scholarly journals Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell

2020 ◽  
Vol 21 (21) ◽  
pp. 8278
Author(s):  
Amparo Pascual-Ahuir ◽  
Josep Fita-Torró ◽  
Markus Proft
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Transient Gene Expression ◽  
Cell Physiology ◽  
Extrinsic Factors ◽  
Intrinsic Factors ◽  
Gene Expression Dynamics ◽  
Transcriptional Memory ◽  
External Stimuli

The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.

scholarly journals Flow-dependent regulation of endothelial Tie2 by GATA3 in vivo

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Temitayo O. Idowu ◽  
Valerie Etzrodt ◽  
Thorben Pape ◽  
Joerg Heineke ◽  
Klaus Stahl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Experimental Models ◽  
Common Denominator ◽  
Dependent Manner ◽  
Pulmonary Endothelium ◽  
Delivery Platform ◽  
Transient Overexpression ◽  
Plasmid Delivery

Abstract Background Reduced endothelial Tie2 expression occurs in diverse experimental models of critical illness, and experimental Tie2 suppression is sufficient to increase spontaneous vascular permeability. Looking for a common denominator among different critical illnesses that could drive the same Tie2 suppressive (thereby leak inducing) phenotype, we identified “circulatory shock” as a shared feature and postulated a flow-dependency of Tie2 gene expression in a GATA3 dependent manner. Here, we analyzed if this mechanism of flow-regulation of gene expression exists in vivo in the absence of inflammation. Results To experimentally mimic a shock-like situation, we developed a murine model of clonidine-induced hypotension by targeting a reduced mean arterial pressure (MAP) of approximately 50% over 4 h. We found that hypotension-induced reduction of flow in the absence of confounding disease factors (i.e., inflammation, injury, among others) is sufficient to suppress GATA3 and Tie2 transcription. Conditional endothelial-specific GATA3 knockdown (B6-Gata3tm1-Jfz VE-Cadherin(PAC)-cerERT2) led to baseline Tie2 suppression inducing spontaneous vascular leak. On the contrary, the transient overexpression of GATA3 in the pulmonary endothelium (jet-PEI plasmid delivery platform) was sufficient to increase Tie2 at baseline and completely block its hypotension-induced acute drop. On the functional level, the Tie2 protection by GATA3 overexpression abrogated the development of pulmonary capillary leakage. Conclusions The data suggest that the GATA3–Tie2 signaling pathway might play a pivotal role in controlling vascular barrier function and that it is affected in diverse critical illnesses with shock as a consequence of a flow-regulated gene response. Targeting this novel mechanism might offer therapeutic opportunities to treat vascular leakage of diverse etiologies.

scholarly journals H2O2 Induces Major Phosphorylation Changes in Critical Regulators of Signal Transduction, Gene Expression, Metabolism and Developmental Networks in Aspergillus nidulans

2021 ◽  
Vol 7 (8) ◽  
pp. 624
Author(s):  
Ulises Carrasco-Navarro ◽  
Jesús Aguirre
Keyword(s):  
Gene Expression ◽  
Protein Phosphorylation ◽  
Aspergillus Nidulans ◽  
Regulation Of Gene Expression ◽  
Antioxidant Response ◽  
Eukaryotic Cell ◽  
Cell Physiology ◽  
Tor Signaling ◽  
Developmental Networks ◽  
General Metabolism

Reactive oxygen species (ROS) regulate several aspects of cell physiology in filamentous fungi including the antioxidant response and development. However, little is known about the signaling pathways involved in these processes. Here, we report Aspergillus nidulans global phosphoproteome during mycelial growth and show that under these conditions, H2O2 induces major changes in protein phosphorylation. Among the 1964 phosphoproteins we identified, H2O2 induced the phosphorylation of 131 proteins at one or more sites as well as the dephosphorylation of a larger set of proteins. A detailed analysis of these phosphoproteins shows that H2O2 affected the phosphorylation of critical regulatory nodes of phosphoinositide, MAPK, and TOR signaling as well as the phosphorylation of multiple proteins involved in the regulation of gene expression, primary and secondary metabolism, and development. Our results provide a novel and extensive protein phosphorylation landscape in A. nidulans, indicating that H2O2 induces a shift in general metabolism from anabolic to catabolic, and the activation of multiple stress survival pathways. Our results expand the significance of H2O2 in eukaryotic cell signaling.

scholarly journals Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

2012 ◽  
Vol 8 (6) ◽  
pp. e1002728 ◽  
Author(s):  
Hermine Mohr ◽  
Christian A. Mohr ◽  
Marlon R. Schneider ◽  
Laura Scrivano ◽  
Barbara Adler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression

scholarly journals Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets

2016 ◽  
Vol 113 (13) ◽  
pp. E1835-E1843 ◽  
Author(s):  
Mina Fazlollahi ◽  
Ivor Muroff ◽  
Eunjee Lee ◽  
Helen C. Causton ◽  
Harmen J. Bussemaker
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Nonsynonymous Mutation ◽  
Gene Regulatory ◽  
Genetic Modulators

Regulation of gene expression by transcription factors (TFs) is highly dependent on genetic background and interactions with cofactors. Identifying specific context factors is a major challenge that requires new approaches. Here we show that exploiting natural variation is a potent strategy for probing functional interactions within gene regulatory networks. We developed an algorithm to identify genetic polymorphisms that modulate the regulatory connectivity between specific transcription factors and their target genes in vivo. As a proof of principle, we mapped connectivity quantitative trait loci (cQTLs) using parallel genotype and gene expression data for segregants from a cross between two strains of the yeast Saccharomyces cerevisiae. We identified a nonsynonymous mutation in the DIG2 gene as a cQTL for the transcription factor Ste12p and confirmed this prediction empirically. We also identified three polymorphisms in TAF13 as putative modulators of regulation by Gcn4p. Our method has potential for revealing how genetic differences among individuals influence gene regulatory networks in any organism for which gene expression and genotype data are available along with information on binding preferences for transcription factors.

scholarly journals A comparative study of the development in vivo and in vitro of rat and rabbit molars

1938 ◽  
Vol 126 (844) ◽  
pp. 315-330 ◽  
Keyword(s):  
Tooth Germ ◽  
The Body ◽  
Extrinsic Factors ◽  
Intrinsic Factors ◽  
Mammalian Teeth ◽  
Tooth Germs ◽  
Developmental Mechanics ◽  
General Influence

Although the normal embryology of mammalian teeth has been carefully studied, little is known of the developmental mechanics of teeth. The present communication is concerned with the problem of cusp formation. The main object of the investigation was to find how far the formation of molar cusps was due to extrinsic factors in the jaw and how far to intrinsic factors in the tooth germ itself. Previous work (Glasstone 1936) had shown that embryonic teeth grown in vitro and removed from the general influence of the body continue to develop. In these earlier experiments the rudiments were explanted when cusps had already appeared but before odontoblasts and dentine had differentiated. In the present experiments the tooth germs were explanted at an earlier stage before the cusps had begun to form, to see whether cusps would develop in vitro in the isolated rudiment and if so whether they would correspond in number, shape and arrangement with those of the normal embryonic tooth.

scholarly journals Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Snigdha Poddar ◽  
Jaclyn Tanaka ◽  
Jamie H. D. Cate ◽  
Brian Staskawicz ◽  
Myeong-Je Cho
Keyword(s):  
Gene Expression ◽  
Suspension Culture ◽  
Genome Editing ◽  
Time Course ◽  
Transient Gene Expression ◽  
Protoplast Isolation ◽  
Transient Transfection ◽  
Study Gene Expression ◽  
Rice Calli

Abstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

scholarly journals A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ligand Binding ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Regulation Of Gene Expression ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

Abstract The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

scholarly journals Hepatic Gene Expression Changes in Hypothyroid Juvenile Mice: Characterization of a Novel Negative Thyroid-Responsive Element

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3932-3940 ◽  
Author(s):  
Hongyan Dong ◽  
Carole L. Yauk ◽  
Andrew Williams ◽  
Alice Lee ◽  
George R. Douglas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Microarrays ◽  
Molecular Mechanisms ◽  
Regulation Of Gene Expression ◽  
Transient Gene Expression ◽  
Early Response ◽  
Response Element ◽  
Gene Expression Studies ◽  
Maternal Thyroid ◽  
Responsive Element

The molecular mechanisms involved in the response of developing mice to disruptions in maternal thyroid hormone (TH) homeostasis are poorly characterized. We used DNA microarrays to examine a broad spectrum of genes from the livers of mice rendered hypothyroid by treating pregnant mice from gestational d 13 to postnatal d 15 with 6-propyl-2-thiouracil in drinking water. Twenty-four individuals (one male and one female pup from six litters of control or 6-propyl-2-thiouracil treatment groups, respectively) were profiled using Agilent oligonucleotide microarrays. MAANOVA identified 96 differentially expressed genes (false discovery rate adjusted P < 0.1 and fold change > 2 in at least one gender). Of these, 72 genes encode proteins of known function, 15 of which had previously been identified as regulated by TH. Pathway analysis revealed these genes are involved in metabolism, development, cell proliferation, apoptosis, and signal transduction. An immediate-early response gene, Nr4a1 (nuclear receptor subfamily 4, group A, member 1), was up-regulated by 3-fold in hypothyroid juvenile mouse liver; treatment of HepG2 cells with T3 resulted in down-regulation of Nr4a1. A potential thyroid response element −1218 to −1188 bp upstream of the promoter region of Nr4a1 was identified and demonstrated to bind TH receptor (TR)-α and TRβ. Point mutation or deletion of the sequence containing the potential Nr4a1-thyroid response element in transient gene expression studies resulted in both higher basal expression and loss of T3 regulatory capacity, suggesting that this site is responsible for the negative regulation of gene expression by TR and TH.

Local Mineral and Matrix Changes Associated with Bone Adaptation and Microdamage

2005 ◽  
Vol 898 ◽  
Author(s):  
David H. Kohn ◽  
Nadder D. Sahar ◽  
Sun Ig Hong ◽  
Kurtulus Golcuk ◽  
Michael D. Morris
Keyword(s):  
Mechanical Loading ◽  
Chemical Properties ◽  
Fatigue Loading ◽  
Bone Adaptation ◽  
Extrinsic Factors ◽  
Intrinsic Factors ◽  
High Incidence ◽  
Insight Into ◽  
Skeletal Fracture

AbstractSkeletal fractures represent a significant medical and economic burden for society. It is generally thought that a high incidence of musculoskeletal fatigue loading results in damage accumulation at too high of a rate to be efficiently remodeled, leading to skeletal fracture. The state of damage in bone at a given time is therefore the net result of damage and repair processes, and is dependent upon extrinsic factors such as mechanical history, but also upon intrinsic factors, such as composition of bone mineral and matrix. In this invited paper, we review investigations on the coupling of Raman spectroscopy with mechanical loading of bone, providing insight into mechanisms of ultrastructural deformation in bone at smaller scales than previously understood. We also present new data showing that in-vivo mechanical loading results in increased resistance to fatigue damage, coupled with an increase in phosphate to amide I ratio and decrease in carbonate to phosphate ratio. Taken together, the data demonstrates the ability to modulate the mechanical and chemical properties of bone via exogenous mechanical stimulation.

Investigation of cell physiology in the animal using transgenic technology

1992 ◽  
Vol 262 (2) ◽  
pp. C261-C275 ◽  
Author(s):  
A. P. Koretsky
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Regulation Of Gene Expression ◽  
Cell Physiology ◽  
Transgenic Technology ◽  
Cell Type ◽  
Mutant Proteins ◽  
The Past ◽  
Targeted Expression ◽  
Made In

Over the past 10 years significant progress has been made in techniques for manipulating the genome of the animal. Production of transgenic mice has led to important insights into the regulation of gene expression, the molecular basis of cancer, immunology, and developmental biology. The tools necessary to generate transgenic mice are becoming widely available, making it possible to study a variety of problems. In this review a description of the strategies being used to address problems of interest in cell physiology using transgenic mice is given. Elucidation of the rules governing the regulation of gene expression now permits the targeted expression of a protein to a particular organ or cell type within an organ. Overexpression of proteins, expression of foreign or mutant proteins, mislocalization of proteins, and directed elimination of proteins are all procedures that can now be used to generate interesting animal models for physiological studies. The applications of these techniques to a variety of problems in normal and abnormal physiology are discussed in this review.

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