Cell Cycle

Centrosomal and microtubule-associated proteins such as CEP170 and WDR62 are essential in regulating mitotic spindle formation and pole orientation during cell division. MAPKBP1, a paralog of WDR62, is also a centrosomal protein, but its function is currently unclear. We have shown here that MAPKBP1 is localised to the subdistal appendages of the mother centriole, the pericentriolar material (PCM) of the centrosomes and the mitotic spindles during metaphase. Furthermore, MAPKBP1, WDR62 and CEP170 exists as a complex, where MAPKBP1 is recruited to the centrosomes by WDR62 and CEP170, and CEP170-MAPKBP1 interaction is mediated by WDR62. In addition, MAPKBP1 depletion leads to mitotic spindle defects and delayed mitosis that were further exacerbated with WDR62 knockout, indicating a possible redundancy between MAPKBP1 and WDR62. MAPKBP1 loss also leads to PCM fragmentation, which supports its role as a subdistal appendages protein vital in maintaining centrosome structure and PCM cohesion for proper anchoring of mitotic spindles. This study provides insight into how subdistal appendages and centrosome and microtubule associated proteins co-operate to tightly regulate mitotic spindle formation and stability.

AO_SCPLOWBSTRACTC_SCPLOWThe cellular transcription factor BCL11b (B-cell CLL/lymphoma 11b) interacts with numerous cellular and viral factors to modulate gene expression positively or negatively. Post-translational modifications of BCL11b, such as SUMOylation and phosphorylation, have been documented to switch its transcriptional activity from a repressor to an activator state. In the present study, we investigated the acetylation of BCL11b and we identified the histone acetyltransferase p300 as able to acetylate BCL11b. Subsequently, we observed that the mutation of the lysine K686 residue of BCL11b (BCL11b K686R) influenced its global acetylation. Furthermore, the BCL11b K686R mutation also modulated the transcriptional regulation of BCL11b, including its activity in regulating the p21 and IL-2 promoters. This effect on transcriptional regulation was due to the importance of the lysine K686 residue for BCL11b nuclear localization. Our results underscore the critical role of the lysine K686 residue in BCL11b for its interaction with p300 and its nuclear localization, suggesting a possible function of p300 in the nuclear transport of BCL11b. Collectively, our findings contribute to a better understanding of BCL11b-mediated gene expression and of the interactions of BCL11b with cellular partners.

Sirtuin 6 (SIRT6) is an important regulator of DNA repair, metabolism, chromatin maintenance and longevity. SIRT6 Serine 10 phosphorylation controls SIRT6 recruitment to the sites of DNA damage. To explore the effect of SIRT6 Serine 10 phosphorylation on lifespan, we generated two SIRT6 mutant mouse strains: phospho-null S10A and phosphomimetic S10E. The S10E mutant mice demonstrated enhanced DNA repair capacity, elevated LINE1 expression and reduced lifespan in male mice compared to the wild-type and S10A mice. This result suggests that SIRT6 S10E mutation enhances DNA repair capacity at the expense of reduced LINE1 silencing leading to shorter lifespan. While both SIRT6 functions in DNA repair and chromatin maintenance are important for longevity, our results suggest that when the balance between these functions is shifted, diminished of LINE1 control has a stronger impact on lifespan than enhanced DNA repair.

Infertility is a complex condition affecting both the male and female population. Influenced by multiple factors, it remains a constant challenge due to limited understanding of endometrial abnormalities. With this study we aim to investigate the molecular basis of infertility using transcriptomic analysis of endometrial tissue from the NCBI GEO dataset GSE92324. We performed exploratory data analysis using Principal Component Analysis (PCA) to find samples variance followed by differential gene expression (DGE) analysis using DESeq2 package where we identified 168 significant genes with adjusted p-value < 0.05 and |log2FC| > 2. Upregulated genes included GPX3, CXCL14, and PPARGC1A and downregulated genes included WNK4, GJB2, and TRPM6. Functional enrichment using KEGG and GO showed that differentially expressed genes (DEGs) are involved in immune-inflammatory pathways, lipid metabolism and steroid biosynthesis pathways. Through Ingenuity Pathway Analysis (IPA) we identified affected canonical pathways such as increased innate immune responses, altered lipid metabolism and inhibition of mitochondrial dysfunction. Upstream regulator analysis highlighted PTEN, PRKAA1, HDAC4, IL10RA, and RAD51, which were impacting metabolic pathways and anti-inflammatory signalling. Further, through Weighted Gene Co-expression Network Analysis (WGCNA) we found a Turquoise module that had very strong and highly significant negative correlation (cor = - 0.84, respectively and P < 0.0001) with traits of interest. This led to the discovery of C7orf50 as a novel insight involved in cholesterol metabolism linked to infertility. This integrative approach reveals crucial genes, co-expression modules, and underlying pathways involved in female infertility. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/701467v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@4418a6org.highwire.dtl.DTLVardef@ae7900org.highwire.dtl.DTLVardef@89f581org.highwire.dtl.DTLVardef@154f1a9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIFrom the dataset GSE92324 total of 168 significant DEGs associated with unexplained infertility were identified using adjusted p-value < 0.05 and |log2FC| > and < 2. C_LIO_LIIn comparison with the CTD list we identified five genes C1orf106, C15orf59, LINC00461, C15orf48, and C10orf99 previously unknown as having direct evidence of involvement in infertility. C_LIO_LIWGCNA analysis highlighted the turquoise module as highly associated and gave the novel gene C7orf50 associated with cholesterol metabolism. C_LIO_LIIPA revealed PTEN, PRKAA1, IL10RA, and RAD51 as potential upstream regulators and inflammatory pathways, mitochondrial dysfunction as canonical pathways. C_LIO_LIThe study highlights a novel link between GI inflammation and endometrial receptivity. C_LI

Cancer-related genomic instability (GI) may cause genetic alterations in spermatozoa, implying health issues not only in cancer survivors, but also in their children [1, 2]. We therefore studied Loss of Y chromosome (LOY), considered as hallmark of GI [3-15], in spermatozoa and blood from survivors of childhood and testicular cancer (CC, TC), and controls (CTRL). We found that LOY was statistically significantly more frequent in spermatozoa from cancer survivors than in controls (Odds Ratio [OR]=2.2 for CC vs. CTRL and OR=2.4 for TC vs. CTRL). Furthermore, LOY was about an order of magnitude more prevalent in spermatozoa than in blood among 18-53-year-old males within all cohorts. Our findings suggest that LOY in spermatozoa might be a clinically useful marker of GI, reduced fertility and disease predisposition in males. Introducing LOY in spermatozoa as a biomarker opens a new research avenue into disease prevention and the causes and consequences of LOY.

BackgroundDDX53 (DEAD-box helicase 53, known also as CAGE) is an intronless gene on the X chromosome, which expression shows strong testis specificity. It belongs to the group of cancer-testis (CT) antigens, with most studies to date focusing on its role in cancer, but the precise biological function of DDX53 remains unclear. Previous reports identifying rare DDX53 variants in infertile men provided the rationale for investigating the role of DDX53 in the context of human spermatogenesis. By using the human seminoma cell line (TCam-2) as an in vitro male germline model, we aimed to investigate the function and molecular targets of DDX53. MethodsIn our study, we used transcriptomic and proteomic approaches (RNA sequencing (RNA-seq), enhanced crosslinking and immunoprecipitation (eCLIP), and Co-immunoprecipitation coupled with Mass Spectrometry (Co-IP-MS)) to investigate the role of DDX53 in the context of human spermatogenesis. By using modified TCam-2 cells to express either DDX53-FLAG or GFP-FLAG, we identified regulated genes, RNA targets, and potential protein interactors of DDX53. In addition, we employed Western Blot, RT-qPCR, immunostaining, and confocal microscopy to gain deeper insight into the DDX53 protein. ResultsOur RNA-seq and eCLIP data provide evidence that DDX53 regulates gene expression changes and directly interacts with a broad spectrum of RNA transcripts. Moreover, for the first time, we described RNAs and protein interactors of DDX53 in the context of spermatogenesis. Subcellular localization analysis by confocal microscopy indicated a predominantly cytoplasmic distribution of DDX53, with partial nuclear presence in TCam-2 cells. We also identified DDX53-positive structures that may correspond to germ granule-like assemblies, although their precise nature remains to be determined. Additionally, we confirmed DDX53 presence in human testis using a specific, commercially available anti-DDX53 antibody. ConclusionsThis studys data indicate that DDX53 protein acts as a regulator of RNA metabolism in human cells. Collectively, we show that it participates in transcriptome regulation (including splicing) in male germ cells and exhibits transcriptome-wide RNA interactions, but its wider biological role remains to be clarified.

Stress Granules (SGs) are cytoplasmic biomolecular condensates that form in response to a variety of stress conditions, though their function remains unclear. "Canonical" SGs - caused by stressors like sodium arsenite - are dynamic and cytoprotective, allowing cells to evade cell death during periods of stress. Ultraviolet (UV) irradiation is known to elicit a "non-canonical" SG subtype, lacking canonical SG components such as eukaryotic initiation factor 3 and polyadenylated mRNAs. The exact function of UV SGs, and the mechanisms driving their formation, remain unknown. Here we report the findings of a comparative analysis of UVA, UVB and UVC exposures on SG formation in three cell types: osteosarcoma (U2OS), keratinocytes (HaCaT), and mouse embryonic fibroblasts (MEF). We observed that SG formation in response to UV is highly cell type dependent. UVB and UVC induce robust SG formation in U2OS cells. However, only UVC exposure induced modest SG formation in MEFs, and none of the wavelengths caused SGs in HaCaT. While UVC-induced SGs in U2OS cells appear to be cell cycle dependent and specific to G1, UVB induced SG formation regardless of cell cycle stage. We tested the hypothesis that oxidative stress triggered by UV may be driving UV SG formation, and that keratin may buffer this effect, by overexpressing keratin in U2OS. Interestingly, we found that keratin and antioxidant treatment efficiently suppressed arsenite-induced SGs but had no effect on UV SGs. Our work confirms that UV SG formation is cell type specific and is not driven by oxidative stress.

Polycystic Ovary Syndrome (PCOS) is known as an endocrine and metabolic disorder; however, emerging molecular evidence suggests a far more complex systems-level pathology. In this study, we performed an integrative transcriptomic and pathway-level analysis of endometrial tissue from women with PCOS to gain a deeper understanding of the underlying mechanism facilitating the disorder. The findings of the study highlighted mitochondrial dysfunction, chronic oxidative stress, and multi-layered immune dysregulation, adding some new insight apart from classical hyperandrogenism and insulin resistance. We identified some novel gene disease associations which involve C15orf48, ODF3B PRR15-DT, LINC01176, and LOC105379193. The upstream regulators such as (NFE2L2, TWNK, ALKBH1, BCOR, SMARCA4) involved in processes including mitochondrial genome, redox balance, and chromatin remodeling provided new insights into regulatory mechanisms. The IPA pathway analysis validated the compromised immune recovery with low grade inflammations and mitochondrial dysfunctionality. The observations emphasize on complex associations discarding its PCOS pure endocrine nature through immunometabolic-mitochondrial dysfunctionalities.

Envelope-Limited Chromatin Sheets (ELCS) can be induced in human promyelocytic HL-60/S4 cells by treatment with retinoic acid (RA). After 4 days, the differentiated granulocytes exhibit multilobed nuclei with outgrowths of the nuclear envelope (NE) and associated heterochromatin extending into the surrounding cytoplasm (ELCS). These fascinating structures reveal a periodic meshwork of 30 nm chromatin fibers, when viewed by Cryo-electron microscopy. Genetic and biochemical evidence indicates that RA increases the synthesis of Lamin B Receptor (LBR), which is a key enzyme for Cholesterol biosynthesis and is an essential bridge between the NE and peripheral heterochromatin. This article is in part a review of our microscopic data on the structure of ELCS, and in part a description of related transcription changes that result in the formation of ELCS. In addition, this article contains a structural and biochemical comparison of RA-induced granulocytes with phorbol ester (TPA) induced HL-60/S4 macrophages, which lack nuclear lobulation, do not form ELCS, and exhibit a reduction in LBR and Cholesterol biosynthesis. From our perspective, ELCS can be viewed as "fabric" outgrowths of the nuclear envelope, frequently connecting nuclear lobes and capable of sustaining the twisting and squeezing distortions imposed upon nuclear shape, as the granulocytes traverse narrow tissue channels.

The cell cycle comprises different phases and is a tightly regulated process at the molecular level. During the cell cycle, two key events occurred: DNA duplication during the S phase and chromosome segregation during mitosis. Accurate cell cycle progression, achieved through faithful chromosome segregation, is essential for maintaining cell fidelity. Long noncoding RNAs are a subclass of noncoding RNA that are longer than 200 bp and form RNA protein complexes (RNPs) to regulate various biological processes. Herein, we demonstrate that lncRNA NORM is involved in regulating the cell cycle by maintaining proper chromosome segregation. NORM exhibited G2 phase-specific expression, and the depletion of NORM resulted in a significant G2/M arrest. NORM-depleted cells failed to progress in mitosis and showed defects in chromosome segregation. We further demonstrated that NORM binds to proteins such as Plk1 and Nsun2. Depletion of NORM hindered the interaction between Plk1 and Bub1, resulting in reduced kinetochore localization of Plk1 during prometaphase. Our results also show that the depletion of NORM affects the binding of Nsun2 protein to CDK1 mRNA and, consequently, the stabilization of CDK1 at the protein level. Altogether, our results demonstrate that NORM regulates chromosome segregation by mediating the interaction between Plk1 and Bub1.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Yin Yang 1 (YY1) is a multifunctional transcription factor and mammalian Polycomb Group (PcG) protein critical for lymphocyte development. While YY1 is essential for early T-cell development and survival, the underlying epigenetic mechanisms by which YY1 regulates early T-cell development are not fully understood. Herein, we utilized the YY1 PcG function conditional knockout mouse model (Yy1-/{Delta}REPO) by CRISPR/Cas9 to further dissect the underlying mechanisms. Yy1-/{Delta}REPO mice show early T cell development blockage at the double-negative (DN) 3 to single positive T cell transition with expansion of the DN3 population. Yy1-/{Delta}REPO DN3 cells are highly proliferative, but more prone to apoptosis, leading to reduced single positive T cells output. The genetic network governing T cell differentiation is deregulated in Yy1-/{Delta}REPO DN3 T cells. The YY1 REPO deletion leads to downregulation of DNA demethylase enzyme Tet1 and Tet2 expressions in DN3 cells with no change of Tet3. Pharmacologic inhibition of TET catalytic activity blocked DN-to-DP progression at the DN3 stage, whereas re-expression of TET2 catalytic domain in Yy1-/{Delta}REPO DN thymocytes partially rescued T cell differentiation. Together, our study demonstrates that YY1-mediated PcG function is essential for the DN3 to SP T cell transition and YY1-TET2 axis promotes proper DN3 differentiation.

Being the basic building blocks of chromatin, nucleosomes and their stability determine the genome accessibility for different DNA-dependent proteins. This characteristic is labile under all cell-life processes. One of the abundant DNA-binding proteins, which is important for genome compaction, is poly(ADP-ribose)polymerase1 (PARP1). Despite the extensive experimental data on the chromatin compaction regulation under ADP-ribosylation, the details of the interplay of nucleosome with PARP1 in the absence of protein activation remain unclear. In this study, we analyzed the changes in the nucleosome wrapping upon PARP1 interaction using a single-molecule approach -- optical tweezers. We demonstrate that PARP1 binding leads to weakening of the contacts that support the nucleosome core.

Medulloblastoma is the most common pediatric brain cancer, but current treatments are largely non-specific, often causing developmental side effects. Genomic sequencing identified the RNA helicase DDX3X as one of the most frequently mutated genes in this cancer and a potential treatment target, yet its role in tumor progression remains elusive. Prior studies have indicated that the mutations cause specific defects in translation; however, both DDX3X and its yeast ortholog Ded1 have also been associated with cellular stress responses, suggesting that the contribution of the DDX3X mutations to medulloblastoma might result from defects in the translational response to stress. Building on our prior study that replicated the DDX3X mutations in yeast DED1 (ded1-mam), we examined the mutants effects following TOR pathway inactivation. First, we demonstrated that ded1-mam displayed substantial rapamycin-resistant growth compared to wild-type cells. In addition, similar to other ded1 mutants, the ded1-mam had decreased degradation of Ded1 and the translation factor eIF4G1 under TOR inactivation. Notably, these differences did not result in increased bulk translation following rapamycin; rather, the growth phenotypes appeared to be driven by translation of specific mRNAs. Reporter assays demonstrated enhanced translation of mRNAs with unstructured 5' UTRs in ded1-mam following TOR inhibition and a decrease in structured reporters. Furthermore, known Ded1 target genes with relatively unstructured 5 UTRs showed upregulated protein levels in rapamycin. We thus hypothesize that mutant DDX3X selectively upregulates translation of unstructured, pro-growth transcripts while downregulating other structured transcripts, allowing tumor cells to bypass stress-induced growth controls and promoting medulloblastoma progression.

The actin polymerization machinery, comprising the ARP2/3 complex and its activators, the WASP family proteins, has been implicated in regulating a broad spectrum of nuclear processes, such as transcriptional regulation and nuclear organization. Here, using clustered protocadherin (cPcdh) and {beta}-globin genes as model systems, we showed that WAVE2, a member of the WASP family, regulates chromatin organization by maintaining heterochromatin dynamics. Specifically, by CRISPR DNA-fragment editing, in conjunction with integrated analyses of ChIP-seq, MeDIP-seq, ATAC-seq, 4C-seq, and RNA-seq, we showed that deposition of H3K9me3, a key heterochromatin mark, is significantly decreased at the cPcdh locus upon WAVE2 deletion, concurrent with aberrant accumulation of CTCF/cohesin complex at promoter regions and spatial reorganization of chromatin architecture around nucleolus. In addition, REST/NRSF exerts a similar heterochromatindependent effect on the cPcdh locus. Finally, genetic and genomic data showed that WAVE2 regulates {beta}-globin gene expression by maintaining heterochromatin status. Together our data suggested that WAVE2 and REST/NRSF regulate clustered gene expression in a heterochromatin-dependent manner.

AimsTo explore exercise-induced fatigue (EIF)s effects on the male reproductive system and MXRA7s regulatory role herein. MethodsWe recruited EIF volunteers for semen/serum tests, established a mouse EIF model via weight-loaded swimming to assess epididymal segmental injury, and constructed pyroptosis models of PC-1/DC-2 cells. Public database transcriptomic analysis identified MXRA7 expression and enriched pathways in epididymitis; MXRA7s function was verified via its knockdown/overexpression in DC-2 cells. PKC-MXRA7 association was explored by phosphorylation assays and CO-IP, and sperm incubation experiments evaluated MXRA7s effect on sperm function. ResultsEIF impaired human sperm motility, reduced mouse sperm quality and induced epididymitis with segment-specific pyroptosis. MXRA7 expression differed in PC-1/DC-2 cells and correlated with pyroptosis; it was phosphorylated by PKC, inhibited the NF-{kappa}B pathway to alleviate inflammation, and mitigated pyroptosis-induced sperm motility damage. ConclusionEIF induces epididymal epithelial pyroptosis and epididymitis, and MXRA7 exerts a protective effect mainly in caudal epididymal cells by alleviating pyroptosis, thus reducing sperm quality damage.

Ferroptosis is linked to various diseases, but the role of transferrin (TF) in endometriosis (EM) remains unclear. Expression levels of ferroptosis-related proteins, including transferrin (TF), transferrin receptor (TFRC), and glutathione peroxidase 4 (GPX4), were analyzed by western blotting. Compared to normal endometrial stromal cells, eutopic and ectopic endometrial stromal cells from EM patients exhibited significantly enhanced proliferative and migratory abilities, accompanied by a marked reduction in glutathione (GSH) levels in both eutopic and ectopic tissues. TF and TFRC expression was upregulated in ectopic endometrium relative to normal controls, while GPX4 expression was downregulated. To evaluate the functional role of TF, siRNA-mediated knockdown was performed in endometrial stromal cells, with knockdown efficiency confirmed by western blotting. Functional assays demonstrated that TF knockdown not only suppressed cell proliferation (CCK-8 and clonogenic assays) and migration (wound healing assay) but also significantly increased apoptosis rate (flow cytometry with Annexin V-FITC/PI staining).These findings implicate TF in the pathogenesis and progression of endometriosis, likely through modulating endometrial stromal cell proliferation, migration, and apoptosis.

BackgroundThe most prevalent kind of oral cancer is oral squamous cell carcinoma (OSCC), which has a poor prognosis because of delayed detection and a lack of molecular indicators. MethodsTranscriptomic data from TCGA were analyzed to identify differentially expressed genes between OSCC and normal samples. Functional enrichment analysis was performed to determine biological pathways. A protein-protein interaction network was constructed using STRING and visualized in Cytoscape to identify hub genes. ResultsA total of 5732 differentially expressed genes were identified, including 2459 upregulated and 3273 downregulated genes. Network analysis revealed several highly connected hub genes such as CDK1, CCNB1, TOP2A, BUB1, and MMP9. Functional enrichment indicated significant involvement of cell cycle regulation and cancer-associated pathways. ConclusionThis integrative analysis identified key regulatory hub genes that may be involved in OSCC progression. These genes may serve as promising biomarkers and therapeutic targets for future studies.

Though whole-genome duplication (WGD) contributes to cancer progression, the mechanism of post-WGD cell proliferation remains unclear. Here, using 6-day live-imaging, we analyzed the proliferation dynamics of more than 150 post-WGD HCT116 cell lineages. A quantitative comparison of mitotic patterns and cell fates between proliferative and non-proliferative lineages revealed that multipolar chromosome segregation in early mitosis is a key factor limiting the proliferative capacity of post-WGD progenies. Multipolar chromosome segregation suppressed post-WGD cell viability, particularly when accompanied by drastic chromosome loss or when it repeatedly occurred. Tracing proliferative lineages elucidated that they proliferated mainly by imposing the risk of multipolar chromosome segregation on one of two sub-lineages that formed after the first bipolar division. Meanwhile, a considerable proportion of proliferative lineages consisted entirely of progeny of early multipolar chromosome segregation events. Our results highlight key cellular events that determine the proliferation dynamics and diversity of post-WGD progenies, providing a fundamental reference for understanding WGD-associated bioprocesses. Summary statementLive image tracing of >150 cell lineages reveals the cross-generation dynamics of multipolar chromosome segregation that determine the fates of post-whole-genome duplication progeny cells.

Centriolar satellites are dynamic pericentrosomal structures implicated in centrosomal protein homeostasis and ciliogenesis. Centriolar satellites have been identified in vertebrates and were only recently described in flies. In C. elegans similar pericentriolar structures were reported for the Sjogrens Syndrome Nuclear Antigen 1 (SSNA-1). However, whether these foci have characteristics resembling centriolar satellites of vertebrates, has not been explored. We show that Spindle Assembly-1 (SAS-1), the interaction partner of SSNA-1 forms similar satellite-like structures that localize to a pericentrosomal space in a cell cycle-dependent manner. SAS-1 satellite-like structures associate with and are dependent on the microtubule cytoskeleton. Furthermore, we demonstrate that they form in a dose dependent manner, are dynamic and sensitive to agents disrupting weak hydrophobic interactions, characteristics of biomolecular condensates. We conclude that C. elegans has bona fide centriolar satellites highlighting their evolutionary conservation and importance across species, and at the same time opening new avenues for future mechanistic studies.