Efficient Regulation Of Gene Expression Research Articles

Electrogenetic control of gene expression in Shewanella oneidensis MR-1 using Arc-dependent transcriptional promoters

Electrochemically active bacteria (EAB) are capable of electrically interacting with electrodes, enabling their application in bioelectrochemical systems (BESs). As the performance of BES is related to the metabolic activities of EAB, the development of methods to control their metabolic activities is important to facilitate BES applications. A recent study found that the EAB Shewanella oneidensis MR-1 uses the Arc system to regulate the expression of catabolic genes in response to electrode potentials, suggesting that a methodology for electrical control of gene expression in EAB, referred to as electrogenetics, can be developed by using electrode potential-responsive, Arc-dependent transcriptional promoters. Here, we explored Arc-dependent promoters in the genomes of S.oneidensis MR-1 and Escherichia coli to identify electrode potential-responsive promoters that are differentially activated in MR-1cells exposed to high- and low-potential electrodes. LacZ reporter assays using electrode-associated cells of MR-1 derivatives revealed that the activities of promoters located upstream of the E.coli feo gene (Pfeo) and the MR-1 nqrA2 (SO_0902) gene (Pnqr2) were significantly increased when S.oneidensis cells were exposed to electrodes poised at+0.7V and-0.4V (versus the standard hydrogen electrode), respectively. Additionally, we developed a microscopic system for in situ monitoring of promoter activity in electrode-associated cells and found that Pnqr2 activity was persistently induced in MR-1cells associated with an electrode poised at-0.4V. Our results indicate that these electrode potential-responsive promoters enable efficient regulation of gene expression in EAB, providing a molecular basis for the development of electrogenetics.

A broad analysis of splicing regulation in yeast using a large library of synthetic introns.

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5' donor site, the branch site, and the 3' acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

Multifunctional Double-Bundle DNA Tetrahedron for Efficient Regulation of Gene Expression.

DNA nanostructures have garnered considerable interest as research tools in the field of cell biology and pathology. Herein, we develop an addressable double-bundle DNA tetrahedron with distinct modification sites to load multiple functional components for efficient regulation of gene expression. In our tailored nanoplatform, nucleic acid drugs (antisense for gene therapy) and protein drugs (KillerRed for photodynamic therapy) are precisely organized in the chemically well-defined DNA tetrahedron. With the attachment of active targeting groups, this functional DNA nanocarrier can efficiently penetrate into the cell membrane and subsequently transport drugs to the target subcellular organelles (mitochondrion and nucleus) for inducing synergistic cell behavior regulation to start the endogenous apoptotic process. This tailored DNA nanocarrier provides unprecedented opportunities for intelligent drug delivery and cell biology research.

DNA Nanosponge for Adsorption and Clearance of Intracellular miR-21 and Enhanced Antitumor Chemotherapy.

Antisense oligonucleotide (ASO)-induced cellular signaling pathway alteration is evolving as a promising therapeutic strategy for improving antitumor chemotherapy. However, the inherent instability and inefficiency of ASO delivery remain major hurdles for its application. Herein, we developed a self-assembled DNA nanosponge (DNS) for adsorption and clearance of intracellular miR-21. The densely packed DNA nanostructure is able to provide large amounts of repeated ASO copies for efficient capturing of miR-21 and inhibiting the miRNAs function in mammalian cells. The cell apoptosis-related protein expression (Bcl-2, Bax, and cleaved caspase-3/9) can be obviously interrupted with the delivery of DNS. Besides, we have shown that the DNS can efficiently carry Dox for chemotherapy and inducing tumor cell (MCF-7) apoptosis meanwhile has little affect to normal cells (Hs578 Bst). These polymeric DNS systems mimic the natural RNA circle-based miRNA sponges and have potential to be applied for specific and efficient regulation of gene expression in tumor cells for synergistic antitumor chemotherapy.

Chemically‐controlled orthogonal regulation of multiple endogenous genes

Genome editing platforms are powerful tools for gene manipulation. These platforms offer a wide variety of promising applications in medical science and biotechnology. Induction of protein domain associations by stimuli such as light or chemicals has been widely used for the temporal control of biomolecular function such as enzymatic activity. Further, the DNA binding specificity of genome editing tools can be applied in gene regulation systems by combining them with transcription‐activator or transcription‐repressor domains. Targeting the gene promoter sequence using multiple artificial transcription factors enables synergistic and efficient regulation of gene expression. In addition, the use of these platforms in combination can expand the scope of targetable sequences by avoiding DNA‐binding issues, such as PAM recognition for CRISPR‐Cas9, 5′‐T recognition for TALE domain, and complementary strand recognition for ZF domain. Furthermore, the guide RNA for Cas9 cannot distinguish alleles because of the commonly used gRNA sequence recognized by Cas9, thereby limiting the utility of this approach for orthogonal regulation of multiple genes. In this study, the rapamycin‐ (R‐) or gibberellin‐ (G‐) inducible system was applied for gene regulation using multiple TALEs and dCas9 to enable orthogonally inducible regulation of endogenous genes. For the R‐inducible system, FKBP‐tagged TALEs, and dCas9, the effector domains with FRB, VP64 for activation and SID×4 for repression, were constructed. GID‐tagged TALEs and dCas9 as well as GAI‐fused VP64 were constructed for the G‐inducible system. The fold change in gene expression was evaluated by luciferase reporter assays. The results showed that gene expression was regulated by addition of chemicals, indicating rapamycin‐induced FKBP‐FRB association and gibberellin‐induced GID‐GAI association. The systems were further utilized for controlling endogenous gene expression in 293A cells. The results revealed that the expression of two genes could be differentially regulated by rapamycin and gibberellin in a time‐dependent manner. This system could achieve the control of gene function in cellular phenomena owing to the programmable timing of expression and differential expression of multiple genes.Support or Funding InformationThis work was supported in part by the New Energy and Industrial Technique Development Organization (NEDO) of Japan, JSPS KAKENHI (25410171), Grant‐in‐Aid for Scientific Research on Innovative Areas (24119506 and 26119703) to W. N.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The deadenylase components Not2p, Not3p, and Not5p promote mRNA decapping.

Decay of mRNA is essential for the efficient regulation of gene expression. A major pathway of mRNA degradation is initiated by the shortening of the poly(A) tail via the CCR4/NOT deadenylase complex. Deadenylation is followed by removal of the 5′ cap (i.e., decapping) and then 5′ to 3′ exonucleolytic decay of the message body. The highly conserved CCR4/NOT deadenylase complex consists of the exonucleases CCR4 and POP2/CAF1, as well as a group of four or five (depending on organism) accessory factors of unknown function, i.e., the NOT proteins. In this study, we find that Saccharomyces cerevisiae Not2p, Not3p, and Not5p (close paralogs of each other) are involved in promoting mRNA decapping. Furthermore, we find that Not3p and Not5p bind to the decapping activator protein Pat1p. Together, these data implicate the deadenylase complex in coordinating the downstream decapping reaction via Not2p, Not3p, and Not5p. This suggests that the coupling of deadenylation with decapping is, in part, a direct consequence of coordinated assembly of decay factors.

CD40 Stimulates a “Feed-Forward” NF-κB–Driven Molecular Pathway That Regulates IFN-β Expression in Carcinoma Cells

IFN-β and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses. In this study, we show that CD40 receptor occupancy results in IFN-β upregulation through an unconventional "feed-forward" mechanism, which is orchestrated by canonical NF-κB and involves the sequential de novo synthesis of IFN regulatory factor (IRF)1 and Viperin (RSAD2), an IRF1 target. RelA (p65) NF-κB, IRF1, and Viperin-dependent IRF7 binding to the IFN-β promoter largely controls its activity. However, full activation of IFN-β also requires the parallel engagement of noncanonical NF-κB2 signaling leading to p52 recruitment to the IFN-β promoter. These data define a novel link between CD40 signaling and IFN-β expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.

Highly Efficient Regulation of Gene Expression by Tetracycline in a Replication-Defective Herpes Simplex Viral Vector

Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx™; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx™-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx™ for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.

Efficient regulation of gene expression using self-contained fiber-modified adenovirus vectors containing the tet-off system

Previously, we developed single adenovirus (Ad) vectors that contained the gene of interest in the E1 deletion region and the transactivator gene for the tetracycline-controllable expression system in the E3 deletion region. In the present study, we improved the Ad vector-mediated tetracycline-controllable expression system by the fiber modification of Ad. We developed fiber-modified Ad vectors containing the tet-off system, which are effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Ad vectors containing the tet-off system with an Arg–Gly–Asp (RGD) peptide in the HI loop of the fiber knob or the Ad type 35 fiber greatly improved transduction efficiency (more than 1–2-log orders) into the cells lacking CAR expression but expressing αv integrin or CD46, respectively. They exhibited vastly higher regulation of gene expression by doxycycline. The combination of fiber-modified Ad vectors and the tetracycline-controllable expression system should offer a powerful tool for gene therapy and gene transfer experiment.

987. Highly Efficient Regulation of Gene Expression by Tetracycline in a Novel Replication-Defective Herpes Simplex Viral Vector

Fetal gene transfer is a promising therapy for the correction of pathology associated with inherited or acquired diseases, however information on the optimal conditions for gene transfer is limited. In this study, we have determined the effect of the time of gene transfer and route of administration on the efficiency of this approach. Fetal rhesus monkeys were injected intraperitoneal (IP) at 40 (N=4) or 60 (N=4) days of gestation (term 165±10 days) with 2 × 107 HIV-1-derived lentiviral vector particles/fetus (HIV/VSV-G/MND/EGFP) and gene marking and expression was assessed in multiple tissues (cerebrum, trachea, esophagus, thymus, lung, heart, pericardium, thorax, lymph nodes, liver, spleen, pancreas, kidneys, adrenals, gonads, stomach, colon, diaphragm, omentum, peritoneum, bone marrow) using quantitative real-time PCR. Results were compared to lentiviral vector gene transfer using the intrahepatic (IH) (N=4) and intraportal (Ipo) (N=4) routes of administration. Analysis revealed that gene transfer at 40 days gestation resulted in high gene marking in omentum (18%), peritoneum (1%) and diaphragm (5%) with levels up to 400-fold higher than in other abdominal organs. The amount of gene expression highly correlated (P < 0.0001) with the extent of gene transduction within the peritoneal cavity. We obtained similar vector biodistribution at 60 days gestation, and no significant differences in the amount of gene transfer (P < 0.005) were found between the groups. However, gene transfer and expression at 40 versus 60 days gestation in nonabdominal tissues were 3 - 20 and 4 - 400 - fold higher, respectively. When comparing IH to IP gene delivery, a similar vector biodistribution was found with no significant differences on the extent of gene transfer (P < 0.05) in all tissues examined. In contrast, Ipo administration resulted in a reduction of vector spread and a 200-fold reduction in the level of gene marking when compared to the IP route of administration. In conclusion, definition of the conditions at the time of fetal transfer will further shape the feasibility and safety of this approach for the treatment of human disease.

The ER Repeat Protein Yt521-B Localizes to a Novel Subnuclear Compartment

The characterization of distinct subnuclear domains suggests a dynamic nuclear framework supporting gene expression and DNA replication. Here, we show that the glutamic acid/arginine-rich domain protein YT521-B localizes to a novel subnuclear structure, the YT bodies. YT bodies are dynamic compartments, which first appear at the beginning of S-phase in the cell cycle and disperse during mitosis. Furthermore, in untreated cells of the human cell line MCF7 they were undetectable and appeared only after drug- induced differentiation. YT bodies contain transcriptionally active sites and are in close contact to other subnuclear structures such as speckles and coiled bodies. YT bodies disperse upon actinomycin D treatment, whereas other transcriptional inhibitors such as alpha-amanitin or DRB have little effect. On the basis of our experiments, we propose that YT521-B may participate in the assembly of genes into transcription centers, thereby allowing efficient regulation of gene expression.

Synthetic oligonucleotides as RNA mimetics: 2'-modified Rnas and N3'-->P5' phosphoramidates.

Significant interest in synthetic DNA and RNA oligonucleotides and their analogues has marked the past two decades of research in chemistry and biochemistry. This attention was largely determined by the great potential of these compounds for various therapeutic applications such as antisense, antigene and ribozyme-based agents. Modified oligonucleotides have also become powerful molecular biological and biochemical research tools that allow fast and efficient regulation of gene expression and gene functions in vitro and in vivo. These applications in turn are based on the ability of the oligonucleotides to form highly sequence-specific complexes with nucleic acid targets of interest. This review summarizes recent advances in the design, synthesis, biochemical and structural properties of various RNA analogues. These comprise 3'-modified oligonucleotide N3'-->P5' phosphoramidates, analogues with modifications at the 2'-position of nucleoside sugar rings, or combinations of the two. Among the properties of the RNA minetics reviewed here are the thermal stability of their duplexes and triplexes, hydrolytic resistance to cellular nucleases and biological activity in in vitro and in vivo systems. In addition, key structural aspects of the complexes formed by the RNA analogues, including interaction with water molecules and ions, are analyzed and presented.

Effect of ligand and DNA binding on the interaction between human transcription intermediary factor 1alpha and estrogen receptors.

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. To achieve efficient regulation of gene expression, these receptors must interact with different type of molecules: 1) the steroid hormone, 2) the DNA response element, and 3) various proteins acting as transcriptional cofactors. In the present study, we have investigated how ligand and DNA binding influence the in vitro interaction between estrogen receptors (ERs) and the transcription intermediary factor hTIF1alpha (human transcriptional intermediary factor 1alpha). We first optimized conditions for the coactivator-dependent receptor ligand assay to lower ED50, and we then analyzed the ability of various natural and synthetic estrogens to allow the binding of the two types of proteins. Results were compared with the respective affinities of these ligands for the receptor. We then developed a protein-protein-DNA assay allowing the quantification of cofactor-ER-estrogen response element (ERE) complex formation in the presence of ligand and used measurements of fluorescence anisotropy to define the equilibrium binding parameters of the interaction. We demonstrated that the leucine-charged domain of hTIF1alpha is sufficient to interact with ERE-bound ERalpha in a ligand-dependent manner and showed that binding of ERalpha onto DNA does not significantly affect its hormone-dependent association with TIF1alpha. Finally, we show that, mainly in the absence of hormone, hTIF1alpha interacts better with ERbeta than with ERalpha independently of the presence of ERE.

Dividing the empire: boundary chromatin elements delimit the territory of enhancers.

Chromatin is the most complex supramolecular organization of the cell. It has a dual role: to compact the chromosomal DNA and to ensure a highly efficient regulation of gene expression. In consequence of the very compact packaging of the DNA, chromatin is highly repressive for transcription. Enhancers are key regulatory elements which can relieve the chromatin‐induced repression. The interplay between the chromatin‐mediated repression and the activating function of enhancers ensures that the difference between the induced or repressed level of gene expression in eukaryotic cells is several orders of magnitude higher than that in prokaryotic cells. The ability of enhancers to activate a gene independently of their distance from a promoter poses a topological problem: how is the territory of enhancer action delimited? It is supposed that the higher‐order chromatin structure divides the genome into topologically constrained loops which are attached to the nuclear matrix. This model implies that the loops are both structural and functional units of the chromatin in the sense that genes under common transcriptional regulation are located within the same loop and regulated by the same set of enhancers (Forrester et al ., 1986; Orkin, 1990; Bonifer et al ., 1991). In this attractive model, the monogamy of enhancers is ensured by the attachment of the bases of the chromatin loops to the nuclear matrix, which insulates the promoters located within the loop from the regulatory influence of enhancers present in the neighboring loops. Two distinct approaches have been applied to prove the validity of this model. In the structural approach, the main goals were to demonstrate that chromatin is indeed anchored at defined sites to a nuclear matrix, and to identify and characterize DNA sequences at these attachment sites. Structural studies have led to the discovery of scaffold attachment region (SAR) sequences (Mirkovitch et al., …

Efficient Regulation of Gene Expression by Adenovirus Vector-Mediated Delivery of the CRE Recombinase

We have constructed an E1-defective adenovirus (Ad) vector designated AdCAG-Cre containing the Cre recombinase gene derived from bacteriophage P1 under control of the cytomegalovirus immediate early enhancer-chicken β-actin hybrid (GAG) promoter. We examined the Cre-loxP-based recombination by this Ad vector in C2C12 cells bearing a reporter gene construct CAG-CAT-Z, which directs expression of the E. colilacZ gene upon Cre-mediated excision of the CAT gene located between the CAG promoter and the lacZ gene. Nearly 100% of these cells were shown to start to produce β-galactosidase after infection with the AdCAG-Cre vector at MOI 100. On the basis of this result, we discuss the possible use of the AdCAG-Cre vector to manipulate the gene expression in mammalian cells.