scholarly journals Visualizing transcription factor dynamics in living cells

2018 ◽  
Vol 217 (4) ◽  
pp. 1181-1191 ◽  
Author(s):  
Zhe Liu ◽  
Robert Tjian
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Imaging Techniques ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Live Cells ◽  
Specific Gene ◽  
Spatiotemporal Resolution ◽  
Functional Relationships

The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type–specific gene expression. However, despite extensive biochemical, genetic, and genomic studies in the past three decades, our understanding of molecular mechanisms underlying enhancer-mediated gene regulation remains incomplete. Recent advances in imaging technologies now enable direct visualization of TF-driven regulatory events and transcriptional activities at the single-cell, single-molecule level. The ability to observe the remarkably dynamic behavior of individual TFs in live cells at high spatiotemporal resolution has begun to provide novel mechanistic insights and promises new advances in deciphering causal–functional relationships of TF targeting, genome organization, and gene activation. In this review, we review current transcription imaging techniques and summarize converging results from various lines of research that may instigate a revision of models to describe key features of eukaryotic gene regulation.

New insights into promoter–enhancer communication mechanisms revealed by dynamic single-molecule imaging

2021 ◽  
Author(s):  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Target Genes ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Single Molecule Imaging ◽  
Cell Type ◽  
Regulatory Information ◽  
Cell Type Specific ◽  
Target Promoters

Establishing cell-type-specific gene expression programs relies on the action of distal enhancers, cis-regulatory elements that can activate target genes over large genomic distances — up to Mega-bases away. How distal enhancers physically relay regulatory information to target promoters has remained a mystery. Here, we review the latest developments and insights into promoter–enhancer communication mechanisms revealed by live-cell, real-time single-molecule imaging approaches.

Transcription in Living Cells: Molecular Mechanisms of Bursting

2020 ◽  
Vol 89 (1) ◽  
pp. 189-212 ◽  
Author(s):  
Joseph Rodriguez ◽  
Daniel R. Larson
Keyword(s):  
Gene Regulation ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Living Cells ◽  
Single Locus ◽  
Individual Gene ◽  
Technological Advances ◽  
Transcriptional Bursting ◽  
Cell Data

Transcription in several organisms from certain bacteria to humans has been observed to be stochastic in nature: toggling between active and inactive states. Periods of active nascent RNA synthesis known as bursts represent individual gene activation events in which multiple polymerases are initiated. Therefore, bursting is the single locus illustration of both gene activation and repression. Although transcriptional bursting was originally observed decades ago, only recently have technological advances enabled the field to begin elucidating gene regulation at the single-locus level. In this review, we focus on how biochemical, genomic, and single-cell data describe the regulatory steps of transcriptional bursts.

scholarly journals Transposable elements as a potent source of diverse cis -regulatory sequences in mammalian genomes

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190347 ◽  
Author(s):  
Vasavi Sundaram ◽  
Joanna Wysocka
Keyword(s):  
Gene Regulation ◽  
Transposable Elements ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Dependent Manner ◽  
Unique Contribution ◽  
Regulatory Potential

Eukaryotic gene regulation is mediated by cis -regulatory elements, which are embedded within the vast non-coding genomic space and recognized by the transcription factors in a sequence- and context-dependent manner. A large proportion of eukaryotic genomes, including at least half of the human genome, are composed of transposable elements (TEs), which in their ancestral form carried their own cis -regulatory sequences able to exploit the host trans environment to promote TE transcription and facilitate transposition. Although not all present-day TE copies have retained this regulatory function, the preexisting regulatory potential of TEs can provide a rich source of cis -regulatory innovation for the host. Here, we review recent evidence documenting diverse contributions of TE sequences to gene regulation by functioning as enhancers, promoters, silencers and boundary elements. We discuss how TE-derived enhancer sequences can rapidly facilitate changes in existing gene regulatory networks and mediate species- and cell-type-specific regulatory innovations, and we postulate a unique contribution of TEs to species-specific gene expression divergence in pluripotency and early embryogenesis. With advances in genome-wide technologies and analyses, systematic investigation of TEs' cis -regulatory potential is now possible and our understanding of the biological impact of genomic TEs is increasing. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

scholarly journals Plenary Lecture 2 Transcription factors, regulatory elements and nutrient–gene communication

2009 ◽  
Vol 69 (1) ◽  
pp. 91-94 ◽  
Author(s):  
Robert J. Cousins ◽  
Tolunay B. Aydemir ◽  
Louis A. Lichten
Keyword(s):  
Transcription Factors ◽  
Histone Acetylation ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Atp Citrate Lyase ◽  
Signalling Molecules ◽  
Citrate Lyase ◽  
Normal Metabolism

Dramatic advances have been made in the understanding of the differing molecular mechanisms used by nutrients to regulate genes that are essential for their biological roles to carry out normal metabolism. Classical studies have focused on nutrients as ligands to activate specific transcription factors. New interest has focused on histone acetylation as a process for either global or limited gene activation and is the first mechanism to be discussed. Nuclear ATP-citrate lyase generates acetyl-CoA, which has been shown to have a role in the activation of specific genes via selective histone acetylation. Transcription factor acetylation may provide a second mode of control of nutrient-responsive gene transcription. The third mechanism relates to the availability of response elements within chromatin, which as well as the location of the elements in the gene may allow or prevent transcription. A fourth mechanism involves intracellular transport of Zn ions, which can orchestrate localized enzyme inhibition–activation. This process in turn influences signalling molecules that regulate gene expression. The examples provided in the present review point to a new level of complexity in understanding nutrient–gene communication.

scholarly journals Obtaining 3D Super-resolution Information from 2D Super-resolution Images through a 2D-to-3D Transformation Algorithm

2017 ◽  
Author(s):  
Andrew Ruba ◽  
Wangxi Luo ◽  
Joseph Kelich ◽  
Weidong Yang
Keyword(s):  
Single Molecule ◽  
Rotational Symmetry ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cells ◽  
Limited Information ◽  
Spatiotemporal Resolution ◽  
Superresolution Microscopy ◽  
Localization Analysis ◽  
Probability Information

AbstractCurrently, it is highly desirable but still challenging to obtain three-dimensional (3D) superresolution information of structures in fixed specimens as well as dynamic processes in live cells with a high spatiotemporal resolution. Here we introduce an approach, without using 3D superresolution microscopy or real-time 3D particle tracking, to achieve 3D sub-diffraction-limited information with a spatial resolution of ≤ 1 nm. This is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability information. The method has been successfully applied to obtain structural and functional information for 25-300 nm sub-cellular organelles that have rotational symmetry. In this article, we will provide a comprehensive analysis of this method by using experimental data and computational simulations.

Organization and Regulation of the Human Genome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-16-SCI-16
Author(s):  
Bing Ren
Keyword(s):  
Gene Regulation ◽  
Long Range ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Target Genes ◽  
Critical Role ◽  
Cell Types ◽  
Regulatory Elements ◽  
Chromatin Organization ◽  
Specific Gene

Abstract The 3-dimensional (3D) chromatin organization plays a critical role in gene regulation. Great strides have been made recently to characterize and identify cis regulatory elements from epigenome profiles in different cell types and tissues, but efforts have just begun to functionally characterize these long-range control elements. Mapping interactions between enhancers and promoters, and understanding how the 3D landscape of the genome constrains such interactions is fundamental to our understanding of genome function. I will present recent findings related to 3D genome organization in mammalian cells, with a particular focus on how chromatin organization contributes to transcriptional regulation. I will describe higher-order organizational features that are observed at the level of both the whole chromosome and individual loci. I will highlight changes in genome organization that occur during the course of differentiation, and discuss the functional relationship between chromatin architecture and gene regulation. Taken together, mounting evidence now shows that the genome organization plays an essential role in orchestrating the lineage-specific gene expression programs through modulating long- range interactions between enhancers and target genes. Disclosures Ren: Arima Genomics, Inc.: Equity Ownership, Patents & Royalties; Eli Lilly: Employment.

scholarly journals A 368-Base-Pair cis-Acting HWP1 Promoter Region, HCR, of Candida albicans Confers Hypha-Specific Gene Regulation and Binds Architectural Transcription Factors Nhp6 and Gcf1p

2007 ◽  
Vol 6 (4) ◽  
pp. 693-709 ◽  
Author(s):  
Samin Kim ◽  
Michael J. Wolyniak ◽  
Janet F. Staab ◽  
Paula Sundstrom
Keyword(s):  
Gene Regulation ◽  
Candida Albicans ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Developmental Regulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Distal Region ◽  
Specific Gene ◽  
Mobility Shift

ABSTRACT To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation.

scholarly journals Flanking HS-62.5 and 3′ HS1, and regions upstream of the LCR, are not required for β-globin transcription

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1395-1401 ◽  
Author(s):  
M. A. Bender ◽  
Rachel Byron ◽  
Tobias Ragoczy ◽  
Agnes Telling ◽  
Michael Bulger ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Globin Gene ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Chromatin Domain ◽  
Open Domain ◽  
Binding Factor ◽  
Necessary And Sufficient ◽  
Globin Gene Regulation ◽  
Endogenous Locus

Abstract The locus control region (LCR) was thought to be necessary and sufficient for establishing and maintaining an open β-globin locus chromatin domain in the repressive environment of the developing erythrocyte. However, deletion of the LCR from the endogenous locus had no significant effect on chromatin structure and did not silence transcription. Thus, the cis-regulatory elements that confer the open domain remain unidentified. The conserved DNaseI hypersensitivity sites (HSs) HS-62.5 and 3′HS1 that flank the locus, and the region upstream of the LCR have been implicated in globin gene regulation. The flanking HSs bind CCCTC binding factor (CTCF) and are thought to interact with the LCR to form a “chromatin hub” involved in β-globin gene activation. Hispanic thalassemia, a deletion of the LCR and 27 kb upstream, leads to heterochromatinization and silencing of the locus. Thus, the region upstream of the LCR deleted in Hispanic thalassemia (upstream Hispanic region [UHR]) may be required for expression. To determine the importance of the UHR and flanking HSs for β-globin expression, we generated and analyzed mice with targeted deletions of these elements. We demonstrate deletion of these regions alone, and in combination, do not affect transcription, bringing into question current models for the regulation of the β-globin locus.

scholarly journals Distribution shapes govern the discovery of predictive models for gene regulation

2018 ◽  
Vol 115 (29) ◽  
pp. 7533-7538 ◽  
Author(s):  
Brian Munsky ◽  
Guoliang Li ◽  
Zachary R. Fox ◽  
Douglas P. Shepherd ◽  
Gregor Neuert
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Single Cells ◽  
Gene Activation ◽  
Mechanistic Modeling ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Accumulation ◽  
Mitogen Activated Protein ◽  
Predictive Understanding ◽  
Computational Analyses

Despite substantial experimental and computational efforts, mechanistic modeling remains more predictive in engineering than in systems biology. The reason for this discrepancy is not fully understood. One might argue that the randomness and complexity of biological systems are the main barriers to predictive understanding, but these issues are not unique to biology. Instead, we hypothesize that the specific shapes of rare single-molecule event distributions produce substantial yet overlooked challenges for biological models. We demonstrate why modern statistical tools to disentangle complexity and stochasticity, which assume normally distributed fluctuations or enormous datasets, do not apply to the discrete, positive, and nonsymmetric distributions that characterize mRNA fluctuations in single cells. As an example, we integrate single-molecule measurements and advanced computational analyses to explore mitogen-activated protein kinase induction of multiple stress response genes. Through systematic analyses of different metrics to compare the same model to the same data, we elucidate why standard modeling approaches yield nonpredictive models for single-cell gene regulation. We further explain how advanced tools recover precise, reproducible, and predictive understanding of transcription regulation mechanisms, including gene activation, polymerase initiation, elongation, mRNA accumulation, spatial transport, and decay.

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications

Lab on a Chip ◽  
2016 ◽  
Vol 16 (11) ◽  
pp. 2014-2024 ◽  
Author(s):  
Lip Ket Chin ◽  
Chau-Hwang Lee ◽  
Bi-Chang Chen
Keyword(s):  
Imaging Techniques ◽  
Super Resolution ◽  
Lab On A Chip ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
Cellular Processes ◽  
High Spatiotemporal Resolution ◽  
Microfluidic Technology ◽  
Resolution Imaging

The synergy of novel super-resolution imaging techniques and microfluidic technology provides new biological and biomedical insights into sub-cellular processes.

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