A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

AbstractCellular systems have evolved numerous mechanisms to finely control signalling pathway activation and properly respond to changing environmental stimuli. This is underpinned by dynamic spatiotemporal patterns of gene expression. Indeed, in addition to gene transcription and translation regulation, modulation of protein levels, dynamics and localization are also essential checkpoints that govern cell functions. The introduction of tetracycline-inducible promoters has allowed gene expression control using orthogonal small molecules, facilitating rapid and reversible manipulation to study gene function in biological systems. However, differing protein stabilities means this solely transcriptional regulation is insufficient to allow precise ON-OFF dynamics, thus hindering generation of temporal profiles of protein levels seen in vivo. We developed an improved Tet-On based system augmented with conditional destabilising elements at the post-translational level that permits simultaneous control of gene expression and protein stability. Integrating these properties to control expression of a fluorescent protein in mouse Embryonic Stem Cells (mESCs), we found that adding protein stability control allows faster response times to changes in small molecules, fully tunable and enhanced dynamic range, and vastly improved microfluidic-based in-silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to finely modulate levels of signalling pathway components in stem cells.

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A tunable dual-input system for on-demand dynamic gene expression regulation

Nature Communications ◽

10.1038/s41467-019-12329-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Elisa Pedone ◽

Lorena Postiglione ◽

Francesco Aulicino ◽

Dan L. Rocca ◽

Sandra Montes-Olivas ◽

...

Keyword(s):

Gene Expression ◽

Protein Stability ◽

Mammalian Cells ◽

Dynamic Range ◽

Embryonic Stem ◽

Cellular Systems ◽

Control Of Gene Expression ◽

Cell Functions ◽

Input System ◽

Gene Expression Control

Abstract Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

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Single cell 3’UTR analysis identifies changes in alternative polyadenylation throughout neuronal differentiation and in autism

10.1101/2020.08.12.247627 ◽

2020 ◽

Author(s):

Manuel Göpferich ◽

Nikhil Oommen George ◽

Ana Domingo Muelas ◽

Alex Bizyn ◽

Rosa Pascual ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Neural Stem Cells ◽

Single Cell ◽

Neuronal Differentiation ◽

Mrna Translation ◽

Autism Spectrum ◽

Adult Brain ◽

Control Of Gene Expression ◽

Risk Genes

SUMMARYAutism spectrum disorder (ASD) is a neurodevelopmental disease affecting social behavior. Many of the high-confident ASD risk genes relate to mRNA translation. Specifically, many of these genes are involved in regulation of gene expression for subcellular compartmentalization of proteins1. Cis-regulatory motifs that often localize to 3’- and 5’-untranslated regions (UTRs) offer an additional path for posttranscriptional control of gene expression. Alternative cleavage and polyadenylation (APA) affect 3’UTR length thereby influencing the presence or absence of regulatory elements. However, APA has not yet been addressed in the context of neurodevelopmental disorders. Here we used single cell 3’end sequencing to examine changes in 3’UTRs along the differentiation from neural stem cells (NSCs) to neuroblasts within the adult brain. We identified many APA events in genes involved in neurodevelopment, many of them being high confidence ASD risk genes. Further, analysis of 3’UTR lengths in single cells from ASD and healthy individuals detected longer 3’UTRs in ASD patients. Motif analysis of modulated 3’UTRs in the mouse adult neurogenic lineage and ASD-patients revealed enrichment of the cytoplasmic and polyadenylation element (CPE). This motif is bound by CPE binding protein 4 (CPEB4). In human and mouse data sets we observed co-regulation of CPEB4 and the CPEB-binding synaptic adhesion molecule amyloid beta precursor-like protein 1 (APLP1). We show that mice deficient in APLP1 show aberrant regulation of APA, decreased number of neural stem cells, and autistic-like traits. Our findings indicate that APA is used for control of gene expression along neuronal differentiation and is altered in ASD patients.

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110 Temporal Changes in Endometrial Gene Expression Between Ipsi- and Contralateral Uterine Horns in Cattle

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab110 ◽

2018 ◽

Vol 30 (1) ◽

pp. 194

Author(s):

J. M. Sánchez ◽

C. Passaro ◽

N. Forde ◽

S. Behura ◽

J. A. Browne ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Luteal Phase ◽

Expression Patterns ◽

Uterine Horn ◽

Oestrous Cycle ◽

Signalling Pathway ◽

Signalling Pathways ◽

Robust Analysis ◽

The Impact

The transfer of an embryo into the uterine horn contralateral to the ovary bearing the corpus luteum has been associated with a decreased pregnancy rate in cattle compared with transfer into the ipsilateral horn. These findings suggest that the environment in the contralateral horn is less conducive to supporting conceptus development than that of the ipsilateral horn. Therefore, this study compared the endometrial transcriptome of the ipsi- and contralateral uterine horns during the luteal phase. Endometrial samples from the ipsi- (IPSI) and contralateral (CONTRA) horns were collected from synchronized nonpregnant beef heifers on Days 5, 7, 13 or 16 post-oestrus (n = 5 heifers per time point). Total RNA was isolated and sequenced. Differences in the transcriptome were determined by edgeR-robust analysis. Principal component analysis found that IPSI and CONTRA have distinct patterns of gene expression on each day, with Day 5 exhibiting the most variation and Day 16 being least variable. Further, the 2 uterine horns had distinct expression patterns on Day 5, with IPSI exhibiting significantly higher variation in gene expression compared twitho CONTRA. EdgeR-robust analysis found 217 (201 up- and 16 down-regulated), 54 (44 up- and 10 down-regulated), 14 (13 up- and 1 down-regulated), and 18 (14 up- and 4 down-regulated) differentially expressed genes (DEG; >2-fold change, false discovery rate P < 0.05) between IPSI and CONTRA endometria on Days 5, 7, 13, and 16 of the oestrous cycle, respectively. The top 5 canonical pathways associated with DEG between IPSI and CONTRA during the luteal phase of the oestrous cycle were involved in signalling pathways regulating pluripotency of stem cells (73/138), progesterone-mediated oocyte maturation (55/89), endometrial cancer (31/51), ErbB signalling pathway (50/87), and mTOR signalling pathway (36/61). The impact of DEG on signalling pathways was assessed using a pathway perturbation algorithm called Signalling Pathway Impact Analysis (SPIA). This topology-based pathway analysis was conducted using the Bioconductor ToPAseq package (https://bioconductor.org/packages/release/bioc/html/ToPASeq.html) and revealed that signalling pathways regulating pluripotency of stem cells showed the highest perturbation score when IPSI was compared with CONTRA irrespective of day. Discovering and cataloguing which pathways are perturbed in each uterine horn throughout the oestrous cycle may contribute to our understanding of the mechanisms underlying early embryonic loss. Ths study was supported by Science Foundation Ireland (13/IA/1983) and the Irish Department of Agriculture, Food and The Marine (13S528).

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MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

Stem Cells International ◽

10.1155/2016/3984937 ◽

2016 ◽

Vol 2016 ◽

pp. 1-17 ◽

Cited By ~ 14

Author(s):

Irma Virant-Klun ◽

Anders Ståhlberg ◽

Mikael Kubista ◽

Thomas Skutella

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Germ Cells ◽

Noncoding Rna ◽

Ovarian Function ◽

Embryonic Stem ◽

Cell Communication ◽

Female Fertility ◽

Control Of Gene Expression

MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells.

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Control of Gene Expression with Small Molecules

Chemistry & Biology ◽

10.1016/j.chembiol.2003.11.007 ◽

2003 ◽

Vol 10 (12) ◽

pp. 1245-1253 ◽

Cited By ~ 18

Author(s):

Sonalee Athavankar ◽

Blake R Peterson

Keyword(s):

Gene Expression ◽

Small Molecules ◽

Control Of Gene Expression

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Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics

10.1101/606863 ◽

2019 ◽

Author(s):

Patrick M. Caveney ◽

Rosemary M. Dabbs ◽

William T. McClintic ◽

C. Patrick Collier ◽

Michael L. Simpson

Keyword(s):

Gene Expression ◽

Membrane Transport ◽

Molecular Species ◽

Lipid Membranes ◽

Molecular Transport ◽

Lipid Vesicles ◽

Cellular Systems ◽

Control Of Gene Expression ◽

External Resources ◽

Synthetic Cell

SummaryEssential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7-fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.

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CTCF and cellular heterogeneity

Cell & Bioscience ◽

10.1186/s13578-019-0347-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Gang Ren ◽

Keji Zhao

Keyword(s):

Gene Expression ◽

Fundamental Property ◽

Cellular Systems ◽

Cellular Heterogeneity ◽

Control Of Gene Expression ◽

Homogeneous Population ◽

Cellular Processes ◽

Underlying Mechanisms ◽

Cell Variation ◽

Promoter Interaction

Abstract Cellular heterogeneity, which was initially defined for tumor cells, is a fundamental property of all cellular systems, ranging from genetic diversity to cell-to-cell variation driven by stochastic molecular interactions involved all cellular processes. Different cells display substantial variation in gene expression and in response to environmental signaling even in an apparently homogeneous population of cells. Recent studies started to reveal the underlying mechanisms for cellular heterogeneity, particularly related to the states of chromatin. Accumulating evidence suggests that CTCF, an important factor regulating chromatin organization, plays a key role in the control of gene expression variation by stabilizing enhancer–promoter interaction.

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A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates

Scientific Reports ◽

10.1038/s41598-020-73656-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Novin Balafkan ◽

Sepideh Mostafavi ◽

Manja Schubert ◽

Richard Siller ◽

Kristina Xiao Liang ◽

...

Keyword(s):

Stem Cells ◽

Small Molecules ◽

Pluripotent Stem Cells ◽

Culture Medium ◽

Electrophysiological Properties ◽

Human Cardiomyocytes ◽

Protein Levels ◽

Incremental Improvement ◽

Induced Pluripotent ◽

The Impact

Abstract The capacity of pluripotent stem cells both for self-renewal and to differentiate into any cell type have made them a powerful tool for studying human disease. Protocols for efficient differentiation towards cardiomyocytes using defined, serum-free culture medium combined with small molecules have been developed, but thus far, limited to larger formats. We adapted protocols for differentiating human pluripotent stem cells to functional human cardiomyocytes in a 96-well microplate format. The resulting cardiomyocytes expressed cardiac specific markers at the transcriptional and protein levels and had the electrophysiological properties that confirmed the presence of functional cardiomyocytes. We suggest that this protocol provides an incremental improvement and one that reduces the impact of heterogeneity by increasing inter-experimental replicates. We believe that this technique will improve the applicability of these cells for use in developmental biology and mechanistic studies of disease.

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