Transcriptional Regulatory System Research Articles

The Whole genome sequence for Acinetobacter baumannii strain in Iraqi Hospitals Outbreak

A study was conducted to identify specialized F plasmid genes and regulatory systems in Acinetobacter baumannii. These genes could be very important for the capacity of these species to coexist with the human host and show a wide range of diversity in genes that contribute to antibiotic resistance. Identification of 50A. baumannii isolates was carried out through morphology and culture on CHROM agar and genotypic identification was performed using blaOxa-51 gene. Out of 50, one AB isolate was chosen for whole genome sequencing (WGS) using Illumina MiSeq technology. The analysis identified specialized genes in these isolates that contribute to antibiotic stress and lipopolysaccharide barrier, including complex sets of partial and complete integrons and transposons. The recent findings, transferring of A. baumannii plasmid genes are inducing under the regulatory system to which the plasmid elements confer Adaptation such as F-plasmid genes transfer in A.baumannii induced by antibiotic stress under the control of transcriptional factor regulation system in order to antimicrobial drug resistance. The selected A. baumannii isolate, IS1- A.baumannii 32 contain five global regulator systems .Our study were provided detailed genomic picture in detected both innate and acquired plasmid-encoded AMR genes. Study aim is the role of genetic regulation to control the transcription, virulence factors and antibiotic resistance related mechanism in A.baumannii .

Regulator DegU can remarkably influence alkaline protease AprE biosynthesis in Bacillus licheniformis 2709

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5′-TAAAT……AAAAT…….AACAT…TAAAA-3′) located upstream −91 to −87 bp, −101 to −97 bp, −195 to −191 bp, −215 to −211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism.

The Competing Endogenous RNAs Regulatory Genes Network Mediates Leaf Shape Variation and Main Effector Gene Function in Mulberry Plant (Morus alba)

Mulberry plants (Morus alba) have leaf shapes, ranging from unlobed to lobed, which are crucial for yield, growth, and adaptability, indicating their ability to adapt to their environment. Competing endogenous RNAs (ceRNAs) constitute a web of RNAs within the organism’s transcriptional regulatory system, including protein-coding genes (mRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and others. In this study, samples for ceRNA sequencing were categorized into two groups: whole leaves and lobed leaves, each group with three replicates. In addition, we isolated, cloned, and characterized the precursor miRNA (miR156x) from the leaves of M. alba. miR156x precursor had a length of 107 base pairs and a minimum folding free energy of 50.27 kcal/mol. We constructed a pCAMBIA-35S-GUS-miR156x dual overexpression vector and established a transient transformation system for mulberry. At an optimal transformation solution (OD600 = 0.7), the GUS gene showed a higher expression in the leaves of transiently transformed mulberry with miR156x overexpression, four days after transformation, while the target genes of miR156x had decreased expression in the same leaves. Investigations into the transgenic mulberry plants uncovered various modifications to physio-chemical parameters including POD, SOD, PRO, MDA, soluble proteins and sugars, and chlorophyl content. miRNAs in the plants were found to act as negative regulators of gene expression in response to changes in leaf shape regulation, which was confirmed in vitro using dual-luciferase reporter assays. Subsequently, we cloned Maspl3 in vitro and conducted GST-Pull down assays, obtaining multiple proteins that interacted with the Maspl3 gene. This indicates that the miR156x/Maspl3/MSTRG.25812.1 regulatory module contributes to the differences in mulberry leaf shape.

Construction and Validation of a CRISPR/dCpf1-Based Transcriptional Regulatory System in Saccharomyces Cerevisiae.

Saccharomyces cerevisiae has been extensively studied and applied as a model microbial platform for efficient and sustainable production of biofuels, chemicals and natural products. In recent years, the application of CRISPR system in genome editing and transcriptional regulation has greatly improved the efficiency of microbial genome editing. To achieve transcriptional regulation the CRISPR/dCpf1-mediated CRISPRa/i transcriptional regulation system was constructed in this study. By constructing crRNA arrays and adding different activation proteins, repression efficiencies from 62% to 177% for the CRISPRi system and activation efficiencies from 154% to 320% for the CRISPRa system were achieved in the eGFP gene reporter system.

RuX: A Novel, Flexible, and Sensitive Mifepristone-Induced Transcriptional Regulation System.

Inducible gene regulation methods are indispensable in diverse biological applications, yet many of them have severe limitations in their applicability. These include inducer toxicity, a limited variety of organisms the given system can be used in, and side effects of the induction method. In this study, a novel inducible system, the RuX system, was created using a mutant ligand-binding domain of the glucocorticoid receptor (CS1/CD), used together with various genetic elements such as the Gal4 DNA-binding domain or Cre recombinase. The RuX system is shown to be capable of over 1000-fold inducibility, has flexible applications, and is offered for use in cell cultures.

Comparative genomics sheds light on transcription factor-mediated regulation in the extreme acidophilic Acidithiobacillia representatives

Extreme acidophiles thrive in acidic environments, confront a multitude of challenges, and demonstrate remarkable adaptability in their metabolism to cope with the ever-changing environmental fluctuations, which encompass variations in temperature, pH levels, and the availability of electron acceptors and donors. The survival and proliferation of members within the Acidithiobacillia class rely on the deployment of transcriptional regulatory systems linked to essential physiological traits. The study of these transcriptional regulatory systems provides valuable insights into critical processes, such as energy metabolism and nutrient assimilation, and how they integrate into major genetic-metabolic circuits. In this study, we examined the transcriptional regulatory repertoires and potential interactions of forty-three Acidithiobacillia complete and draft genomes, encompassing nine species. To investigate the function and diversity of Transcription Factors (TFs) and their DNA Binding Sites (DBSs), we conducted a genome-wide comparative analysis, which allowed us to identify these regulatory elements in representatives of Acidithiobacillia. We classified TFs into gene families and compared their occurrence among all representatives, revealing conservation patterns across the class. The results identified conserved regulators for several pathways, including iron and sulfur oxidation, the main pathways for energy acquisition, providing new evidence for viable regulatory interactions and branch-specific conservation in Acidithiobacillia. The identification of TFs and DBSs not only corroborates existing experimental information for selected species, but also introduces novel candidates for experimental validation. Moreover, these promising candidates have the potential for further extension to new representatives within the class.

Genome editing for biodiesel production in oleaginous microalga, Nannochloropsis species

Algae are oxygen-producing photosynthetic aquatic organisms. Algal biodiesels have attracted attention because these fuels are produced via photosynthesis, which assimilates CO2. Algal biodiesels are sustainable, not competitive with food production, and have higher productivity compared with terrestrial plants. However, the production costs of algal biodiesels are much higher than those of fossil fuels. Therefore, improvement of algal lipid productivity is essential for the practical use of algal biodiesels. To achieve this, the application of genome-editing systems for the molecular breeding of algae is expected to generate “high-performance algae.” Here, we review the genome-editing technologies developed for the oleaginous microalgae, Nannochloropsis species, which are the most promising algae for producing algal-biodiesel feedstock. In this review, we discuss the development of genome-editing systems for gene disruption, transgene-free genome-editing systems, transcriptional regulation systems using nuclease-deficient Cas proteins, and the applications of genome editing in Nannochloropsis species.

G-quadruplexes in the evolution of hepatitis B virus.

Hepatitis B virus (HBV) is one of the most dangerous human pathogenic viruses found in all corners of the world. Recent sequencing of ancient HBV viruses revealed that these viruses have accompanied humanity for several millenia. As G-quadruplexes are considered to be potential therapeutic targets in virology, we examined G-quadruplex-forming sequences (PQS) in modern and ancient HBV genomes. Our analyses showed the presence of PQS in all 232 tested HBV genomes, with a total number of 1258 motifs and an average frequency of 1.69 PQS per kbp. Notably, the PQS with the highest G4Hunter score in the reference genome is the most highly conserved. Interestingly, the density of PQS motifs is lower in ancient HBV genomes than in their modern counterparts (1.5 and 1.9/kb, respectively). This modern frequency of 1.90 is very close to the PQS frequency of the human genome (1.93) using identical parameters. This indicates that the PQS content in HBV increased over time to become closer to the PQS frequency in the human genome. No statistically significant differences were found between PQS densities in HBV lineages found in different continents. These results, which constitute the first paleogenomics analysis of G4 propensity, are in agreement with our hypothesis that, for viruses causing chronic infections, their PQS frequencies tend to converge evolutionarily with those of their hosts, as a kind of 'genetic camouflage' to both hijack host cell transcriptional regulatory systems and to avoid recognition as foreign material.

Transcriptional activation of ompA in Neisseria gonorrhoeae mediated by the XRE family member protein NceR.

Increasing antibiotic resistance of Neisseria gonorrhoeae, the causative agent of gonorrhea, is a growing global concern that has renewed vaccine development efforts. The gonococcal OmpA protein was previously identified as a vaccine candidate due to its surface exposure, conservation, stable expression, and involvement in host-cell interactions. We previously demonstrated that the transcription of ompA can be activated by the MisR/MisS two-component system. Interestingly, earlier work suggested that the availability of free iron also influences ompA expression, which we confirmed in this study. In the present study, we found that iron regulation of ompA was independent of MisR and searched for additional regulators. A DNA pull-down assay with the ompA promoter from gonococcal lysates obtained from bacteria grown in the presence or absence of iron identified an XRE (Xenobiotic Response Element) family member protein encoded by NGO1982. We found that an NGO1982 null mutant of N. gonorrhoeae strain FA19 displayed a reduced level of ompA expression compared to the wild-type (WT) parent strain. Given this regulation, and the capacity of this XRE-like protein to regulate a gene involved in peptidoglycan biosynthesis (ltgA), along with its presence in other Neisseria sp., we termed the NGO1982-encoded protein as NceR (Neisseria cell envelope regulator). Critically, results from DNA-binding studies indicated that NceR regulates ompA through a direct mechanism. Thus, ompA expression is subject to both iron-dependent (NceR) and -independent (MisR/MisS) pathways. Hence, levels of the vaccine antigen candidate OmpA in circulating gonococcal strains could be influenced by transcriptional regulatory systems and the availability of iron. IMPORTANCE Herein, we report that the gene encoding a conserved gonococcal surface-exposed vaccine candidate (OmpA) is activated by a heretofore undescribed XRE family transcription factor, which we term NceR. We report that NceR regulation of ompA expression in N. gonorrhoeae is mediated by an iron-dependent mechanism, while the previously described MisR regulatory system is iron-independent. Our study highlights the importance of defining the complexity of coordinated genetic and physiologic systems that regulate genes encoding vaccine candidates to better understand their availability during infection.

Bioinformatics Analysis of the Molecular Networks Associated with the Amelioration of Aberrant Gene Expression by a Tyr-Trp Dipeptide in Brains Treated with the Amyloid-β Peptide.

Short-chain peptides derived from various protein sources have been shown to exhibit diverse bio-modulatory and health-promoting effects in animal experiments and human trials. We recently reported that the oral administration of the Tyr-Trp (YW) dipeptide to mice markedly enhances noradrenaline metabolism in the brain and ameliorates the working-memory deficits induced by the β-amyloid 25-35 peptide (Aβ25-35). In the current study, we performed multiple bioinformatics analyses of microarray data from Aβ25-35/YW-treated brains to determine the mechanism underlying the action of YW in the brain and to infer the molecular mechanisms and networks involved in the protective effect of YW in the brain. We found that YW not only reversed inflammation-related responses but also activated various molecular networks involving a transcriptional regulatory system, which is mediated by the CREB binding protein (CBP), EGR-family proteins, ELK1, and PPAR, and the calcium-signaling pathway, oxidative stress tolerance, and an enzyme involved in de novo l-serine synthesis in brains treated with Aβ25-35. This study revealed that YW has a neuroprotective effect against Aβ25-35 neuropathy, suggesting that YW is a new functional-food-material peptide.

The role of replication-induced chromosomal copy numbers in spatio-temporal gene regulation and evolutionary chromosome plasticity.

For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but on replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in Escherichia coli. Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen Dickeya dadantii and the biotechnologically relevant organism Vibrio natriegens. The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. A further extension to the bacterial kingdom indicates that chromosome evolution is governed by growth rate related transient copy numbers.

Characterization of radiation-resistance mechanism in Spirosoma montaniterrae DY10T in terms of transcriptional regulatory system

To respond to the external environmental changes for survival, bacteria regulates expression of a number of genes including transcription factors (TFs). To characterize complex biological phenomena, a biological system-level approach is necessary. Here we utilized six computational biology methods to infer regulatory network and to characterize underlying biologically mechanisms relevant to radiation-resistance. In particular, we inferred gene regulatory network (GRN) and operons of radiation-resistance bacterium Spirosoma montaniterrae DY10^T and identified the major regulators for radiation-resistance. Our results showed that DNA repair and reactive oxygen species (ROS) scavenging mechanisms are key processes and Crp/Fnr family transcriptional regulator works as a master regulatory TF in early response to radiation.

Combinatorial protein dimerization enables precise multi-input synthetic computations

Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein–protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.

Live cell single molecule imaging reveals concentration dependent changes to dynamic behavior of nuclear receptors RAR and RXR.

Type 2 Nuclear Receptors (T2NRs) like Retinoic Acid Receptor (RAR) are believed to require heterodimerization with a common factor, the Retinoid X Receptor (RXR), to bind chromatin and regulate its target genes. Here, we deploy stroboscopic photo-activation SPT (spaSPT) to understand the molecular dynamics of the RAR/RXR system and infer different diffusion states acquired by RAR and RXR in the nucleoplasm. These states occupied by RAR and RXR presumably exist in dynamic equilibrium. Next, we ask in a complex heterodimer driven transcriptional regulatory system such as this, how is the dynamic equilibrium shifted when either of the heterodimeric partner is in excess. We find that in live cells, increasing the number of RAR molecules and not RXR molecules leads to altered chromatin binding of RAR. While increasing the number of molecules of RAR and RXR both alter the chromatin binding of RXR. Since pharmacological modulation of the RAR/RXR system is an important avenue for cancer treatments, this study constitutes a step toward understanding how perturbations of this system affect chromatin binding and possibly downstream transcriptional regulatory function.

Stochastic resonance in gene transcriptional regulatory system driven by Gaussian noise and Lévy noise

The impacts of noise parameters and input signal parameters on stochastic resonance are explored in a gene transcriptional regulatory model driven by a combination of multiplicative Gaussian noise and additive Lévy noise. The response time series and signal-to-noise ratios are obtained by numerical simulations to quantify the occurrence of stochastic resonance. It is shown that the Gaussian noise intensity can cause the stochastic resonance, while the Lévy noise intensity can cause both the stochastic resonance and inverse stochastic resonance. The increases of Gaussian noise intensity inhibits these two phenomena and enhances system stability. The changes of the Lévy noise intensity parameter can cause the system to switch between low and high concentration state. In addition, the stability index and skewness parameter of Lévy noise can also result in stochastic resonance. Therefore, the optimum stochastic resonance can be achieved and useful genetic information can be easily acquired by adjusting the noise parameters. Our findings should contribute in the selection of appropriate parameters to achieve stochastic resonance in gene transcriptional regulation models, laying the foundation for the selection of stochastic resonance parameter ranges in actual gene transcriptional engineering.

A dual-regulation inducible switch system for microRNA detection and cell type-specific gene activation.

Rationale: MicroRNAs (miRNAs) play key roles in multiple biological processes, many of which exhibit distinct cell type-specific expression patterns. A miRNA-inducible expression system can be adapted as a signal-on reporter for detecting miRNA activity or as a cell type-specific gene activation tool. However, due to the inhibitory properties of miRNAs on gene expression, few miRNA-inducible expression systems are available, and the available systems are only transcriptional or post-transcriptional regulatory system with obvious leaky expression. Methods: To address this limitation, a miRNA-inducible expression system that can tightly control target gene expression is desirable. Here, by taking advantage of an enhanced LacI repression system and the translational repressor L7Ae, a miRNA-inducible dual transcriptional-translational switch system was designed called the miR-ON-D system. Luciferase activity assay, western blotting, CCK-8 assay and flow cytometry analysis were performed to characterize and validate this system. Results: The results demonstrated that leakage expression was strongly suppressed in the miR-ON-D system. It was also validated that the miR-ON-D system could be used to detect exogenous and endogenous miRNAs in mammalian cells. Moreover, it was shown that the miR-ON-D system could be triggered by cell type-specific miRNAs to regulate the expression of biologically relevant proteins (e.g., p21 and Bax) to achieve cell type-specific reprogramming. Conclusion: This study established a tight miRNA-inducible expression switch system for miRNA detection and cell type-specific gene activation.

Regulon: An overview of plant abiotic stress transcriptional regulatory system and role in transgenic plants.

Population growth is increasing rapidly around the world, in these consequences we need to produce more foods to full fill the demand of increased population. The world is facing global warming due to urbanizations and industrialization and in this concerns plants exposed continuously to abiotic stresses which is a major cause of crop hammering every year. Abiotic stresses consist of Drought, Salt, Heat, Cold, Oxidative and Metal toxicity which damage the crop yield continuously. Drought and salinity stress severally affected in similar manner to plant and the leading cause of reduction in crop yield. Plants respond to various stimuli under abiotic or biotic stress condition and express certain genes either structural or regulatory genes which maintain the plant integrity. The regulatory genes primarily the transcription factors that exert their activity by binding to certain cis DNA elements and consequently either up regulated or down regulate to target expression. These transcription factors are known as masters regulators because its single transcript regulate more than one gene, in this context the regulon word is fascinating more in compass of transcription factors. Progress has been made to better understand about effect of regulons (AREB/ABF, DREB, MYB, and NAC) under abiotic stresses and a number of regulons reported for stress responsive and used as a better transgenic tool of Arabidopsis and Rice.

Heat-Induced Conformational Transition Mechanism of Heat Shock Factor 1 Investigated by Tryptophan Probe.

A transcriptional regulatory system called heat shock response (HSR) has been developed in eukaryotic cells to maintain proteome homeostasis under various stresses. Heat shock factor-1 (Hsf1) plays a central role in HSR, mainly by upregulating molecular chaperones as a transcription factor. Hsf1 forms a complex with chaperones and exists as a monomer in the resting state under normal conditions. However, upon heat shock, Hsf1 is activated by oligomerization. Thus, oligomerization of Hsf1 is considered an important step in HSR. However, the lack of information about Hsf1 monomer structure in the resting state, as well as the structural change via oligomerization at heat response, impeded the understanding of the thermosensing mechanism through oligomerization. In this study, we applied solution biophysical methods, including fluorescence spectroscopy, nuclear magnetic resonance, and circular dichroism spectroscopy, to investigate the heat-induced conformational transition mechanism of Hsf1 leading to oligomerization. Our study showed that Hsf1 forms an inactive closed conformation mediated by intramolecular contact between leucine zippers (LZs), in which the intermolecular contact between the LZs for oligomerization is prevented. As the temperature increases, Hsf1 changes to an open conformation, where the intramolecular LZ interaction is dissolved so that the LZs can form intermolecular contacts to form oligomers in the active form. Furthermore, since the interaction sites with molecular chaperones and nuclear transporters are also expected to be exposed in the open conformation, the conformational change to the open state can lead to understanding the regulation of Hsf1-mediated stress response through interaction with multiple cellular components.

Establishment of a human embryonic stem cell line, WAe009-A-99, with constitutive expression of the dCas9-p300 fusion protein

CRISPR/Cas9-based transcriptional regulation systems can induce the site-specific activation or repression of endogenous genes. p300 is a transcriptional co-activator that functions as a histone acetyltransferase that regulates gene transcription via chromatin remodeling. Here, we generated a human embryonic stem cell line stably expressing catalytically dead Cas9 (dCas9) fused to the catalytic core domain of human p300 via lentiviral transduction. This cell line can be used for locus-specific histone acetylation in combination with guide RNAs, and is a valuable tool for gene regulation in stem cell research.

ER Stress and the Unfolded Protein Response: Homeostatic Regulation Coordinate Plant Survival and Growth

The endoplasmic reticulum (ER), a eukaryotic organelle, is the major site of protein biosynthesis. The disturbance of ER function by biotic or abiotic stress triggers the accumulation of misfolded or unfolded proteins in the ER. The unfolded protein response (UPR) is the best-studied ER stress response. This transcriptional regulatory system senses ER stress, activates downstream genes that function to mitigate stress, and restores homeostasis. In addition to its conventional role in stress responses, recent reports indicate that the UPR is involved in plant growth and development. In this review, we summarize the current knowledge of ER stress sensing and the activation and downstream regulation of the UPR. We also describe how the UPR modulates both plant growth and stress tolerance by maintaining ER homeostasis. Lastly, we propose that the UPR is a major component of the machinery that balances the trade-off between plant growth and survival in a dynamic environment.