Search Results (59)

Gene delivery with cationic polymers requires balancing DNA compaction, colloidal stability, and intracellular release, yet for imine-linked low-molecular-weight polyethyleneimine (PEI) vectors, quantitative relationships connecting the N/P ratio with the full property–transfection cascade remain undefined. Here, two amphiphilic non-viral vectors were prepared by linking a hydrophobic benzene–siloxane core (TAS) to hyperbranched PEI (800 or 2000 Da) through reversible imine bonds and complexed with DNA across a broad N/P range (10–600). Polyplexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), ζ-potential, agarose gel electrophoresis, transfection via green fluorescent protein (GFP) imaging and luciferase assay in HeLa cells. Both vectors formed spherical nano-entities (AFM diameters ~30 nm for TAS-PEI800; ~100 nm for TAS-PEI2000). TAS-PEI2000 achieved complete DNA retardation at N/P ≈ 30 versus N/P ≈ 150 for TAS-PEI800, consistent with its higher charge density (ζ = +37.59 vs. +18.35 mV). Transfection efficiency was superior for TAS-PEI2000 across most N/P ratios; however, TAS-PEI2000 displayed an optimal transfection efficiency at N/P ≈ 100 (ζ ≈ 3.84 mV), beyond which efficiency declined, indicating a binding–release trade-off. Cell viability remained >77% across the N/P range for TAS-PEI800, but dropped below 25% at N/P ≥ 400 for TAS-PEI2000. A phenomenological logistic model identified characteristic transition thresholds (θ ≈ 60 for TAS-PEI800; θ ≈ 40 for TAS-PEI2000), capturing the onset of cooperative self-assembly; however, the post-optimum decline observed for TAS-PEI2000 requires additional inhibitory terms. These findings demonstrate that PEI molecular weight governs both the N/P threshold required for efficient transfection and the width of the therapeutic window, thereby providing structure–activity descriptors for the rational design of imine-linked polyplex systems. Full article

Erythropoiesis is tightly regulated by lineage-specific transcription factors that govern erythroid commitment, proliferation, and differentiation. A core erythroid transcriptional network, together with non-DNA-binding cofactors, occupies regulatory regions of genes essential for erythroid development. This process is further shaped by epigenetic mechanisms, including histone post-translational modifications and long-range chromatin interactions. CCCTC-binding factor (CTCF) is a multifunctional regulator with a central role in three-dimensional chromatin organization. Although CTCF has been implicated in hematopoietic differentiation and leukemogenesis, its specific function in erythropoiesis remains poorly defined. Here, we investigated the role of CTCF during erythroid differentiation using two complementary models: pluripotent K562 leukemia cells and primary human CD34⁺ hematopoietic stem/progenitor cells, each induced toward the erythroid lineage by distinct stimuli. In both systems, CTCF silencing impaired erythroid differentiation by repression of key erythroid transcription factor genes, including LMO2, KLF1, MYB, and ETS1. This repression was associated with enrichment of repressive histone marks at CTCF-binding sites within their regulatory regions. Moreover, CTCF cooperated with cohesin to establish and stabilize long-range chromatin interactions at these loci. These results provide new insight into how CTCF-dependent chromatin regulation contributes to normal erythroid development and suggest that perturbation of this regulatory axis may have implications for hematopoietic disorders and malignancies. Full article

Cyclic GMP-AMP synthase (cGAS) functions as a DNA sensor in the cytoplasm, triggering immune responses, but it is also translocated to the nucleus, where it is kept catalytically inactive. It consists of an unstructured N-terminal domain of around 160 amino acids, and a larger C-terminal fold comprising the catalytic and DNA-binding domains. Subcellular localization of cGAS is thought to play a key role in its regulation. Here, we make use of heterologous expression in the eukaryotic model Saccharomyces cerevisiae to study cGAS localization in a neutral cellular environment. cGAS-eGFP was mostly found in aggregates at the endoplasmic reticulum–mitochondria encounter structure (ERMES) and juxtanuclear protein quality compartments (JUNQs), although some cells displayed an association between cGAS-eGFP and the plasma membrane. The N-terminus of cGAS fused to eGFP was unable to associate with the plasma membrane by itself, but its deletion dramatically promoted nuclear localization of cGAS-eGFP and decreased cytoplasmic aggregates. A mutant in the DNA-binding Zn-thumb motif of cGAS also showed a more prominent nuclear localization. Thus, both the N-terminal and C-terminal domains of cGAS seem to cooperate to prevent nuclear localization and to maintain cytoplasmic reservoirs of the protein. Heterologous cGAS expression in yeast is a valuable tool for modeling aspects of its subcellular localization and aggregative features. Full article

DprA (also known as Smf) is a conserved RecA mediator originally characterized by its role in natural chromosomal transformation, yet its widespread presence across bacteria hints at broader DNA metabolic functions. Here, we demonstrate that Bacillus subtilis DprA enhances the frequency of Escherichia coli Hfr conjugation in vivo. In vitro, RecA·ATP binds and cooperatively polymerizes in a 50-nucleotide (nt) polydeoxy T (dT)₅₀ ssDNA to form dynamic filaments that SSB inhibits, an effect fully reversed by Bacillus subtilis DprA. Escherichia coli RecA bound to (dT)₂₁ exhibits minimal dATPase activity, but the addition of B. subtilis DprA significantly stimulates RecA dATP hydrolysis. B. subtilis RecA·dATP readily assembles on (dT)₂₀ complexes, and DprA allosterically activates RecA on even shorter (dT)₁₅ substrates. Combining biochemical assays with a fully atomic model of the RecA–DprA–ssDNA complex, we proposed that only one DNA binding site of the DprA dimer engages the ssDNA during RecA loading, owing to steric constraints. This work refines the mechanism of DprA-mediated RecA nucleation and defines the minimal ssDNA footprint required for mediator activity. Full article

Transcription factors (TFs) are the main regulators of eukaryotic gene expression. The cooperative binding of at least two TFs to genomic DNA is a major mechanism of transcription regulation. Massive analysis of the co-occurrence of overrepresented pairs of motifs for different target TFs studied in ChIP-seq experiments can clarify the mechanisms of TF cooperation. We categorized the target TFs from M. musculus ChIP-seq and A. thaliana ChIP-seq/DAP-seq experiments according to the structure of their DNA-binding domains (DBDs) into classes. We studied homotypic pairs of motifs, using the same recognition model for each motif. Asymmetric and symmetric pairs consist of motifs of remote and close recognition scores. We found that asymmetric pairs of motifs predominate for all TF classes. TFs from the murine/plant ‘Basic helix–loop–helix (bHLH)’, ‘Basic leucine zipper (bZIP)’, and ‘Tryptophan cluster’ classes and murine ‘p53 domain’ and ‘Rel homology region’ classes showed the highest enrichment of asymmetric homotypic pairs of motifs. Pioneer TFs, despite their DBD types, have a higher significance of asymmetry within homotypic pairs of motifs compared to other TFs. Asymmetry within homotypic CEs is a promising new feature decrypting the mechanisms of gene transcription regulation. Full article

Mycobacteriophages are viruses that specifically infect bacterial species within the genera Mycobacterium and Mycolicibacterium. Over 2400 mycobacteriophages have been isolated on the host Mycolicibacterium smegmatis and sequenced. This wealth of genomic data indicates that mycobacteriophage genomes are diverse, mosaic, and contain numerous (35–60%) genes for which there is no predicted function based on sequence similarity to characterized orthologs, many of which are essential to lytic growth. To fully understand the molecular aspects of mycobacteriophage–host interactions, it is paramount to investigate the function of these genes and gene products. Here we show that the temperate mycobacteriophage, Alexphander, makes stable lysogens with a frequency of 2.8%. Alexphander gene 94 is essential for lytic infection and encodes a protein predicted to contain a C-terminal MerR family helix–turn–helix DNA-binding motif (HTH) and an N-terminal DinB/YfiT motif, a putative metal-binding motif found in stress-inducible gene products. Full-length and C-terminal gp94 constructs form high-order nucleoprotein complexes on 100–500 base pair double-stranded DNA fragments and full-length phage genomic DNA with little sequence discrimination for the DNA fragments tested. Maximum gene 94 mRNA levels are observed late in the lytic growth cycle, and gene 94 is transcribed in a message with neighboring genes 92 through 96. We hypothesize that gp94 is an essential DNA-binding protein for Alexphander during lytic growth. We proposed that gp94 forms multiprotein complexes on DNA through cooperative interactions involving its HTH DNA-binding motif at sites throughout the phage chromosome, facilitating essential DNA transactions required for lytic propagation. Full article

Glutathione transferases (GSTs) are the primary catalysts protecting from reactive electrophile attack. In this review, the quantitative levels and distribution of glutathione transferases in relation to physiological function are discussed. The catalytic properties (random sequential) tell us that these enzymes have evolved to intercept reactive intermediates. High concentrations of enzymes (up to several hundred micromolar) ensure efficient protection. Individual enzyme molecules, however, turn over only rarely (estimated as low as once daily). The protection of intracellular protein and DNA targets is linearly proportional to enzyme levels. Any lowering of enzyme concentration, or inhibition, would thus result in diminished protection. It is well established that GSTs also function as binding proteins, potentially resulting in enzyme inhibition. Here the relevance of ligand inhibition and catalytic mechanisms, such as negative co-operativity, is discussed. There is a lack of knowledge pertaining to relevant ligand levels in vivo, be they exogenous or endogenous (e.g., bile acids and bilirubin). The stoichiometry of active sites in GSTs is well established, cytosolic enzyme dimers have two sites. It is puzzling that a third of the site’s reactivity is observed in trimeric microsomal glutathione transferases (MGSTs). From a physiological point of view, such sub-stoichiometric behavior would appear to be wasteful. Over the years, a substantial amount of detailed knowledge on the structure, distribution, and mechanism of purified GSTs has been gathered. We still lack knowledge on exact cell type distribution and levels in vivo however, especially in relation to ligand levels, which need to be determined. Such knowledge must be gathered in order to allow mathematical modeling to be employed in the future, to generate a holistic understanding of reactive intermediate protection. Full article

A robust predictive model was developed using 136 novel peroxisome proliferator-activated receptor delta (PPARδ) agonists, a distinct subtype of lipid-activated transcription factors of the nuclear receptor superfamily that regulate target genes by binding to characteristic sequences of DNA bases. The model employs various structural descriptors and docking calculations and provides predictions of the biological activity of PPARδ agonists, following the criteria of the Organization for Economic Co-operation and Development (OECD) for the development and validation of quantitative structure–activity relationship (QSAR) models. Specifically focused on small molecules, the model facilitates the identification of highly potent and selective PPARδ agonists and offers a read-across concept by providing the chemical neighbours of the compound under study. The model development process was conducted on Isalos Analytics Software (v. 0.1.17) which provides an intuitive environment for machine-learning applications. The final model was released as a user-friendly web tool and can be accessed through the Enalos Cloud platform’s graphical user interface (GUI). Full article

Characterization of gene regulatory mechanisms in cancer is a key task in cancer genomics. CCCTC-binding factor (CTCF), a DNA binding protein, exhibits specific binding patterns in the genome of cancer cells and has a non-canonical function to facilitate oncogenic transcription programs by cooperating with transcription factors bound at flanking distal regions. Identification of DNA sequence features from a broad genomic region that distinguish cancer-specific CTCF binding sites from regular CTCF binding sites can help find oncogenic transcription factors in a cancer type. However, the presence of long DNA sequences without localization information makes it difficult to perform conventional motif analysis. Here, we present DNAResDualNet (DARDN), a computational method that utilizes convolutional neural networks (CNNs) for predicting cancer-specific CTCF binding sites from long DNA sequences and employs DeepLIFT, a method for interpretability of deep learning models that explains the model’s output in terms of the contributions of its input features. The method is used for identifying DNA sequence features associated with cancer-specific CTCF binding. Evaluation on DNA sequences associated with CTCF binding sites in T-cell acute lymphoblastic leukemia (T-ALL) and other cancer types demonstrates DARDN’s ability in classifying DNA sequences surrounding cancer-specific CTCF binding from control constitutive CTCF binding and identifying sequence motifs for transcription factors potentially active in each specific cancer type. We identify potential oncogenic transcription factors in T-ALL, acute myeloid leukemia (AML), breast cancer (BRCA), colorectal cancer (CRC), lung adenocarcinoma (LUAD), and prostate cancer (PRAD). Our work demonstrates the power of advanced machine learning and feature discovery approach in finding biologically meaningful information from complex high-throughput sequencing data. Full article

Seryl-tRNA synthetases (SerRSs), members of the aminoacyl-tRNA synthetase family, interact with diverse proteins, enabling SerRSs to enhance their role in the translation of the genetic message or to perform alternative functions in cellular processes beyond translation. Atypical archaeal SerRS interacts with arginyl-tRNA synthetase and proteins of the ribosomal P-stalk to optimize translation through tRNA channeling. The complex between yeast SerRS and peroxin Pex21p provides a connection between translation and peroxisome function. The partnership between Arabidopsis SerRS and BEN1 indicates a link between translation and brassinosteroid metabolism and may be relevant in plant stress response mechanisms. In Drosophila, the unusual heterodimeric mitochondrial SerRS coordinates mitochondrial translation and replication via interaction with LON protease. Evolutionarily conserved interactions of yeast and human SerRSs with m³C32 tRNA methyltransferases indicate coordination between tRNA modification and aminoacylation in the cytosol and mitochondria. Human cytosolic SerRS is a cellular hub protein connecting translation to vascular development, angiogenesis, lipogenesis, and telomere maintenance. When translocated to the nucleus, SerRS acts as a master negative regulator of VEGFA gene expression. SerRS alone or in complex with YY1 and SIRT2 competes with activating transcription factors NFκB1 and c-Myc, resulting in balanced VEGFA expression important for proper vascular development and angiogenesis. In hypoxia, SerRS phosphorylation diminishes its binding to the VEGFA promoter, while the lack of nutrients triggers SerRS glycosylation, reducing its nuclear localization. Additionally, SerRS binds telomeric DNA and cooperates with the shelterin protein POT1 to regulate telomere length and cellular senescence. As an antitumor and antiangiogenic factor, human cytosolic SerRS appears to be a promising drug target and therapeutic agent for treating cancer, cardiovascular diseases, and possibly obesity and aging. Full article

C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT/enhancer-binding protein (C/EBP) family. CHOP expression is induced by unfolded protein response (UPR), and sustained CHOP activation acts as a pivotal trigger for ER stress-induced apoptosis. MicroRNA-616 is located within an intron of the CHOP gene. However, the regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. Here we show that the expression of miR-616 and CHOP (host gene of miR-616) is downregulated in human breast cancer. Both miR-5p/-3p arms of miR-616 are expressed with levels of the 5p arm higher than the 3p arm. During conditions of ER stress, the expression of miR-616-5p and miR-616-3p arms was concordantly increased primarily through the PERK pathway. Our results show that ectopic expression of miR-616 significantly suppressed cell proliferation and colony formation, whereas knockout of miR-616 increased it. We found that miR-616 represses c-MYC expression via binding sites located in its protein coding region. Furthermore, we show that miR-616 exerted growth inhibitory effects on cells by suppressing c-MYC expression. Our results establish a new role for the CHOP locus by providing evidence that miR-616 can inhibit cell proliferation by targeting c-MYC. In summary, our results suggest a dual function for the CHOP locus, where CHOP protein and miR-616 can cooperate to inhibit cancer progression. Full article

Aldosterone and cortisol serve important roles in the pathogenesis of cardiovascular diseases and metabolic disorders. Epigenetics is a mechanism to control enzyme expression by genes without changing the gene sequence. Steroid hormone synthase gene expression is regulated by transcription factors specific to each gene, and methylation has been reported to be involved in steroid hormone production and disease. Angiotensin II or potassium regulates the aldosterone synthase gene, CYP11B2. The adrenocorticotropic hormone controls the 11b-hydroxylase, CYP11B1. DNA methylation negatively controls the CYP11B2 and CYP11B1 expression and dynamically changes the expression responsive to continuous stimulation of the promoter gene. Hypomethylation status of the CYP11B2 promoter region is seen in aldosterone-producing adenomas. Methylation of recognition sites of transcription factors, including cyclic AMP responsive element binding protein 1 or nerve growth factor-induced clone B, diminish their DNA-binding activity. A methyl-CpG-binding protein 2 cooperates directly with the methylated CpG dinucleotides of CYP11B2. A low-salt diet, treatment with angiotensin II, and potassium increase the CYP11B2 mRNA levels and induce DNA hypomethylation in the adrenal gland. A close association between a low DNA methylation ratio and an increased CYP11B1 expression is seen in Cushing’s adenoma and aldosterone-producing adenoma with autonomous cortisol secretion. Epigenetic control of CYP11B2 or CYP11B1 plays an important role in autonomic aldosterone or cortisol synthesis. Full article

Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis. Full article

Ag⁺-bridged T-Ag⁺-T was recently discovered in a Ag⁺-DNA nanowire crystal, but it was reported that Ag⁺ had little to no affinity to T nucleobases and T-rich oligonucleotides in solution. Therefore, the binding mode for the formation of this type of novel metallo base pair in solution is elusive. Herein, we demonstrate that Ag⁺ can interact with polyT oligonucleotides once the concentration of Ag⁺ in solution exceeds a threshold value. The threshold value is independent of the concentration of the polyT oligonucleotide but is inversely proportional to the length of the polyT oligonucleotide. The polyT oligonucleotides are intramolecularly folded due to their positively cooperative formation and the stack of T-Ag⁺-T base pairs, resulting in the 5′- and 3′-ends being in close proximity to each other. The intramolecular Ag⁺-folded polyT oligonucleotide has a higher thermal stability than the duplex and can be reversibly modulated by cysteine. Full article

An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and ND WT for binding a consensus DNA sequence is −3.0 kcal/mol at physiological ionic strength. ΔΔG increased to −1.03 kcal/mol when acidic residues in TAD2 were changed to alanine (ND DE) and to −1.13 kcal/mol when all the nonpolar residues, including W53/F54, were changed to alanine (ND NP). These results indicate there is some cooperation between acidic, nonpolar, and aromatic residues from TAD2 to inhibit DNA binding. The dependence of DNA binding affinity on ionic strength was used to predict excess counterion release for binding both consensus and scrambled DNA sequences, which was smaller for ND WT and ND NP with consensus DNA and smaller for scrambled DNA overall. Using size exclusion chromatography, we show that the ND mutants have similar Stokes radii to ND WT suggesting the mutants disrupt autoinhibition without changing the global structure. Full article