scholarly journals Regulatory mechanisms are revealed by the distribution of transcription initiation times in single microbial cells

2017 ◽  
Author(s):  
Sandeep Choubey ◽  
Jane Kondev ◽  
Alvaro Sanchez
Keyword(s):  
Transcription Initiation ◽  
Single Cells ◽  
Quantitative Information ◽  
Rna Polymerases ◽  
Live Cells ◽  
Control Of Gene Expression ◽  
Fluorescent Reporters ◽  
Transcriptional Dynamics ◽  
General Stochastic Model ◽  
State Of Affairs

AbstractTranscription is the dominant point of control of gene expression. Biochemical studies have revealed key molecular components of transcription and their interactions, but the dynamics of transcription initiation in cells is still poorly understood. This state of affairs is being remedied with experiments that observe transcriptional dynamics in single cells using fluorescent reporters. Quantitative information about transcription initiation dynamics can also be extracted from experiments that use electron micrographs of RNA polymerases caught in the act of transcribing a gene (Miller spreads). Inspired by these data we analyze a general stochastic model of transcription initiation and elongation, and compute the distribution of transcription initiation times. We show that different mechanisms of initiation leave distinct signatures in the distribution of initiation times that can be compared to experiments. We analyze published micrographs of RNA polymerases transcribing ribosomal RNA genes inE.coliand compare the observed distributions of inter-polymerase distances with the predictions from previously hypothesized mechanisms for the regulation of these genes. Our analysis demonstrates the potential of measuring the distribution of time intervals between initiation events as a probe for dissecting mechanisms of transcription initiation in live cells.

scholarly journals Simultaneous quantification of mRNA and protein in single cells reveals post-transcriptional effects of genetic variation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Christian Brion ◽  
Sheila M Lutz ◽  
Frank Wolfgang Albert
Keyword(s):  
Statistical Power ◽  
Single Cells ◽  
Live Cells ◽  
Kinase Gene ◽  
Fluorescent Reporters ◽  
Protein Levels ◽  
Simultaneous Quantification ◽  
Specific Effects ◽  
Recombinant Cells ◽  
Large Populations

Trans-acting DNA variants may specifically affect mRNA or protein levels of genes located throughout the genome. However, prior work compared trans-acting loci mapped in separate studies, many of which had limited statistical power. Here, we developed a CRISPR-based system for simultaneous quantification of mRNA and protein of a given gene via dual fluorescent reporters in single, live cells of the yeast Saccharomyces cerevisiae. In large populations of recombinant cells from a cross between two genetically divergent strains, we mapped 86 trans-acting loci affecting the expression of ten genes. Less than 20% of these loci had concordant effects on mRNA and protein of the same gene. Most loci influenced protein but not mRNA of a given gene. One locus harbored a premature stop variant in the YAK1 kinase gene that had specific effects on protein or mRNA of dozens of genes. These results demonstrate complex, post-transcriptional genetic effects on gene expression.

Imaging mRNA In Vivo, from Birth to Death

2018 ◽  
Vol 47 (1) ◽  
pp. 85-106 ◽  
Author(s):  
Evelina Tutucci ◽  
Nathan M. Livingston ◽  
Robert H. Singer ◽  
Bin Wu
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Information Transfer ◽  
Gene Expression Regulation ◽  
Single Cells ◽  
Quantitative Information ◽  
Live Cells ◽  
Messenger Rnas ◽  
Study Gene Expression

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

scholarly journals ZipSeq : Barcoding for Real-time Mapping of Single Cell Transcriptomes

2020 ◽  
Author(s):  
Kenneth H. Hu ◽  
John P. Eichorst ◽  
Chris S. McGinnis ◽  
David M. Patterson ◽  
Eric D. Chow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Real Time ◽  
Dimensional Space ◽  
Single Cells ◽  
Expression Patterns ◽  
Live Cells ◽  
Glass Substrates ◽  
Complete Mapping

ABSTRACTSpatial transcriptomics seeks to integrate single-cell transcriptomic data within the 3-dimensional space of multicellular biology. Current methods use glass substrates pre-seeded with matrices of barcodes or fluorescence hybridization of a limited number of probes. We developed an alternative approach, called ‘ZipSeq’, that uses patterned illumination and photocaged oligonucleotides to serially print barcodes (Zipcodes) onto live cells within intact tissues, in real-time and with on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in-vitro wound healing, live lymph node sections and in a live tumor microenvironment (TME). In all cases, we discovered new gene expression patterns associated with histological structures. In the TME, this demonstrated a trajectory of myeloid and T cell differentiation, from periphery inward. A variation of ZipSeq efficiently scales to the level of single cells, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

scholarly journals Control of gene expression by glucocorticoid hormones

1984 ◽  
Vol 224 (1) ◽  
pp. 1-12 ◽  
Author(s):  
G G Rousseau
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Control Of Gene Expression ◽  
Mouse Mammary Tumour Virus ◽  
Virus Rna ◽  
Molecular Determinants ◽  
Chromatin Proteins ◽  
Modulation Of Gene Expression ◽  
Inducible Genes ◽  
Heterologous Cell

Glucocorticoids control the expression of a small number of transcriptionally active genes by increasing or decreasing mRNA concentration. Either effect can result from a transcriptional or a post-transcriptional mechanism. Induction of mouse mammary tumour virus RNA results from a stimulation of transcription initiation and depends on the presence of defined regions in proviral DNA. These regions bind the glucocorticoid receptor and behave functionally as proto-enhancers. Glucocorticoid-inducible genes can retain their sensitivity to the hormone after transfer to a heterologous cell by transfection techniques. Non-inducible genes can become inducible when linked to the promoter region of an inducible gene. The mechanisms by which the receptor-steroid complex stimulates or inhibits transcription or influences mRNA stability are unknown. Receptor binding to nucleic acids appears to be a necessary but not sufficient condition. It is likely that the receptor also interacts with chromatin proteins. This might lead to a catalytic modification of these proteins, resulting in a modulation of gene expression. Development of glucocorticoid-sensitive, biochemically defined, cell-free transcription systems should provide a tool to delineate the molecular determinants of this essential regulatory mechanism.

Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II

2018 ◽  
Vol 47 (1) ◽  
pp. 425-446 ◽  
Author(s):  
Christoph Engel ◽  
Simon Neyer ◽  
Patrick Cramer
Keyword(s):  
Transcription Initiation ◽  
Messenger Rna ◽  
Rna Polymerases ◽  
Chain Elongation ◽  
Structural Studies ◽  
Pol Ii ◽  
Initiation Factors ◽  
Initiation System ◽  
Pol I ◽  
Promoter Dna

RNA polymerases I and II (Pol I and Pol II) are the eukaryotic enzymes that catalyze DNA-dependent synthesis of ribosomal RNA and messenger RNA, respectively. Recent work shows that the transcribing forms of both enzymes are similar and the fundamental mechanisms of RNA chain elongation are conserved. However, the mechanisms of transcription initiation and its regulation differ between Pol I and Pol II. Recent structural studies of Pol I complexes with transcription initiation factors provided insights into how the polymerase recognizes its specific promoter DNA, how it may open DNA, and how initiation may be regulated. Comparison with the well-studied Pol II initiation system reveals a distinct architecture of the initiation complex and visualizes promoter- and gene-class-specific aspects of transcription initiation. On the basis of new structural studies, we derive a model of the Pol I transcription cycle and provide a molecular movie of Pol I transcription that can be used for teaching.

scholarly journals A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

2017 ◽  
Vol 30 (12) ◽  
pp. 771-780 ◽  
Author(s):  
M Hinrichsen ◽  
M Lenz ◽  
J M Edwards ◽  
O K Miller ◽  
S G J Mochrie ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Single Cells ◽  
Covalent Bond ◽  
Subcellular Location ◽  
Turnover Time ◽  
Target Protein ◽  
Live Cells ◽  
General Applicability ◽  
Novel Approach ◽  
And Function

AbstractWe present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein’s turnover time from such data.

scholarly journals Fibronectin-Based Nanomechanical Biosensors to Map 3D Strains in Live Cells and Tissues

2020 ◽  
Author(s):  
Daniel J. Shiwarski ◽  
Joshua W. Tashman ◽  
Alkiviadis Tsamis ◽  
Jacqueline M. Bliley ◽  
Malachi A. Blundon ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Square Lattice ◽  
Live Cells ◽  
3D Analysis ◽  
Wide Range ◽  
And Function ◽  
Analysis Platform

AbstractMechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

scholarly journals Multiplexed dynamic imaging of genomic loci in single cells by combined CRISPR imaging and DNA sequential FISH

2017 ◽  
Author(s):  
Yodai Takei ◽  
Sheel Shah ◽  
Sho Harvey ◽  
Lei S. Qi ◽  
Long Cai
Keyword(s):  
Single Cells ◽  
Embryonic Stem ◽  
Dynamic Imaging ◽  
Live Cells ◽  
Chromosome Dynamics ◽  
Cellular Processes ◽  
Sequential Fish ◽  
Telomere Dynamics ◽  
Subtelomeric Regions

ABSTRACTVisualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labelled and uniquely identified. Here we describe an approach we call “track first and identify later” for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR labeling and DNA sequential fluorescence in situ hybridization (DNA seqFISH). Our approach first labels and tracks chromosomal loci in live cells with the CRISPR system, then barcodes those loci by DNA seqFISH in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

scholarly journals Characterizing the temporal dynamics of gene expression in single cells with sci-fate

2019 ◽  
Author(s):  
Junyue Cao ◽  
Wei Zhou ◽  
Frank Steemers ◽  
Cole Trapnell ◽  
Jay Shendure
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Temporal Dynamics ◽  
Single Cells ◽  
Receptor Activation ◽  
State Transitions ◽  
Proof Of Concept ◽  
Transcriptional Dynamics ◽  
A New Technique ◽  
Normal Differentiation

AbstractGene expression programs are dynamic, e.g. the cell cycle, response to stimuli, normal differentiation and development, etc. However, nearly all techniques for profiling gene expression in single cells fail to directly capture the dynamics of transcriptional programs, which limits the scope of biology that can be effectively investigated. Towards addressing this, we developed sci-fate, a new technique that combines S4U labeling of newly synthesized mRNA with single cell combinatorial indexing (sci-), in order to concurrently profile the whole and newly synthesized transcriptome in each of many single cells. As a proof-of-concept, we applied sci-fate to a model system of cortisol response and characterized expression dynamics in over 6,000 single cells. From these data, we quantify the dynamics of the cell cycle and glucocorticoid receptor activation, while also exploring their intersection. We furthermore use these data to develop a framework for inferring the distribution of cell state transitions. We anticipate sci-fate will be broadly applicable to quantitatively characterize transcriptional dynamics in diverse systems.

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