scholarly journals Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator

2016 ◽  
Author(s):  
Alexandra M. Westbrook ◽  
Julius B. Lucks
Keyword(s):  
Gene Expression ◽  
Antisense Rna ◽  
Regulatory Networks ◽  
Dynamic Range ◽  
Transcriptional Regulators ◽  
Genetic Circuit ◽  
Control Of Gene Expression ◽  
Antisense Rnas ◽  
Transcriptional Termination

ABSTRACTRNA transcriptional regulators are emerging as versatile components for genetic circuit construction. However, RNA transcriptional regulators suffer from incomplete repression, making their dynamic range less than that of their protein counterparts. This incomplete repression can cause expression leak, which impedes the construction of larger RNA synthetic regulatory networks. Here we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and RBS sequestration increases repression from 85% to 98% and activation from 10 fold to over 900 fold in response to cognate antisense RNAs. We also show that orthogonal versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this dual control mechanism, we use it to reduce circuit leak in an RNA-only transcriptional cascade that activates gene expression as a function of a small molecule input. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of larger and more sophisticated circuits.

scholarly journals A versatile genetic tool for post-translational control of gene expression in Drosophila melanogaster

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sachin Sethi ◽  
Jing W Wang
Keyword(s):  
Gene Expression ◽  
Side Effects ◽  
Translational Control ◽  
High Efficiency ◽  
Developmental Stages ◽  
Dynamic Range ◽  
In Vivo Studies ◽  
Control Of Gene Expression ◽  
Regulate Protein

Several techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations.

scholarly journals A versatile genetic tool for post-translational control of gene expression in Drosophila melanogaster

2017 ◽  
Author(s):  
Sachin Sethi ◽  
Jing W. Wang
Keyword(s):  
Gene Expression ◽  
Side Effects ◽  
Translational Control ◽  
High Efficiency ◽  
Developmental Stages ◽  
Dynamic Range ◽  
Control Of Gene Expression ◽  
Neuronal Populations ◽  
Regulate Protein

AbstractSeveral techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

Abstract 286: Long Noncoding RNAs Are Differentially Expressed in Heart Failure

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Emma L Robinson ◽  
Syed Haider ◽  
Hillary Hei ◽  
Richard T Lee ◽  
Roger S Foo
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Significant Proportion ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Control Of Gene Expression ◽  
Failing Heart ◽  
Novel Transcripts ◽  
Non Coding Rnas

Heart failure comprises of clinically distinct inciting causes but a consistent pattern of change in myocardial gene expression supports the hypothesis that unifying biochemical mechanisms underlie disease progression. The recent RNA-seq revolution has enabled whole transcriptome profiling, using deep-sequencing technologies. Up to 70% of the genome is now known to be transcribed into RNA, a significant proportion of which is long non-coding RNAs (lncRNAs), defined as polyribonucleotides of ≥200 nucleotides. This project aims to discover whether the myocardium expression of lncRNAs changes in the failing heart. Paired end RNA-seq from a 300-400bp library of ‘stretched’ mouse myocyte total RNA was carried out to generate 76-mer sequence reads. Mechanically stretching myocytes with equibiaxial stretch apparatus mimics pathological hypertrophy in the heart. Transcripts were assembled and aligned to reference genome mm9 (UCSC), abundance determined and differential expression of novel transcripts and alternative splice variants were compared with that of control (non-stretched) mouse myocytes. Five novel transcripts have been identified in our RNA-seq that are differentially expressed in stretched myocytes compared with non-stretched. These are regions of the genome that are currently unannotated and potentially are transcribed into non-coding RNAs. Roles of known lncRNAs include control of gene expression, either by direct interaction with complementary regions of the genome or association with chromatin remodelling complexes which act on the epigenome.Changes in expression of genes which contribute to the deterioration of the failing heart could be due to the actions of these novel lncRNAs, immediately suggesting a target for new pharmaceuticals. Changes in the expression of these novel transcripts will be validated in a larger sample size of stretched myocytes vs non-stretched myocytes as well as in the hearts of transverse aortic constriction (TAC) mice vs Sham (surgical procedure without the aortic banding). In vivo investigations will then be carried out, using siLNA antisense technology to silence novel lncRNAs in mice.

scholarly journals Synthetic gene regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

2019 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Regulatory Networks ◽  
Expression Patterns ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Expression Control ◽  
Gene Expression Control

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

scholarly journals Effect of destrin mutations on the gene expression profile in vivo

2008 ◽  
Vol 34 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Angela M. Verdoni ◽  
Natsuyo Aoyama ◽  
Akihiro Ikeda ◽  
Sakae Ikeda
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Target Genes ◽  
Actin Dynamics ◽  
In Vivo Model ◽  
Strong Impact ◽  
Filamentous Actin ◽  
Control Of Gene Expression

Remodeling of the actin cytoskeleton through actin dynamics (assembly and disassembly of filamentous actin) is known to be essential for numerous basic biological processes. In addition, recent studies have provided evidence that actin dynamics participate in the control of gene expression. A spontaneous mouse mutant, corneal disease 1 ( corn1), is deficient for a regulator of actin dynamics, destrin (DSTN, also known as ADF), which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn corn1 mice exhibit an actin dynamics defect in the corneal epithelial cells, offering an in vivo model to investigate cellular mechanisms affected by the Dstn mutation and resultant actin dynamics abnormalities. To examine the effect of the Dstn corn1 mutation on the gene expression profile, we performed a microarray analysis using the cornea from Dstn corn1 and wild-type mice. A dramatic alteration of the gene expression profile was observed in the Dstn corn1 cornea, with 1,226 annotated genes differentially expressed. Functional annotation of these genes revealed that the most significantly enriched functional categories are associated with actin and/or cytoskeleton. Among genes that belong to these categories, a considerable number of serum response factor target genes were found, indicating the possible existence of an actin-SRF pathway of transcriptional regulation in vivo. A comparative study using an allelic mutant strain with milder corneal phenotypes suggested that the level of filamentous actin may correlate with the level of gene expression changes. Our study shows that Dstn mutations and resultant actin dynamics abnormalities have a strong impact on the gene expression profile in vivo.

Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function

2000 ◽  
Vol 113 (11) ◽  
pp. 1841-1849 ◽  
Author(s):  
T. Misteli
Keyword(s):  
Gene Expression ◽  
Cell Nucleus ◽  
Cell Biology ◽  
Nuclear Architecture ◽  
Molecular Techniques ◽  
Mrna Splicing ◽  
Cellular Organization ◽  
Control Of Gene Expression ◽  
Expression Of Genes

Gene expression is a fundamental cellular process. The basic mechanisms involved in expression of genes have been characterized at the molecular level. A major challenge is now to uncover how transcription, RNA processing and RNA export are organized within the cell nucleus, how these processes are coordinated with each other and how nuclear architecture influences gene expression and regulation. A significant contribution has come from cell biological approaches, which combine molecular techniques with microscopy methods. These studies have revealed that the mammalian cell nucleus is a complex but highly organized organelle, which contains numerous subcompartments. I discuss here how two essential nuclear processes - transcription and pre-mRNA splicing - are spatially organized and coordinated in vivo, and how this organization might contribute to the control of gene expression. The dynamic nature of nuclear proteins and compartments indicates a high degree of plasticity in the cellular organization of nuclear functions. The cellular organization of transcription and splicing suggest that the morphology of nuclear compartments is largely determined by the activities of the nucleus.

Engineering Gene Control Circuits with Allosteric Ribozymes in Human Cells as a Medicine of the Future

2013 ◽  
pp. 860-883
Author(s):  
Robert Penchovsky
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Control Of Gene Expression ◽  
Practical Applications ◽  
Gene Therapies ◽  
Sequencing Technologies ◽  
The Future ◽  
Control Circuits ◽  
Exogenous Control

Systems and synthetic biology promise to develop new approaches for analysis and design of complex gene expression regulatory networks in living cells with many practical applications to the pharmaceutical and biotech industries. In this chapter the development of novel universal strategies for exogenous control of gene expression is discussed. They are based on designer allosteric ribozymes that can function in the cell. The synthetic riboswitches are obtained by a patented computational procedure that provides fast and accurate modular designs with various Boolean logic functions. The riboswitches can be designed to sense in the cell either the presence or the absence of disease indicative RNA(s) or small molecules, and to switch on or off the gene expression of any exogenous protein. In addition, the riboswitches can be engineered to induce RNA interference or microRNA pathways that can conditionally down regulate the expression of key proteins in the cell. That can prevent a disease’s development. Therefore, the presented synthetic riboswitches can be used as truly universal cellular biosensors. Nowadays, disease indicative RNA(s) can be precisely identified by employing next-generation sequencing technologies with high accuracy . The methods can be employed not only for exogenous control of gene expression but also for re-programming the cell fate, anticancer, and antiviral gene therapies. Such approaches may be employed as potent molecular medicines of the future.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Biology Open ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. bio055343 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

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.

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