The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.

Spp1 is the H3K4me3 reader subunit of the Set1 complex (COMPASS/Set1C) that contributes to the mechanism by which meiotic DNA break sites are mechanistically selected. We previously proposed a model in which Spp1 interacts with H3K4me3 and the chromosome axis protein Mer2 that leads to DSB formation. Here we show that spatial interactions of Spp1 and Mer2 occur independently of Set1C. Spp1 exhibits dynamic chromatin binding features during meiosis, with many de novo appearing and disappearing binding sites. Spp1 chromatin binding dynamics depends on its PHD finger and Mer2-interacting domain and on modifiable histone residues (H3R2/K4). Remarkably, association of Spp1 with Mer2 axial sites reduces the effective turnover rate and diffusion coefficient of Spp1 upon chromatin binding, compared with other Set1C subunits. Our results indicate that "chromosomal turnover rate" is a major molecular determinant of Spp1 function in the framework of meiotic chromatin structure that prepares recombination initiation sites for break formation.

Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18-22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G-C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae.

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3'OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3'OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.

Adaptation to different types of environmental stress is a common part of life for today's fungi. A deeper understanding of the organization, regulation and evolution of fungal stress response systems may lead to the development of novel antifungal drugs and technologies or the engineering of industrial strains with elevated stress tolerance. Here we present the Fungal Stress Response Database (http://internal.med.unideb.hu/fsrd) aimed to stimulate further research on stress biology of fungi. The database incorporates 1985 fungal stress response proteins with verified physiological function(s) and their orthologs identified and annotated in 28 species including human and plant pathogens, as well as important industrial fungi. The database will be extended continuously to cover other fully sequenced fungal species. Our database, as a starting point for future stress research, facilitates the analysis of literature data on stress and the identification of ortholog groups of stress response proteins in newly sequenced fungal genomes. Database URL: http://internal.med.unideb.hu/fsrd

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chromosome arms. Here we show that NDX is primarily a heterochromatin regulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericentromeric het-siRNA loci that mediate transposon silencing, and are antagonistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericentromeres where NDX is enriched in wild-type plants, and increased interchromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key regulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways.

Industrial fungi need a strong environmental stress tolerance to ensure acceptable efficiency and yields. Previous studies shed light on the important role that Aspergillus nidulans gfdB, putatively encoding a NAD+-dependent glycerol-3-phosphate dehydrogenase, plays in the oxidative and cell wall integrity stress tolerance of this filamentous fungus model organism. The insertion of A. nidulans gfdB into the genome of Aspergillus glaucus strengthened the environmental stress tolerance of this xerophilic/osmophilic fungus, which may facilitate the involvement of this fungus in various industrial and environmental biotechnological processes. On the other hand, the transfer of A. nidulans gfdB to Aspergillus wentii, another promising industrial xerophilic/osmophilic fungus, resulted only in minor and sporadic improvement in environmental stress tolerance and meanwhile partially reversed osmophily. Because A. glaucus and A. wentii are phylogenetically closely related species and both fungi lack a gfdB ortholog, these results warn us that any disturbance of the stress response system of the aspergilli may elicit rather complex and even unforeseeable, species-specific physiological changes. This should be taken into consideration in any future targeted industrial strain development projects aiming at the fortification of the general stress tolerance of these fungi. KEY POINTS: • A. wentii c' gfdB strains showed minor and sporadic stress tolerance phenotypes. • The osmophily of A. wentii significantly decreased in the c' gfdB strains. • Insertion of gfdB caused species-specific phenotypes in A. wentii and A. glaucus.

Arabidopsis NODULIN HOMEOBOX (NDX) is a plant-specific transcriptional regulator whose role in small RNA biogenesis and heterochromatin homeostasis has recently been described. Here we extend our previous transcriptomic analysis to the flowering stage of development. We performed mRNA-seq and small RNA-seq measurements on inflorescence samples of wild-type and ndx1-4 mutant (WiscDsLox344A04) Arabidopsis plants. We identified specific groups of differentially expressed genes and noncoding heterochromatic siRNA (hetsiRNA) loci/regions whose transcriptional activity was significantly changed in the absence of NDX. In addition, data obtained from inflorescence were compared with seedling transcriptomics data, which revealed development-specific changes in gene expression profiles. Overall, we provide a comprehensive data source on the coding and noncoding transcriptomes of NDX-deficient Arabidopsis flowers to serve as a basis for further research on NDX function.

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these "domesticated", transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an HsMar1 transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression. Herein, we mapped the genomic landscape of SETMAR binding in a near-haploid human leukemia cell line (HAP1) in order to identify on-target and off-target binding sites at high resolution and to elucidate their role in terms of gene expression. Our analysis revealed a perfect correlation between SETMAR and inverted terminal repeats (ITRs) of HsMar1 transposon remnants, which are considered as natural target sites for SETMAR binding. However, we did not detect any untargeted events at non-ITR sequences, calling into question previously proposed off-target binding sites. We identified sequence fidelity of the ITR motif as a key factor for determining the binding affinity of SETMAR for chromosomes, as higher conservation of ITR sequences resulted in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci. These associations highlight how SETMAR's chromatin binding fine-tune gene regulatory networks in human tumour cells.

Uveal melanoma (UM) is the most common malignant tumor of the eye with extremely high metastatic potential. UM tumor cells can disseminate only hematogenously, thus, angiogenic signals have a particular role in the prognosis of the disease. Although the presence of cancer stem cells (CSCs) in densely vascularized UMs has been reported previously, their role in the process of hematogenous spread of UM has not been studied. In this study, we investigated the regulation of angiogenesis in UM in correlation with the presence of CSCs. Seventy UM samples were collected to analyze the expression of CSC markers and angiogenic factors. The expression of CSC markers was studied by RT-PCR, Western blotting techniques and IHC-TMA technique. RT-PCR showed high expression of CSC markers, particularly nestin, FZD6 and SOX10 and somewhat lower expression of NGFR. The protein expression of FZD6, HIF-1α and VEGFA was further evaluated in 52 UM samples by the IHC-TMA technique. We report here for the first time a significant correlation between FZD6 and VEGFA expression in UM samples. The observed correlation between FZD6 and VEGFA suggests the presence of CSCs in UM that are associated with the vascularization process.

The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

Cardiovascular diseases are among the leading causes of mortality worldwide. Hypertension is a preventable risk factor leading to major cardiovascular events. We have not found a comprehensive study investigating Central and Eastern European hypertensive patients' complex metabolic status. Therefore, our goal was to calculate the prevalence of hypertension and associated metabolic abnormalities using data-mining methods in our region. We assessed the data of adults who visited the University of Debrecen Clinical Center's hospital (n = 937,249). The study encompassed data from a period of 20 years (2001-2021). We detected 292,561 hypertensive patients. The calculated prevalence of hypertension was altogether 32.2%. Markedly higher body mass index values were found in hypertensive patients as compared to non-hypertensives. Significantly higher triglyceride and lower HDL-C levels were found in adults from 18 to 80 years old. Furthermore, significantly higher serum glucose and uric acid levels were measured in hypertensive subjects. Our study confirms that the calculated prevalence of hypertension is akin to international findings and highlights the extensive association of metabolic alterations. These findings emphasize the role of early recognition and immediate treatment of cardiometabolic abnormalities to improve the quality of life and life expectancy of hypertensive patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the death of motor neurons, the aetiology of which is essentially unknown. Here, we present an integrative epigenomic study in blood samples from seven clinically characterised sporadic ALS patients to elucidate molecular factors associated with the disease. We used clinical exome sequencing (CES) to study DNA variants, DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) to assess R-loop distribution, and reduced representation bisulfite sequencing (RRBS) to examine DNA methylation changes. The above datasets were combined to create a comprehensive repository of genetic and epigenetic changes associated with the ALS cases studied. This repository is well-suited to unveil new correlations within individual patients and across the entire patient cohort. The molecular attributes described here are expected to guide further mechanistic studies on ALS, shedding light on the underlying genetic causes and facilitating the development of new epigenetic therapies to combat this life-threatening disease.

In breast cancer patients, the diversity of the microbiome decreases, coinciding with decreased production of cytostatic bacterial metabolites like lithocholic acid (LCA). We hypothesized that LCA can modulate oxidative stress to exert cytostatic effects in breast cancer cells. Treatment of breast cancer cells with LCA decreased nuclear factor-2 (NRF2) expression and increased Kelch-like ECH associating protein 1 (KEAP1) expression via activation of Takeda G-protein coupled receptor (TGR5) and constitutive androstane receptor (CAR). Altered NRF2 and KEAP1 expression subsequently led to decreased expression of glutathione peroxidase 3 (GPX3), an antioxidant enzyme, and increased expression of inducible nitric oxide synthase (iNOS). The imbalance between the pro- and antioxidant enzymes increased cytostatic effects via increased levels of lipid and protein oxidation. These effects were reversed by the pharmacological induction of NRF2 with RA839, tBHQ, or by thiol antioxidants. The expression of key components of the LCA-elicited cytostatic pathway (iNOS and 4HNE) gradually decreased as the breast cancer stage advanced. The level of lipid peroxidation in tumors negatively correlated with the mitotic index. The overexpression of iNOS, nNOS, CAR, KEAP1, NOX4, and TGR5 or the downregulation of NRF2 correlated with better survival in breast cancer patients, except for triple negative cases. Taken together, LCA, a metabolite of the gut microbiome, elicits oxidative stress that slows down the proliferation of breast cancer cells. The LCA-oxidative stress protective pathway is lost as breast cancer progresses, and the loss correlates with poor prognosis.