Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin binding protein Barrier-to-autointegration factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to deformation of NL structure and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf mis-regulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained mis-expressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global mis-expression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found up-regulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

ABSTRACTSARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase of PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Time is of the essence: the molecular mechanisms of primary microcephaly

Primary microcephaly is a brain growth disorder characterized by a severe reduction of brain size and thinning of the cerebral cortex. Many primary microcephaly mutations occur in genes that encode centrosome proteins, highlighting an important role for centrosomes in cortical development. Centrosomes are microtubule organizing centers that participate in several processes, including controlling polarity, catalyzing spindle assembly in mitosis, and building primary cilia. Understanding which of these processes are altered and how these disruptions contribute to microcephaly pathogenesis is a central unresolved question. In this review, we revisit the different models that have been proposed to explain how centrosome dysfunction impairs cortical development. We review the evidence supporting a unified model in which centrosome defects reduce cell proliferation in the developing cortex by prolonging mitosis and activating a mitotic surveillance pathway. Finally, we also extend our discussion to centrosome-independent microcephaly mutations, such as those involved in DNA replication and repair.

Depletion of NK6 Homeobox 3 (NKX6.3) causes gastric carcinogenesis through copy number alterations by inducing impairment of DNA replication and repair regulation

Genomic stability maintenance requires correct DNA replication, chromosome segregation, and DNA repair, while defects of these processes result in tumor development or cell death. Although abnormalities in DNA replication and repair regulation are proposed as underlying causes for genomic instability, the detailed mechanism remains unclear. Here, we investigated whether NKX6.3 plays a role in the maintenance of genomic stability in gastric epithelial cells. NKX6.3 functioned as a transcription factor for CDT1 and RPA1, and its depletion increased replication fork rate, and fork asymmetry. Notably, we showed that abnormal DNA replication by the depletion of NKX6.3 caused DNA damage and induced homologous recombination inhibition. Depletion of NKX6.3 also caused copy number alterations of various genes in the vast chromosomal region. Hence, our findings underscore NKX6.3 might be a crucial factor of DNA replication and repair regulation from genomic instability in gastric epithelial cells.

Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology

The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated “promoter and pre-rRNA antisense” RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

DNA topoisomerase II (TOPII) plays a very important role in DNA topology and in different biological processes such as DNA replication, transcription, repair, and chromosome condensation in higher eukaryotes. TOPII has been found to interact directly with a protein called topoisomerase II binding protein 1 (TopBP1) which also seems to have important roles in DNA replication and repair. In this study, we conducted different experiments to assess the roles of TopBP1 in DNA repair, mitosis, and meiosis, exploring the relationship between TOPII activity and TopBP1. We found that topbp1 mutant seedlings of Arabidopsis thaliana were hypersensitive to cisplatin treatment and the inhibition of TOPII with etoposide produced similar hypersensitivity levels. Furthermore, we recognised that there were no significant differences between the WT and topbp1 seedlings treated with cisplatin and etoposide together, suggesting that the hypersensitivity to cisplatin in the topbp1 mutant could be related to the functional interaction between TOPII and TopBP1. Somatic and meiotic anaphase bridges appeared in the topbp1 mutant at similar frequencies to those when TOPII was inhibited with merbarone, etoposide, or ICFR-187. The effects on meiosis of TOPII inhibition were produced at S phase/G2 stage, suggesting that catenanes could be produced at the onset of meiosis. Thus, if the processing of the catenanes is impaired, some anaphase bridges can be formed. Also, the appearance of anaphase bridges at first and second division is discussed.

Maternal Vaginal Fluids Play A Major Role In The Colonization of The Neonatal Intestinal Microbiota

Background: Caesarean delivery (CD) is associated with newborns’ health risks due to the blocking of microbiome transfer. To understand the vertical bacterial seeding and reduce CD disadvantages, microbiome transmissions via anal and genital routes were investigated, and the efficiency of vaginal fluid swabbing treatment was evaluated using 16s rDNA sequencing-based techniques.Results: Pregnant women were recruited in the Women and Children’s Hospital, School of Medicine, Xiamen University from June 1st to August 15th, 2017. Maternal faeces (n = 26), maternal vaginal fluids (n = 26), and neonatal transitional stools (n = 26) were collected, while the participants underwent natural delivery (ND) (n = 6), CD (n = 4) and CD with vaginal fluid swabbing (CS) on their newborns (n = 16). 26 mothers with the median age 26.50 (25.00-27.25) years showed no substantial clinical differences. The newborns’ gut microbiota altered among ND, CD and CS, and clustered into two groups (PERMANOVA P < 0.01) of swabbing and no-swabbing exposure. Gut colonization of ND babies majorly originated from maternal vaginal microbes (PERMANOVA P = 0.08), no vertical transmission was observed via anal route in any group. The vaginal transfer partially occurred by swabbing, in which the phyla Firmicutes and Proteobacteria and the genera Lactobacillus, Bacteroides, and Escherichia-Shigella in newborns were similar to their mothers. The recovered taxa predicted KEGG functions of biosynthesis, metabolism, and DNA replication and repair with benefits of low risks of digestive, cardiovascular, and immune diseases.Conclusions: The newborn’s gut microbiota is mainly shaped by maternal vaginal microbiota, and aberrant colonization initiated by CD is partly mitigated by the swabbing treatment.

Comparison of the transcriptome in circulating leukocytes in early lactation between primiparous and multiparous cows provides evidence for age-related changes

Background Previous studies have identified many immune pathways which are consistently altered in humans and model organisms as they age. Dairy cows are often culled at quite young ages due to an inability to cope adequately with metabolic and infectious diseases, resulting in reduced milk production and infertility. Improved longevity is therefore a desirable trait which would benefit both farmers and their cows. This study analysed the transcriptome derived from RNA-seq data of leukocytes obtained from Holstein cows in early lactation with respect to lactation number. Results Samples were divided into three lactation groups for analysis: i) primiparous (PP, n = 53), ii) multiparous in lactations 2–3 (MP 2–3, n = 121), and iii) MP in lactations 4–7 (MP > 3, n = 55). Leukocyte expression was compared between PP vs MP > 3 cows with MP 2–3 as background using DESeq2 followed by weighted gene co-expression network analysis (WGCNA). Seven modules were significantly correlated (r ≥ 0.25) to the trait lactation number. Genes from the modules which were more highly expressed in either the PP or MP > 3 cows were pooled, and the gene lists subjected to David functional annotation cluster analysis. The top three clusters from modules more highly expressed in the PP cows all involved regulation of gene transcription, particularly zinc fingers. Another cluster included genes encoding enzymes in the mitochondrial beta-oxidation pathway. Top clusters up-regulated in MP > 3 cows included the terms Glycolysis/Gluconeogenesis, C-type lectin, and Immunity. Differentially expressed candidate genes for ageing previously identified in the human blood transcriptome up-regulated in PP cows were mainly associated with T-cell function (CCR7, CD27, IL7R, CAMK4, CD28), mitochondrial ribosomal proteins (MRPS27, MRPS9, MRPS31), and DNA replication and repair (WRN). Those up-regulated in MP > 3 cows encoded immune defence proteins (LYZ, CTSZ, SREBF1, GRN, ANXA5, ADARB1). Conclusions Genes and pathways associated with lactation number in cows were identified for the first time to date, and we found that many were comparable to those known to be associated with ageing in humans and model organisms. We also detected changes in energy utilization and immune responses in leukocytes from older cows.

Overexpression of Pyruvate Dehydrogenase Kinase-3 Predicts Poor Prognosis in Urothelial Carcinoma

BackgroundThe mitochondrial pyruvate dehydrogenase complex (PDC) link glycolysis to the tricarboxylic acid cycle by decarboxylating pyruvate to acetyl coenzyme A irreversibly. Cancer cells are characterized by a shift in cellular metabolism from mitochondrial respiration to glycolysis. PDC activity inhibition mediated by phosphorylation via pyruvate dehydrogenase kinase (PDK) has been linked to cancer. However, the clinical significance of PDKs in urothelial cancer prognosis is not clear. We investigated the role and prognostic value of PDK3 expression in patients with upper urinary tract urothelial carcinoma (UTUC) and urinary bladder urothelial carcinoma (UBUC).Patients and MethodsWe retrospectively analyzed clinical data and pathological features. Formalin-fixed urothelial carcinoma (UC) tissues were collected and embedded in paraffin. The correlation of PDK3 expression with clinical characteristics, pathological findings and patient outcomes, including metastasis-free survival (MFS) and disease-specific survival (DSS) were analyzed by Pearson’s chi-square test, Kaplan–Meier analysis, and the multivariate Cox proportional hazards model.ResultsData from 295 patients with UBUC and 340 patients with UTUC were evaluated. High PDK3 expression significantly correlated with several pathologic variables such as high T stage, lymph node metastases, high tumor grade, vascular invasion, and high mitotic rate (all P &lt; 0.001). High PDK3 expression was associated with poor disease-specific survival (DSS) (P &lt; 0.0001) and metastatic free survival (MFS) (P &lt; 0.0001) in a Kaplan–Meier analysis. Additionally, multivariate analysis demonstrated increased PDK3 expression is a significant predictive risk factor for DSS [hazard ratio (HR) in UBUC, 2.79, P = 0.009; in UTUC, 2.561, P = 0.03] and MFS (HR in UBUC, 1.907, P = 0.024; in UTUC, 1.793, P = 0.044). The gene co-expression analysis showed abundant PDK3 co-upregulated genes were involved in the processes of DNA replication and repair through the Gene Ontology classification system.ConclusionHigh PDK3 expression has been linked to negative pathologic characteristics and poor oncological outcomes, suggesting that it could be used as a predictive biomarker for UC. PDK3 mRNA levels and its co-upregulated genes were strongly associated with DNA replication and repair. These results suggest that PDK3 may play a key role in tumor proliferation and development.

SAMHD1 in cancer: curse or cure?

Human sterile α motif and HD domain-containing protein 1 (SAMHD1), originally described as the major cellular deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) balancing the intracellular deoxynucleotide (dNTP) pool, has come recently into focus of cancer research. As outlined in this review, SAMHD1 has been reported to be mutated in a variety of cancer types and the expression of SAMHD1 is dysregulated in many cancers. Therefore, SAMHD1 is regarded as a tumor suppressor in certain tumors. Moreover, it has been proposed that SAMHD1 might fulfill the requirements of a driver gene in tumor development or might promote a so-called mutator phenotype. Besides its role as a dNTPase, several novel cellular functions of SAMHD1 have come to light only recently, including a role as negative regulator of innate immune responses and as facilitator of DNA end resection during DNA replication and repair. Therefore, SAMHD1 can be placed at the crossroads of various cellular processes. The present review summarizes the negative role of SAMHD1 in chemotherapy sensitivity, highlights reported SAMHD1 mutations found in various cancer types, and aims to discuss functional consequences as well as underlying mechanisms of SAMHD1 dysregulation potentially involved in cancer development.