scholarly journals Precise tuning of gene expression output levels in mammalian cells

2018 ◽  
Author(s):  
Yale S. Michaels ◽  
Mike B. Barnkob ◽  
Hector Barbosa ◽  
Toni A. Baeumler ◽  
Mary K. Thompson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Throughput Sequencing ◽  
Regulation Of Gene Expression ◽  
Antigen Expression ◽  
Control Of Gene Expression ◽  
Precise Control ◽  
Mouse Melanoma Model

ABSTRACTPrecise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

scholarly journals Genome expression dynamics reveals parasitism regulatory landscape of the root-knot nematode Meloidogyne incognita and a promoter motif associated with effector genes

2021 ◽  
Author(s):  
Martine Da Rocha ◽  
Caroline Bournaud ◽  
Julie Dazeniere ◽  
Peter Thorpe ◽  
Clement Pellegrin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Meloidogyne Incognita ◽  
Life Stage ◽  
Regulation Of Gene Expression ◽  
Root Knot Nematode ◽  
Root Knot Nematodes ◽  
Control Of Gene Expression ◽  
Precise Control ◽  
Effector Genes ◽  
Spatio Temporal

Root-knot nematodes are the major contributor to the crop losses caused by nematodes. Root-knot nematodes secrete effectors into the plant, derived from two sets of pharyngeal gland cells, to manipulate host physiology and immunity. Successful completion of the life cycle, involving successive molts from egg to adult, covers morphologically and functionally distinct stages and will require precise control of gene expression, including effectors. The details of how root-knot nematodes regulate transcription remain sparse. Here, we report a life stage-specific transcriptome of Meloidogyne incognita. Combined with an available annotated genome, we explore the spatio-temporal regulation of gene expression. We reveal gene expression clusters and predicted functions that accompany the major developmental transitions. Focusing on effectors, we identify a putative cis-regulatory motif associated with expression in the dorsal glands: providing an insight into effector regulation. We combine the presence of this motif with several other criteria to predict a novel set of putative dorsal gland effectors. Finally, we show this motif, and thereby its utility, is broadly conserved across the Meloidogyne genus and termed it Mel-DOG. Taken together, we provide the first genome-wide analysis of spatio-temporal gene expression in a root-knot nematode, and identify a new set of candidate effector genes that will guide future functional analyses.

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

Target-independent regulation of a novel growth associated protein in the visual system of the chicken

Development ◽  
1990 ◽  
Vol 109 (2) ◽  
pp. 395-409 ◽  
Author(s):  
B. Schlosshauer ◽  
D. Dutting ◽  
M. Wild
Keyword(s):  
Ganglion Cell ◽  
Ganglion Cells ◽  
Antigen Expression ◽  
Subcellular Fractionation ◽  
Axonal Outgrowth ◽  
Control Of Gene Expression ◽  
Chick Retina ◽  
Retinal Axons

Using an immunosuppression technique, the monoclonal antibody 2A1 has been generated specific for a 140 × 10(3) Mr cytoplasmic-membrane-associated protein as shown by subcellular fractionation and Western blot analysis. The antigen is initially confined to perikarya of postmitotic migratory ganglion cells of the embryonic chick retina as revealed by bromodeoxyuridine labeling. During the subsequent period of axon outgrowth, the antigen becomes restricted to ganglion cell axons but disappears during the innervation of the tectum opticum, suggesting a tectal inhibition of antigen expression in retinal axons. To analyse whether the tectum suppresses 2A1-antigen expression, optic nerves of chick embryos were severed to prevent tectal innervation. 2A1-immunoreactivity was determined in deflected axons in comparison to control axons. In addition, retinal axons were grown in vitro on a substratum consisting of alternating stripes of laminin and tectal membranes, in order to investigate whether retinal axons become devoid of the 2A1-antigen once they cross from laminin to tectal membranes. However, neither prevention of target innervation by optic nerve transection in vivo nor exposure of retinal axons to soluble or particulate tectal components in vitro modify 2A1-antigen regulation in ganglion cell axons, suggesting a retina inherent-control of gene expression. Antigen expression is essentially restricted to the period of axonal outgrowth and therefore the 2A1-protein is likely to be involved in processes essential for neurite extension, independent of the synaptic target.

DEFECTIVE STEROIDAL SYNTHESIS BY ADRENAL TISSUE: THE CONTROL OF GENE EXPRESSION CAUSING LOSS OF ACTIVE STEROIDOGENIC ENZYMES

1974 ◽  
Vol 77 (2) ◽  
pp. 325-336
Author(s):  
K. Williams
Keyword(s):  
Gene Expression ◽  
Adrenal Glands ◽  
Hydroxysteroid Dehydrogenase ◽  
Ascites Cell ◽  
Control Of Gene Expression ◽  
Adrenocortical Hyperplasia ◽  
Normal Human ◽  
Adrenocortical Tumour

ABSTRACT RNA was isolated from normal human adrenal glands and found to cause the formation of Δ5-3β-hydroxysteroid dehydrogenase-isomerase and steroid 21-hydroxylase activities by a Krebs II ascites cell-free protein synthesising system. Although no functional steroid 21-hydroxylase in vivo or in vitro was found in a gland from a patient with virilism due to congenital adrenocortical hyperplasia the RNA would still give steroid 21-hydroxylase-like activity in the protein synthesising system which suggests that the inherited defect was not in the structural gene. Activity could not be induced by RNA from a 'non-functioning' adrenocortical tumour or rat liver.

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 Progressive lengthening of 3′ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development

2009 ◽  
Vol 106 (17) ◽  
pp. 7028-7033 ◽  
Author(s):  
Zhe Ji ◽  
Ju Youn Lee ◽  
Zhenhua Pan ◽  
Bingjun Jiang ◽  
Bin Tian
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Posttranscriptional Regulation ◽  
Regulation Of Gene Expression ◽  
Alternative Polyadenylation ◽  
Untranslated Regions ◽  
C2c12 Myoblast ◽  
Control Of Gene Expression ◽  
Differentiation And Proliferation

The 3′ untranslated regions (3′ UTRs) of mRNAs containcis-acting elements for posttranscriptional regulation of gene expression. Here, we report that mouse genes tend to express mRNAs with longer 3′ UTRs as embryonic development progresses. This global regulation is controlled by alternative polyadenylation and coordinates with initiation of organogenesis and aspects of embryonic development, including morphogenesis, differentiation, and proliferation. Using myogenesis of C2C12 myoblast cells as a model, we recapitulated this process in vitro and found that 3′ UTR lengthening is likely caused by weakening of mRNA polyadenylation activity. Because alternative 3′ UTR sequences are typically longer and have higher AU content than constitutive ones, our results suggest that lengthening of 3′ UTR can significantly augment posttranscriptional control of gene expression during embryonic development, such as microRNA-mediated regulation.

HMGN proteins play roles in DNA repair and gene expression in mammalian cells

2004 ◽  
Vol 32 (6) ◽  
pp. 918-919 ◽  
Author(s):  
K.L. West
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Knockout Mice ◽  
Mammalian Cells ◽  
High Mobility ◽  
Genetically Engineered ◽  
Expression In Mammalian Cells ◽  
Transcription And Replication

HMGN (high-mobility-group N) family members are vertebrate proteins that unfold chromatin and promote transcription and replication of chromatin templates in vitro. However, their precise roles in vivo have been elusive until recently. This paper summarizes recent advances from studies of Hmgn1 knockout mice and genetically engineered cell lines that are beginning to reveal the diverse roles that HMGN proteins play in DNA repair and transcription within mammalian cells.

scholarly journals Physical and Functional Interactions between Cellular Retinoic Acid Binding Protein II and the Retinoic Acid-Dependent Nuclear Complex

1999 ◽  
Vol 19 (10) ◽  
pp. 7158-7167 ◽  
Author(s):  
Laurent Delva ◽  
Jean-Noël Bastie ◽  
Cécile Rochette-Egly ◽  
Radhia Kraïba ◽  
Nicole Balitrand ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Nuclear Receptors ◽  
Mammalian Cells ◽  
Specific Response ◽  
Independent Manner ◽  
Control Of Gene Expression ◽  
Steady State Levels ◽  
And Control

ABSTRACT Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARα and RXRα in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRα-RARα heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.

scholarly journals Epigenetics and maternal nutrition: nature v. nurture

2010 ◽  
Vol 70 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Rebecca Simmons
Keyword(s):  
Gene Expression ◽  
Maternal Nutrition ◽  
Current Knowledge ◽  
Regulation Of Gene Expression ◽  
Epigenetic Modifications ◽  
Epigenetic Gene Regulation ◽  
Diabetes And Obesity

Under- and over-nutrition during pregnancy has been linked to the later development of diseases such as diabetes and obesity. Epigenetic modifications may be one mechanism by which exposure to an altered intrauterine milieu or metabolic perturbation may influence the phenotype of the organism much later in life. Epigenetic modifications of the genome provide a mechanism that allows the stable propagation of gene expression from one generation of cells to the next. This review highlights our current knowledge of epigenetic gene regulation and the evidence that chromatin remodelling and histone modifications play key roles in adipogenesis and the development of obesity. Epigenetic modifications affecting processes important to glucose regulation and insulin secretion have been described in the pancreatic β-cells and muscle of the intrauterine growth-retarded offspring, characteristics essential to the pathophysiology of type-2 diabetes. Epigenetic regulation of gene expression contributes to both adipocyte determination and differentiation in in vitro models. The contributions of histone acetylation, histone methylation and DNA methylation to the process of adipogenesis in vivo remain to be evaluated.

scholarly journals Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Nobutoshi Yamaguchi
Keyword(s):  
Gene Expression ◽  
Plant Development ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Histone H3 ◽  
Control Of Gene Expression ◽  
Precise Control ◽  
Repressive Histone Modification ◽  
Environmental Responses

Trimethylation of histone H3 lysine 27 (H3K27me3) is a highly conserved repressive histone modification that signifies transcriptional repression in plants and animals. In Arabidopsis thaliana, the demethylation of H3K27 is regulated by a group of JUMONJI DOMAIN-CONTANING PROTEIN (JMJ) genes. Transcription of JMJ genes is spatiotemporally regulated during plant development and in response to the environment. Once JMJ genes are transcribed, recruitment of JMJs to target genes, followed by demethylation of H3K27, is critically important for the precise control of gene expression. JMJs function synergistically and antagonistically with transcription factors and/or other epigenetic regulators on chromatin. This review summarizes the latest advances in our understanding of Arabidopsis H3K27me3 demethylases that provide robust and flexible epigenetic regulation of gene expression to direct appropriate development and environmental responses in plants.

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