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

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.

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Epigenetics and maternal nutrition: nature v. nurture

Proceedings of The Nutrition Society ◽

10.1017/s0029665110003988 ◽

2010 ◽

Vol 70 (1) ◽

pp. 73-81 ◽

Cited By ~ 69

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.

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Applications of cell-penetrating peptides in regulation of gene expression

Biochemical Society Transactions ◽

10.1042/bst0350770 ◽

2007 ◽

Vol 35 (4) ◽

pp. 770-774 ◽

Cited By ~ 30

Author(s):

P. Järver ◽

K. Langel ◽

S. El-Andaloussi ◽

Ü. Langel

Keyword(s):

Gene Expression ◽

Lipid Bilayers ◽

Regulation Of Gene Expression ◽

Cell Penetrating Peptides ◽

Remarkable Feature ◽

Cell Penetrating ◽

Plasmid Delivery ◽

Interfering Rna

CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.

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Signal-Induced Transcriptional Activation by Dif Requires the dTRAP80 Mediator Module

Molecular and Cellular Biology ◽

10.1128/mcb.23.4.1358-1367.2003 ◽

2003 ◽

Vol 23 (4) ◽

pp. 1358-1367 ◽

Cited By ~ 18

Author(s):

Jin Mo Park ◽

Jung Mo Kim ◽

Lark Kyun Kim ◽

Se Nyun Kim ◽

Jeongsil Kim-Ha ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Specific Binding ◽

Regulation Of Gene Expression ◽

Transcriptional Activator ◽

Mediator Complex ◽

Activator Proteins ◽

In Vitro Interactions

ABSTRACT The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-κB-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.

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Gene Codon Composition Determines Differentiation-Dependent Expression of a Viral Capsid Gene in Keratinocytes In Vitro and InVivo

Molecular and Cellular Biology ◽

10.1128/mcb.25.19.8643-8655.2005 ◽

2005 ◽

Vol 25 (19) ◽

pp. 8643-8655 ◽

Cited By ~ 41

Author(s):

Kong-Nan Zhao ◽

WenYi Gu ◽

Ning Xia Fang ◽

Nicholas A. Saunders ◽

Ian H. Frazer

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Viral Capsid ◽

Virion Assembly ◽

Codon Composition ◽

L1 Protein ◽

First Time ◽

Expression Plasmids

ABSTRACT By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism.

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ΔNp63α is a super enhancer-enriched master factor controlling the basal-to-luminal differentiation transcriptional program and gene regulatory networks in nasopharyngeal carcinoma

Carcinogenesis ◽

10.1093/carcin/bgz203 ◽

2019 ◽

Vol 41 (9) ◽

pp. 1282-1293 ◽

Cited By ~ 3

Author(s):

Jing Cai ◽

Shengnan Chen ◽

Mei Yi ◽

Yixin Tan ◽

Qian Peng ◽

...

Keyword(s):

Gene Expression ◽

Nasopharyngeal Carcinoma ◽

Basal Cell ◽

Regulatory Networks ◽

Regulation Of Gene Expression ◽

Gene Expression Signature ◽

Molecular Signatures ◽

Chip Sequencing

Abstract Nasopharyngeal carcinoma (NPC) originates via malignant transformation of the pseudostratified nasopharyngeal epithelium, composed of basal and luminal cells. Super enhancers (SEs) are large clusters of cis-elements involved in the regulation of gene expression through epigenetic regulatory mechanisms. In this study, we demonstrated that basal cell-specific proteins are highly expressed, whereas luminal cell proteins are downregulated in NPC, implying a perturbation of basal-to-luminal differentiation during NPC development. We characterized NPC cell models according to different molecular signatures associated with their differentiation status and found that distinct SE landscapes are tightly associated with basal or luminal-like molecular signatures in NPC cells. Furthermore, the transcription of ΔNP63α, a prominent isoform of TP63, was found to be driven by SEs in NPC cells. Data from chromatin immunoprecipitation (ChIP)-sequencing showed that ΔNP63α largely occupied regions of SEs associated with basal cell-specific genes. Silencing of ΔNP63α led to a loss of H3K27ac occupancy at basal-type SEs and triggered a basal-to-luminal gene expression signature switch, suggesting that ΔNP63α is a master factor contributing to the perturbation of luminal differentiation. Integrative transcriptomics analysis also revealed that ΔNP63α acts as a core factor involved in the dysregulation of gene expression in NPC. Furthermore, ΔNP63α enhanced EGF-stimulated NF-κB activation in NPC cells by activating SE-mediated EGFR transcription. Finally, depletion of ΔNP63α in NPC cells induced robust growth inhibition of NPC cells in vitro and in vivo. Our data revealed that ΔNP63α-dependent SE reprogramming contributes to the blockade of luminal differentiation and uncontrolled proliferation in NPC.

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Casein Kinase II Phosphorylates the Fragile X Mental Retardation Protein and Modulates Its Biological Properties

Molecular and Cellular Biology ◽

10.1128/mcb.22.24.8438-8447.2002 ◽

2002 ◽

Vol 22 (24) ◽

pp. 8438-8447 ◽

Cited By ~ 57

Author(s):

Mikiko C. Siomi ◽

Kyoko Higashijima ◽

Akira Ishizuka ◽

Haruhiko Siomi

Keyword(s):

Gene Expression ◽

Rna Binding ◽

Fragile X ◽

Regulation Of Gene Expression ◽

Phosphorylation Site ◽

Casein Kinase Ii ◽

Biological Properties ◽

Casein Kinase

ABSTRACT Fragile X syndrome is caused by loss of FMR1 protein expression. FMR1 binds RNA and associates with polysomes in the cytoplasm; thus, it has been proposed to function as a regulator of gene expression at the posttranscriptional level. Posttranslational modification of FMR1 had previously been suggested to regulate its activity, but no experimental support for this model has been reported to date. Here we report that FMR1 in Drosophila melanogaster (dFMR1) is phosphorylated in vivo and that the homomer formation and the RNA-binding activities of dFMR1 are modulated by phosphorylation in vitro. Identification of a protein phosphorylating dFMR1 showed it to be Drosophila casein kinase II (dCKII). dCKII directly interacts with and phosphorylates dFMR1 in vitro. The phosphorylation site in dFMR1 was identified as Ser406, which is highly conserved among FMR1 family members from several species. Using mass spectrometry, we established that Ser406 of dFMR1 is indeed phosphorylated in vivo. Furthermore, human FMR1 (hFMR1) is also phosphorylated in vivo, and alteration of the conserved Ser500 in hFMR1 abolishes phosphorylation by CKII in vitro. These studies support the model that the biological functions of FMR1, such as regulation of gene expression, are likely regulated by its phosphorylation.

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A histone demethylase inhibitor, methylstat, inhibits angiogenesis in vitro and in vivo

RSC Advances ◽

10.1039/c4ra07154a ◽

2014 ◽

Vol 4 (72) ◽

pp. 38230-38233 ◽

Cited By ~ 2

Author(s):

Yumi Cho ◽

Ki Hyun Kim ◽

Yoon Sun Cho ◽

Wenqing Xu ◽

Xiang Wang ◽

...

Keyword(s):

Gene Expression ◽

Histone Methylation ◽

Regulation Of Gene Expression ◽

Histone Demethylase ◽

Chemical Probe

Histone methylation has been highlighted in the regulation of gene expression. Methylstat, a Jumonji C domain containing histone demethylase inhibitor, inhibitedin vitroandin vivoangiogenesis at nontoxic dose. Collectively, methylstat could be a promising chemical probe for addressing its role in angiogenesis

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Precise tuning of gene expression output levels in mammalian cells

10.1101/352377 ◽

2018 ◽

Cited By ~ 1

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.

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A Systems Biology Approach To Modeling Vibrio cholerae Gene Expression under Virulence-Inducing Conditions

Journal of Bacteriology ◽

10.1128/jb.00182-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4300-4310 ◽

Cited By ~ 19

Author(s):

Sanjat Kanjilal ◽

Robert Citorik ◽

Regina C. LaRocque ◽

Marco F. Ramoni ◽

Stephen B. Calderwood

Keyword(s):

Gene Expression ◽

Systems Biology ◽

Vibrio Cholerae ◽

Regulation Of Gene Expression ◽

Virulence Gene ◽

Temporal Separation ◽

New Members ◽

Virulence Gene Expression

ABSTRACT Vibrio cholerae is a Gram-negative bacillus that is the causative agent of cholera. Pathogenesis in vivo occurs through a series of spatiotemporally controlled events under the control of a gene cascade termed the ToxR regulon. Major genes in the ToxR regulon include the master regulators toxRS and tcpPH, the downstream regulator toxT, and virulence factors, the ctxAB and tcpA operons. Our current understanding of the dynamics of virulence gene expression is limited to microarray analyses of expression at selected time points. To better understand this process, we utilized a systems biology approach to examine the temporal regulation of gene expression in El Tor V. cholerae grown under virulence-inducing conditions in vitro (AKI medium), using high-resolution time series genomic profiling. Results showed that overall gene expression in AKI medium mimics that of in vivo studies but with less clear temporal separation between upstream regulators and downstream targets. Expression of toxRS was unaffected by growth under virulence-inducing conditions, but expression of toxT was activated shortly after switching from stationary to aerating conditions. The tcpA operon was also activated early during mid-exponential-phase growth, while the ctxAB operon was turned on later, after the rise in toxT expression. Expression of ctxAB continued to rise despite an eventual decrease in toxT. Cluster analysis of gene expression highlighted 15 hypothetical genes and six genes related to environmental information processing that represent potential new members of the ToxR regulon. This study applies systems biology tools to analysis of gene expression of V. cholerae in vitro and provides an important comparator for future studies done in vivo.

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