Here we exploit the qualitative difference in the patterns of covariance between these two sources to quantify their relative contributions to expression variance in mammalian cells. Using multiplexed error robust RNA fluorescent in situ hybridization (MERFISH) we measured the multivariate gene expression distribution of 150 genes related to Ca2+ signaling coupled with the dynamic Ca2+ response of live cells to ATP. We show that after controlling for cellular phenotypic states such as size, cell cycle stage, and Ca2+ response to ATP, the remaining variability is effectively at the Poisson limit for most genes. These findings demonstrate that the majority of expression variability results from cell state differences and that the contribution of transcriptional bursting is relatively minimal.

scholarly journals Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2323-2328 ◽  
Author(s):  
David A. Hume
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Mammalian Cells ◽  
Cell Lineage ◽  
Monoallelic Expression ◽  
Stem Cell Lineage ◽  
The Stability ◽  
Stochastic Phenomena ◽  
Inducible Genes ◽  
Probabilistic Nature

The phenotype of individual hematopoietic cells, like all other differentiated mammalian cells, is determined by selective transcription of a subset of the genes encoded within the genome. This overview summarizes the recent evidence that transcriptional regulation at the level of individual cells is best described in terms of the regulation of the probability of transcription rather than the rate. In this model, heterogeneous gene expression among populations of cells arises by chance, and the degree of heterogeneity is a function of the stability of the mRNA and protein products of individual genes. The probabilistic nature of transcriptional regulation provides one explanation for stochastic phenomena, such as stem cell lineage commitment, and monoallelic expression of inducible genes, such as lymphokines and cytokines.

scholarly journals Polysome arrest restricts miRNA turnover by preventing exosomal export of miRNA in growth-retarded mammalian cells

2015 ◽  
Vol 26 (6) ◽  
pp. 1072-1083 ◽  
Author(s):  
Souvik Ghosh ◽  
Mainak Bose ◽  
Anirban Ray ◽  
Suvendra N. Bhattacharyya
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Subcellular Distribution ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Mature Mirnas ◽  
Regulatory Rnas ◽  
The Stability ◽  
External Stimuli

MicroRNAs (miRNAs) are tiny posttranscriptional regulators of gene expression in metazoan cells, where activity and abundance of miRNAs are tightly controlled. Regulated turnover of these regulatory RNAs is important to optimize cellular response to external stimuli. We report that the stability of mature miRNAs increases inversely with cell proliferation, and the increased number of microribonucleoproteins (miRNPs) in growth-restricted mammalian cells are in turn associated with polysomes. This heightened association of miRNA with polysomes also elicits reduced degradation of target mRNAs and impaired extracellular export of miRNA via exosomes. Overall polysome sequestration contributes to an increase of cellular miRNA levels but without an increase in miRNA activity. Therefore miRNA activity and turnover can be controlled by subcellular distribution of miRNPs that may get differentially regulated as a function of cell growth in mammalian cells.

scholarly journals Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC

2016 ◽  
Vol 113 (33) ◽  
pp. 9369-9374 ◽  
Author(s):  
Hongchun Yang ◽  
Martin Howard ◽  
Caroline Dean
Keyword(s):  
Rna Polymerase Ii ◽  
Histone Modifications ◽  
Transcriptional Activity ◽  
Epigenetic Mechanisms ◽  
Functional Importance ◽  
Development And Differentiation ◽  
Paf1 Complex ◽  
Transcriptional State ◽  
Silent State

Establishment and maintenance of gene expression states is central to development and differentiation. Transcriptional and epigenetic mechanisms interconnect in poorly understood ways to determine these states. We explore these mechanisms through dissection of the regulation of Arabidopsis thaliana FLOWERING LOCUS C (FLC). FLC can be present in a transcriptionally active state marked by H3K36me3 or a silent state marked by H3K27me3. Here, we investigate the trans factors modifying these opposing histone states and find a physical coupling in vivo between the H3K36 methyltransferase, SDG8, and the H3K27me3 demethylase, ELF6. Previous modeling has predicted this coupling would exist as it facilitates bistability of opposing histone states. We also find association of SDG8 with the transcription machinery, namely RNA polymerase II and the PAF1 complex. Delivery of the active histone modifications is therefore likely to be through transcription at the locus. SDG8 and ELF6 were found to influence the localization of each other on FLC chromatin, showing the functional importance of the interaction. In addition, both influenced accumulation of the associated H3K27me3 and H3K36me3 histone modifications at FLC. We propose the physical coupling of activation and derepression activities coordinates transcriptional activity and prevents ectopic silencing.

Drugs affecting microtubule dynamics increase alpha-tubulin mRNA accumulation via transcription in Tetrahymena thermophila

1992 ◽  
Vol 12 (4) ◽  
pp. 1443-1450
Author(s):  
L A Stargell ◽  
D P Heruth ◽  
J Gaertig ◽  
M A Gorovsky
Keyword(s):  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Actinomycin D ◽  
Tetrahymena Thermophila ◽  
Rapid Decline ◽  
Mrna Accumulation ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Tubulin Mrna ◽  
The Stability

In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene. These results indicate that tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.

scholarly journals Multiplex genomic recording of enhancer and signal transduction activities in mammalian cells

2021 ◽  
Author(s):  
Jay Shendure ◽  
Wei Chen ◽  
Junhong Choi ◽  
Jenny Nathans ◽  
Vikram Agarwal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Mammalian Cells ◽  
Biological Sample ◽  
Transcriptional Activity ◽  
Relative Activity ◽  
Signal Transduction Pathways ◽  
High Fidelity ◽  
Molecular Events ◽  
Recording Unit

Abstract Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

scholarly journals Drugs affecting microtubule dynamics increase alpha-tubulin mRNA accumulation via transcription in Tetrahymena thermophila.

1992 ◽  
Vol 12 (4) ◽  
pp. 1443-1450 ◽  
Author(s):  
L A Stargell ◽  
D P Heruth ◽  
J Gaertig ◽  
M A Gorovsky
Keyword(s):  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Actinomycin D ◽  
Tetrahymena Thermophila ◽  
Rapid Decline ◽  
Mrna Accumulation ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Tubulin Mrna ◽  
The Stability

In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene. These results indicate that tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.

scholarly journals Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells

2020 ◽  
Vol 6 (25) ◽  
pp. eaaz6699 ◽  
Author(s):  
Hiroshi Ochiai ◽  
Tetsutaro Hayashi ◽  
Mana Umeda ◽  
Mika Yoshimura ◽  
Akihito Harada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Mammalian Cells ◽  
Transcription Elongation ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Kinetic Properties ◽  
Mrna Levels ◽  
Transcriptional Bursting

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body–binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9–based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

scholarly journals Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

2019 ◽  
Author(s):  
Hiroshi Ochiai ◽  
Tetsutaro Hayashi ◽  
Mana Umeda ◽  
Mika Yoshimura ◽  
Akihito Harada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Transcription Elongation ◽  
Mapk Signaling ◽  
Kinetic Properties ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Transcriptional Bursting

AbstractTranscriptional bursting is stochastic activation and inactivation of promoters, leading to discontinuous production of mRNA, and is considered to be a contributing factor to cell-to-cell heterogeneity in gene expression. However, it remains elusive how the kinetic properties of transcriptional bursting (e.g., burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. In this study, we performed a genome-wide analysis of transcriptional bursting in mouse embryonic stem cells (mESCs) using single-cell RNA-sequencing. We found that the kinetics of transcriptional bursting was determined by a combination of promoter and gene body binding proteins, including polycomb repressive complex 2 and transcription elongation-related factors. Furthermore, large-scale CRISPR-Cas9-based screening and functional analysis revealed that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncover key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

scholarly journals Multiplex genomic recording of enhancer and signal transduction activity in mammalian cells

2021 ◽  
Author(s):  
Wei Chen ◽  
Junhong Choi ◽  
Jenny F. Nathans ◽  
Vikram Agarwal ◽  
Beth Martin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Mammalian Cells ◽  
Biological Sample ◽  
Transcriptional Activity ◽  
Relative Activity ◽  
Signal Transduction Pathways ◽  
High Fidelity ◽  
Molecular Events ◽  
Recording Unit

Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

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