Том 58, № 5 (2024)

Age-associated transformation of the methylation pattern is considered to be an important predictor of human biological age. Changes in the level of CpG-dinucleotide methylation contribute to a shift in the function of a number of genes, including those associated with the functioning of the immune system. One of such gene is CSF1. The protein product of this gene is associated with inflammatory aging, making it an important biomarker of age-related diseases. We studied the methylation profile of the promoter-associated CpG island of the CSF1 gene by MALDI-TOF mass spectrometry. The dependences between the character of CpG-site methylation within the investigated regions and the relative level of the gene mRNA and its protein product in people of different age groups were searched. For two CpG sites, a high level of correlation with the studied parameters is shown. A search for the landing sites of transcription factor binding sites associated with gene transcription showed that these CpG dinucleotides are part of motifs for the NFI family transcription factors and the EGR1 factor. We hypothesize that these CpG sites play an important role in the regulation of CSF1 gene expression.

The serine/threonine protein kinase ATM (ataxia-telangiectasia mutated) performs a number of functions in the cell that are interrelated with the aging process. In addition to regulating the cellular response to DNA damage, ATM phosphorylates vacuolar ATPase, leading to lysosome degradation and cellular senescence. In this work, we analysed the geroprotective potential of KU-60019, a selective ATM inhibitor, using individuals of three Drosophila species with different lifespans. KU-60019 was shown to increase the lifespan of individuals of the long-lived species D. virilis and individuals of a species with moderate lifespan D. melanogaster. However, in individuals of the short-lived species D. kikkawai, longevity is reduced after KU-60019 treatment. At the same time, KU-60019 treatment increases survival of Drosophila individuals of the three species under hyperthermia, oxidative stress and starvation, but has no effect on age-dependent changes in the level of locomotor activity. Suppression of tefu gene expression (ATM homologue) by RNA interference also causes an increase in longevity and stress tolerance of D. melanogaster individuals compared to individuals of control lines. Thus, the effect of KU-60019 on longevity varies depending on the Drosophila species, which may be related to the previously established differences of transcriptomes in the studied species and requires further experimental study.

Transposable elements (TE) increase the frequency of spontaneous mutations in the genome and are also capable of altering function and affecting gene expression, so it is important to have an idea of their activity and position in the genome. The paper demonstrates the advantage of combining the analysis of two sequencing methods of searching TE insertions and chromosomal rearrangements: full-genome nanopore sequencing allows the detection of TE insertions, and the use of transcriptome sequencing evaluates the effect of insertions on gene expression. The results are presented using SS (w¹, flamenco mutant) and MS (w¹, flamenco mutant, active copy of gypsy) strains with the flamenco phenotype as an example to investigate the causes of impaired control of TE activity. The laboratory wild type strain D32 was used as a control. Insertions and deletions of TE into the euchromatin regions of the genome and into the introns of genes relative to the reference genome were found in the studied strains and wild-type strains. In the analyzed genomes, a search for insertions and deletions in RNA interference system genes and in differentially expressed genes in SS and MS strains with flamenco phenotype was performed. We have detected TE insertions in various structures of AGO3, CG17147, Su(var)3-3, Gasz, CG43348, moody, CG17752 genes. For most of the analyzed genes, no correlation between a change in the TE position and a decrease or increase in gene transcription was found. A chromosomal rearrangement affecting the 3’-untranslated region has been detected for the vig gene. Based on the results of long-read sequencing, a de novo genome assembly for the MS strain was obtained. The increased expression in SS and MS strains for CR45822 and pst genes was found to be associated with triplication, but not with changes in gene regulatory sequences or TE insertion.

Pancreatic Ductal AdenoCarcinoma (PDAC) is characterized by a poor prognosis and is poorly amenable to modern therapies. A range of cell cultures reflecting different degrees of tumor differentiation and malignancy can serve as a model of PDAC development. Highly differentiated low malignant cells are characterized by increased expression of the KLF5 gene. The KLF5 protein is a vivid representative of multifunctional transcription factors, and its involvement in a variety of cellular processes, particularly in the pathology of various cancers, has been demonstrated. We investigated the spatial organization of chromatin of regulatory regions of KLF5 gene using highly differentiated Capan2 cells PDAC with a high level of KLF5 expression and poorly differentiated MIA PaCa2 PDAC cells with a low level of this gene expression by circular chromosome conformation capture (4C-seq). It was shown that the number and distribution of contacts of the KLF5 regulatory region with other chromatin regions are significantly different for these cell types; the number of contacts is significantly higher for Capan2 cells. There is a correlation between the expression level of genes close to KLF5 and the intensity of their sequence contacts with the KLF5 regulatory region, indicating that their expression is coordinated, possibly within the transcriptional factory. Only Capan2 is characterized by a high level of contacts of the KLF5 regulatory region with the gene free region containing a cluster of PDAC-associated single nucleotide polymorphisms (SNP). Thus, the total number of contacts of the promoter region of the KLF5 gene and the expression level of most of the surrounding KLF5 genes decrease as the grade of cell malignancy increases.

Klebsiella oxytoca is a causative agent of various community-acquired and nosocomial infections in humans, including urinary tract infections, nosocomial pneumonia, antibiotic-associated diarrhea, etc. However, the virulence factors of this species have not been sufficiently studied. In this study, we characterized the adhesive potential of the urological isolate К. oxytoca NK-1 using different substrates. Our findings indicate that this strain effectively adheres to epithelial cell lines, glycosylated and non-glycosylated proteins and polystyrene. Furthermore, it induces yeast agglutination, indicating the presence of type 1 and type 3 fimbriae – organelles that facilitate the adhesion of enterobacteria to a diverce range of substrates. Both type 1 and type 3 fimbrial operons were identified in the strain´s genome, with the latter presented in two copies. Mutants with inactivated genes encoding these fimbriae were constructed. It has been shown that the inactivation of type 1 fimbrial genes does not affect bacterial adhesion, while the inactivation of type 3 fimbrial genes increases the adhesion of К. oxytoca NK-1 to lung epithelial cells (line H1299), with mannose serving as an additional inducer of the increased adhesion. At the same time, the adhesion of this mutant to other substrates is not affected. These findings lead us to the conclusion that the adhesive apparatus of К. oxytoca is multifactorial. Additionally, they suggest the possibility of compensatory expression or overexpression of genes encoding alternative adhesins in the absence of types 1 and/or 3 fimbriae.

Human papillomavirus type 16 (HPV16) belongs to the high-risk type viruses and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the reactive oxygen species (ROS) production level and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that in HPV-16-positive SiHa cells of cervical squamous cell carcinoma, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.g., decreases ROS and GSH levels, suppresses the expression of E6 oncoprotein, activates the expression of p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the mitochondrial membrane integrity is a signal for the mitochondrial apoptosis pathway activation.

The human primase-polymerase PrimPol is a key participant of the mechanism of DNA synthesis restart during replication fork stalling at DNA damaged sites. PrimPol has a DNA primase activity and synthesizes DNA primers that are used by processive DNA polymerases to continue replication. Recruitment of PrimPol to the sites of DNA damage, as well as catalytic activity stimulation depends on interaction with the replicative protein RPA, which binds single-stranded DNA. The C-terminal domain of PrimPol contains a negatively charged RPA-binding motif (RBM), which mutations disrupt the interaction between two proteins. The RBM motif also plays a role in the negative regulation of PrimPol interaction with DNA. Deletion of RBM dramatically increases PrimPol affinity to DNA and stimulates PrimPol activity. The mechanism of RBM-mediated regulation of PrimPol activity is unclear. The relatively strong negative charge of RBM potentially may contribute to the interaction of PrimPol with RPA and DNA. RBM contains two negatively charged regions RBM-A and RBM-B. In this work, we additionally added (substitution V546E) or decreased (substitution D547H) the negative charge in RBM-B PrimPol and characterized these mutant variants biochemically. It was shown that the local change of RBM-B charge has no effect on the interaction of PrimPol with DNA and RPA, as well as the catalytic activity of the enzyme.