scholarly journals De novo Design of Translational RNA Repressors

2018 ◽  
Author(s):  
Paul D. Carlson ◽  
Cameron J. Glasscock ◽  
Julius B. Lucks
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Genetic Circuit ◽  
Bacterial Cells ◽  
Control Of Gene Expression ◽  
Sequence Characterization ◽  
Mrna Targets ◽  
Modern Drug ◽  
And Function

ABSTRACTA central goal of synthetic biology is the development of methods for the predictable control of gene expression. RNA is an attractive substrate by which to achieve this goal because the relationship between its sequence, structure, and function is being uncovered with increasing depth. In addition, design approaches that use this relationship are becoming increasingly effective, as evidenced by significant progress in the de novo design of RNA-based gene regulatory mechanisms that activate transcription and translation in bacterial cells. However, the design of synthetic RNA mechanisms that are efficient and versatile repressors of gene expression has lagged, despite their importance for gene regulation and genetic circuit construction. We address this gap by developing two new classes of RNA regulators, toehold repressors and looped antisense oligonucleotides (LASOs), that repress translation of a downstream gene in response to an arbitrary input RNA sequence. Characterization studies show that these designed RNAs robustly repress translation, are highly orthogonal, and can be multiplexed with translational activators. We show that our LASO design can repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity and predictability. These results demonstrate significant yet easy-to-implement improvements in the design of synthetic RNA repressors for synthetic biology, and point more broadly to design principles for repressive RNA interactions relevant to modern drug design.

scholarly journals Roles of HIF and 2-Oxoglutarate-Dependent Dioxygenases in Controlling Gene Expression in Hypoxia

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 350
Author(s):  
Julianty Frost ◽  
Mark Frost ◽  
Michael Batie ◽  
Hao Jiang ◽  
Sonia Rocha
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Regulator ◽  
Hydroxylase Activity ◽  
Control Of Gene Expression ◽  
Prolyl Hydroxylases ◽  
Chromatin Organisation ◽  
Sense And Respond ◽  
Almost All ◽  
And Function

Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.

scholarly journals Dynamic changes in chromatin accessibility, altered adipogenic gene expression, and total versus de novo fatty acid synthesis in subcutaneous adipose stem cells of normal-weight polycystic ovary syndrome (PCOS) women during adipogenesis: evidence of cellular programming

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Karen L. Leung ◽  
Smriti Sanchita ◽  
Catherine T. Pham ◽  
Brett A. Davis ◽  
Mariam Okhovat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
De Novo ◽  
Polycystic Ovary ◽  
Total Content ◽  
Chromatin Accessibility ◽  
Normal Weight ◽  
Triacylglycerol Synthesis ◽  
And Function

Abstract Background Normal-weight polycystic ovary syndrome (PCOS) women exhibit adipose resistance in vivo accompanied by enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with accelerated lipid accumulation per cell in vitro. The present study examines chromatin accessibility, RNA expression and fatty acid (FA) synthesis during SC abdominal ASC differentiation into adipocytes in vitro of normal-weight PCOS versus age- and body mass index-matched normoandrogenic ovulatory (control) women to study epigenetic/genetic characteristics as well as functional alterations of PCOS and control ASCs during adipogenesis. Results SC abdominal ASCs from PCOS women versus controls exhibited dynamic chromatin accessibility during adipogenesis, from significantly less chromatin accessibility at day 0 to greater chromatin accessibility by day 12, with enrichment of binding motifs for transcription factors (TFs) of the AP-1 subfamily at days 0, 3, and 12. In PCOS versus control cells, expression of genes governing adipocyte differentiation (PPARγ, CEBPα, AGPAT2) and function (ADIPOQ, FABP4, LPL, PLIN1, SLC2A4) was increased two–sixfold at days 3, 7, and 12, while that involving Wnt signaling (FZD1, SFRP1, and WNT10B) was decreased. Differential gene expression in PCOS cells at these time points involved triacylglycerol synthesis, lipid oxidation, free fatty acid beta-oxidation, and oxidative phosphorylation of the TCA cycle, with TGFB1 as a significant upstream regulator. There was a broad correspondence between increased chromatin accessibility and increased RNA expression of those 12 genes involved in adipocyte differentiation and function, Wnt signaling, as well as genes involved in the triacylglycerol synthesis functional group at day 12 of adipogenesis. Total content and de novo synthesis of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and oleic (C18:1) acid increased from day 7 to day 12 in all cells, with total content and de novo synthesis of FAs significantly greater in PCOS than controls cells at day 12. Conclusions In normal-weight PCOS women, dynamic chromatin remodeling of SC abdominal ASCs during adipogenesis may enhance adipogenic gene expression as a programmed mechanism to promote greater fat storage.

scholarly journals De novo design of programmable inducible promoters

2019 ◽  
Vol 47 (19) ◽  
pp. 10452-10463 ◽  
Author(s):  
Xiangyang Liu ◽  
Sanjan T P Gupta ◽  
Devesh Bhimsaria ◽  
Jennifer L Reed ◽  
José A Rodríguez-Martínez ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput Screening ◽  
De Novo ◽  
Vital Role ◽  
Control Of Gene Expression ◽  
Core Motif ◽  
Expression Levels ◽  
Synthetic Promoters ◽  
The Core ◽  
Rich Diversity

Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

Platelet microparticles reprogram macrophage gene expression and function

2016 ◽  
Vol 115 (02) ◽  
pp. 311-323 ◽  
Author(s):  
Matthieu Rousseau ◽  
Anne-Claire Duchez ◽  
Chan Ho C. Lee ◽  
Eric Boilard ◽  
Benoit Laffont ◽  
...  
Keyword(s):  
Actinomycin D ◽  
De Novo ◽  
Phagocytic Capacity ◽  
Human Macrophages ◽  
Mrna Targets ◽  
De Novo Gene ◽  
Supplementary Material ◽  
And Function ◽  
Microarray Analyses ◽  
Platelet Microparticles

SummaryPlatelet microparticles (MPs) represent the most abundant MPs subtype in the circulation, and can mediate intercellular communication through delivery of bioactives molecules, such as cytokines, proteins, lipids and RNAs. Here, we show that platelet MPs can be internalised by primary human macrophages and deliver functional miR-126–3p. The increase in macrophage miR-126–3p levels was not prevented by actinomycin D, suggesting that it was not due to de novo gene transcription. Platelet MPs dose-dependently downregulated expression of four predicted mRNA targets of miR-126–3p, two of which were confirmed also at the protein level. The mRNA downregulatory effects of platelet MPs were abrogated by expression of a neutralising miR-126–3p sponge, implying the involvement of miR-126–3p. Transcriptome-wide, microarray analyses revealed that as many as 66 microRNAs and 653 additional RNAs were significantly and differentially expressed in macrophages upon exposure to platelet MPs. More specifically, platelet MPs induced an upregulation of 34 microRNAs and a concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1 and TNF. These changes were associated with reduced CCL4, CSF1 and TNF cytokine/chemokine release by macrophages, and accompanied by a marked increase in their phagocytic capacity. These findings demonstrate that platelet MPs can modify the transcriptome of macrophages, and reprogram their function towards a phagocytic phenotype.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Critical Role of Types 2 and 3 Deiodinases in the Negative Regulation of Gene Expression by T3 in the Mouse Cerebral Cortex

Endocrinology ◽  
2012 ◽  
Vol 153 (6) ◽  
pp. 2919-2928 ◽  
Author(s):  
Arturo Hernandez ◽  
Beatriz Morte ◽  
Mónica M. Belinchón ◽  
Ainhoa Ceballos ◽  
Juan Bernal
Keyword(s):  
Gene Expression ◽  
Cerebral Cortex ◽  
Thyroid Hormone ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Control Of Gene Expression ◽  
High Doses ◽  
And Function ◽  
Type 3

Thyroid hormones regulate brain development and function through the control of gene expression, mediated by binding of T3 to nuclear receptors. Brain T3 concentration is tightly controlled by homeostatic mechanisms regulating transport and metabolism of T4 and T3. We have examined the role of the inactivating enzyme type 3 deiodinase (D3) in the regulation of 43 thyroid hormone-dependent genes in the cerebral cortex of 30-d-old mice. D3 inactivation increased slightly the expression of two of 22 positively regulated genes and significantly decreased the expression of seven of 21 negatively regulated genes. Administration of high doses of T3 led to significant changes in the expression of 12 positive genes and three negative genes in wild-type mice. The response to T3 treatment was enhanced in D3-deficient mice, both in the number of genes and in the amplitude of the response, demonstrating the role of D3 in modulating T3 action. Comparison of the effects on gene expression observed in D3 deficiency with those in hypothyroidism, hyperthyroidism, and type 2 deiodinase (D2) deficiency revealed that the negative genes are more sensitive to D2 and D3 deficiencies than the positive genes. This observation indicates that, in normal physiological conditions, D2 and D3 play critical roles in maintaining local T3 concentrations within a very narrow range. It also suggests that negatively and positively regulated genes do not have the same physiological significance or that their regulation by thyroid hormone obeys different paradigms at the molecular or cellular levels.

Centromere identity and function put to use: construction and transfer of mammalian artificial chromosomes to animal models

2020 ◽  
Vol 64 (2) ◽  
pp. 185-192
Author(s):  
Ye Yang ◽  
Michael A. Lampson ◽  
Ben E. Black
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Animal Model ◽  
Synthetic Biology ◽  
Animal Models ◽  
Model Systems ◽  
Expression Vectors ◽  
Artificial Chromosomes ◽  
Conventional Expression ◽  
And Function

Abstract Mammalian artificial chromosomes (MACs) are widely used as gene expression vectors and have various advantages over conventional expression vectors. We review and discuss breakthroughs in MAC construction, initiation of functional centromeres allowing their faithful inheritance, and transfer from cell culture to animal model systems. These advances have contributed to advancements in synthetic biology, biomedical research, and applications in industry and in the clinic.

scholarly journals Genome-Wide Gene Expression in relation to Age in Large Laboratory Cohorts of Drosophila melanogaster

2015 ◽  
Vol 2015 ◽  
pp. 1-19 ◽  
Author(s):  
Kimberly A. Carlson ◽  
Kylee Gardner ◽  
Anjeza Pashaj ◽  
Darby J. Carlson ◽  
Fang Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Immune Function ◽  
Expression Patterns ◽  
Self Organizing Map ◽  
Control Of Gene Expression ◽  
Genome Wide ◽  
Complex Process ◽  
Temporal Variance ◽  
And Function

Aging is a complex process characterized by a steady decline in an organism’s ability to perform life-sustaining tasks. In the present study, two cages of approximately 12,000 mated Drosophila melanogaster females were used as a source of RNA from individuals sampled frequently as a function of age. A linear model for microarray data method was used for the microarray analysis to adjust for the box effect; it identified 1,581 candidate aging genes. Cluster analyses using a self-organizing map algorithm on the 1,581 significant genes identified gene expression patterns across different ages. Genes involved in immune system function and regulation, chorion assembly and function, and metabolism were all significantly differentially expressed as a function of age. The temporal pattern of data indicated that gene expression related to aging is affected relatively early in life span. In addition, the temporal variance in gene expression in immune function genes was compared to a random set of genes. There was an increase in the variance of gene expression within each cohort, which was not observed in the set of random genes. This observation is compatible with the hypothesis that D. melanogaster immune function genes lose control of gene expression as flies age.

scholarly journals Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Simone Balzer Le ◽  
Ingerid Onsager ◽  
Jon Andreas Lorentzen ◽  
Rahmi Lale
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Design Concept ◽  
Fine Tuning ◽  
The Novel ◽  
Wide Range ◽  
Application Potential

Abstract Bacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

scholarly journals Roles Of HIF and 2-Oxoglutarate Dependent Enzymes in Controlling Gene Expression In Hypoxia

2020 ◽  
Author(s):  
Julianty Frost ◽  
Mark Frost ◽  
Michael Batie ◽  
Hao Jiang ◽  
Sonia Rocha
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Regulator ◽  
Hydroxylase Activity ◽  
Control Of Gene Expression ◽  
Prolyl Hydroxylases ◽  
Chromatin Organisation ◽  
Sense And Respond ◽  
Almost All ◽  
And Function

Hypoxia — reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OG dioxyenases in the control of gene expression in response to hypoxia and their relevance to human cancers.

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.

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