In the 40 years since the discovery of the CENP proteins, many studies have examined the role of these proteins and their interactions with other chromosomal proteins of the centromere and beyond. Together, these studies have yielded vast amounts of sequencing and proteomics data. Typically, each study has focused on a single question and the majority of each dataset remains largely unexplored. Often the interesting details of publicly deposited data are left behind, buried in archives online, while more and more new data are generated. Reanalysing these databases can represent a new paradigm for investigating diverse biological pathways in unprecedented detail. Here, we explore two publicly available pan-cancer proteomic datasets to compare proteins whose abundance correlates with CENP proteins, with a particular focus on CENP-C. Our analysis confirms an expected link between CENP-C and cohesin levels but reveals a surprising and unexpected correlation between CENP-C and proteins of the inner nuclear membrane and the NuMA protein. This guilt-by-association analysis has the potential to identify proteins that act in common pathways but never associate or colocalize and may not even be expressed at the same time in cells. As an example, we show here that it can reveal unexpected links that expand our conception of centromeric chromatin beyond chromosome segregation.
Various long non-coding RNAs (lncRNAs) have indicated their role in different regulatory processes and therapeutics in cervical cancer (CC). This study aims to assess the gene expression and methylation status of LINC00518 MAFG-AS1 in CC patients. Methylation-specific PCR (MS-PCR) and quantitative real-time PCR (qRT-PCR) were performed on 81 patients. The association of the promoter methylation status of cancer tissues was studied with HPV infection and clinicopathological factors. The Kaplan-Meier curves were used from the GEPIA and TANRIC databases to analyze the overall survival of CC patients. The bioinformatics analysis of relative gene expression was carried out using the GEPIA database. The RNAinter database was also explored to find out the potential interacting partners. This is the first-ever research revealing that hypomethylation of the LINC00518 gene promoter may be relevant to its oncogenic behavior in CC (p < 0.05). However, no significant difference was observed between the MAFG-AS1 methylation status of cancerous and normal tissues. A notable association between the methylation status of LINC00518 promoter and clinicopathological factors, including age (p < 0.001), histological subtypes (p < 0.00001), and differentiation degree (p < 0.00001), has been observed, indicating its possible role in predicting the severity and prognosis of this disease. Overall survival analysis showed a significant value for LINC00518 using GEPIA (p < 0.05). Our findings about the gene expression of LINC00518 and its hypomethylated status in cancerous tissues suggest a potential mechanism that might contribute to its dysregulation in CC and could serve as a potential clinical biomarker.
In Drosophila melanogaster females, as in most organisms, the segregation of homologous chromosomes in meiosis depends on the formation of crossovers between them. In most cases, crossovers require the synaptonemal complex (SC), a conserved multi-protein structure that forms between homologous chromosomes in early meiosis. Recent studies leveraging partial-loss-of-function alleles suggest that the SC plays a more direct role in crossover formation. One SC protein that is involved in crossover formation is SYP-4 in nematodes, which we found is a likely ortholog of the D. melanogaster SC protein Corolla. To create a hypomorphic allele of corolla in D. melanogaster, we used CRISPR/Cas9 to replace it with its D. mauritiana ortholog, yielding corollamau. Since SC protein sequences are rapidly diverging while maintaining the SC’s structure, we hypothesized that this replacement would enable SC assembly but show defects in crossover formation. Indeed, at 25 °C corollamau homozygous females exhibited defects in SC maintenance and crossover formation, resulting in moderate levels of chromosome missegregation. At 18 °C, SC maintenance was rescued, and recombination rates were improved, although they remained significantly lower than observed in wild type. Unexpectedly, in homozygotes we also observed unique polycomplexes composed of the SC proteins Corolla and Corona but lacking the transverse filament protein C(3)G. Overall, we report a novel hypomorphic allele of corolla that suggests Corolla regulates crossover formation. Further, the unique polycomplexes found in mutant flies may provide new insights into SC architecture and protein-protein interactions.
The Heterochromatin Protein 1 (HP1) family proteins are key regulators of chromatin structure and genome function, acting as “reader” proteins that recognize and bind to histone H3 lysine 9 methylation (H3K9me). Beyond their canonical role in heterochromatin formation and transcriptional repression, HP1 proteins exhibit functional versatility, participating in transcriptional activation, RNA processing, DNA repair, and chromosome segregation. This multifunctionality is mediated partially by post-translational modifications (PTMs), with phosphorylation emerging as a central regulatory mechanism. This review explores the diverse effects of HP1 phosphorylation on protein function and chromatin interactions, focusing on Drosophila melanogaster HP1a and its orthologs, mammalian HP1α and S. pombe Swi6. Phosphorylation in the N-terminal tail enhances HP1’s affinity for H3K9me, promoting transcriptional silencing. Mitotic phosphorylation of serine residues in the hinge region, regulated by kinases such as AURKB and NDR1/2, leads to chromatin release and relocalization to the kinetochore, enabling proper chromosome segregation. Additionally, phosphorylation modulates HP1 phase separation dynamics, influencing nuclear compartmentalization and chromatin condensation. These findings highlight phosphorylation as a versatile molecular switch that enables HP1 proteins to transition between structural and regulatory roles, contributing to their evolutionary conserved multifunctionality in genome regulation and cell division. Further investigation into HP1 phosphorylation across species and contexts is essential to fully understand its contributions to chromatin biology.
BACKGROUND:Esophageal cancer (EC) is still a difficult problem in medicine, depriving many patients of their lives every year. RAD18 and ATM were implicated in cancers including esophageal squamous cell carcinoma (ESCC). However, whether RAD18/ATM axis influences ESCC progression remains unclear. METHODS:The abundance of genes and proteins was evaluated using RT-qPCR and western blot. Cell proliferation, migration and invasion were examined using clone formation, scratch test and transwell. The level of ATM ubiquitination was verified and experimented using Co-IP. RESULTS:Our findings found that RAD18 expression was enhanced in TCGA database, in ESCC patients and ESCC cells. Similarly, ATM expression was declined in ESCC patients and ESCC cells. RAD18 silencing resulted in suppression of cell proliferation, migration and invasion of ESCC cells, which were abolished by ATM silencing. In addition, ATM silencing promoted malignant behaviors of ESCC cells by activating STAT3/PD-L1 axis, which was reversed by PD-L1 knockdown. Moreover, RAD18 could reduce ATM protein levels. CONCLUSION:RAD18 mediated ATM ubiquitination to reduce ATM protein level, thereby activating STAT3/PD-L1 axis and strengthening cell proliferation, migration and invasion of ESCC cells.
Overexpression or knockdown of a specific gene is usually helpful in understanding its underlying molecular mechanism. PVT1 gene is regarded as an oncogenic long non-coding RNA (lncRNA) in many cancers, including breast invasive carcinoma (BRCA). We investigated some of the underlying molecular mechanisms of PVT1 in human invasive breast cancer MDA-MB-231 cells. Differentially expressed genes (DEGs) were obtained after PVT1 overexpression and knockdown in MDA-MB-231 cells from the gene expression profiles GSE175736 and GSE97587. RNAInter database was used to predict miRNAs and TFs that have interactions with PVT1. Competing endogenous RNA (ceRNA) and transcription regulatory networks visualized using Cytoscape software. It was found that HLA-G, GBP4, SERPINE1, DHRS2, MT1X, and PRLR were common PVT1 co-upregulated and co-downregulated genes in the two datasets. SERPINE1 was identified as the most positively correlated gene with PVT1 expression in MDA-MB-231 cells. DEGs in overexpressed and silenced PVT1 cells were enriched in the cell adhesion process and JAK-STAT signaling pathway, respectively. In the ceRNA network, PVT1 acts as a competing endogenous RNA for downregulated miR-145-5p, miR-17-5p, and miR-20a-5p. PVT1/miR-145-5p/SERPINE1 was a common axis in ceRNA networks in the two datasets. SERPINIE1 was also a common node between ceRNA and transcription regulatory networks. RT-qPCR validated the anticipated levels of PVT1, miR-145-5p, and SERPINE1 in MDA-MB-231 cancer compared to MCF-10 A noncancerous cells. Taken together, the results of this work shed light on the several possible oncogenic mechanisms of PVT1, including its closely related genes and signaling pathways.
Sex chromosomes have independently evolved in various species, displaying unique evolutionary patterns, including differentiation, degeneration, and repetitive DNA accumulation. Pentatomidae hemipterans are characterized by a highly conserved diploid number of 2n = 14 with a XX/XY sex chromosome system, i.e. 2n = 14, 12 A + XY. Thus, it represents an interesting group for investigating the reorganization of repeats in conserved karyotypes, i.e. the absence of large chromosomal rearrangements. Using comparative genomic hybridization (CGH) with male and female genomic DNAs (gDNA), this study examined a total of 25 Pentatomidae species to uncover repetitive DNA dynamics and their role in chromosome differentiation, especially sex chromosome differentiation. New karyotype data for nine species reinforces the chromosomal stasis in Pentatomidae for macro-chromosomal structure. However, significant variability in repetitive DNA patterns on autosomes and sex chromosomes has been revealed despite the karyotypic conservation. Autosomal signals varied in intensity and distribution, with some species exhibiting terminal enrichment of repeats, while others displayed dispersed patterns. Sex chromosomes showed distinct hybridization patterns, with the Y chromosome exhibiting more significant variability compared to the X. These findings emphasize the dynamic nature of sex chromosomes and suggest further studies combining genomic sequencing and cytogenetics to uncover sequences and the mechanisms behind their evolution.
Tetraploidisation plays a crucial role in evolution, development, stress adaptation, and disease, but its beneficial or pathological effects in different tissues remain unclear. This study aims to compare physiological and unphysiological tetraploidy in eight steps: 1) mechanisms of diploidy-to-tetraploidy transition, 2) induction and elimination of unphysiological tetraploidy, 3) tetraploid cell characteristics, 4) stress-induced unphysiological tetraploidy, 5) comparison of physiological vs. unphysiological tetraploidy, 6) consequences of unphysiological stress-induced tetraploidy, 7) nutritional or pharmacological prevention strategies of tetraploidisation, and 8) knowledge gaps and future perspectives. Unphysiological tetraploidy is an adaptive stress response at a given threshold, often involving mitotic slippage. If tetraploid cells evade elimination through apoptosis or immune surveillance, they may re-enter the cell cycle, causing genetic instability, micronuclei formation, aneuploidy, modification of the epigenome and the development of diseases. The potential contributions of unphysiological tetraploidy to neurodegenerative, cardiovascular and diabetes related diseases are summarized in schematic figures and contrasted with its role in cancer development. The mechanisms responsible for the transition from physiological to unphysiological tetraploidy and the tolerance to tetraploidisation in unphysiological tetraploidy are not fully understood. Understanding these mechanisms is of critical importance to allow the development of targeted nutritional and pharmacological prevention strategies and therapies.
Some insects have holocentric chromosomes, with multiple kinetochores rather than a single centromere. They also lack the CENP-A and CENP-C proteins, suggesting a kinetochore assembly process different from that of monocentric chromosomes. The homolog of CENP-T was recently shown to bind silent chromatin and to play a key role in kinetochore assembly in Bombyx mori, but its role in other insects with holocentric chromosomes is unknown. We identified kinetochore genes and analyzed their expression in Spodoptera frugiperda. We silenced the kinetochore genes cenp-L, cenp-S, cenp-X and ndc80 and searched for chromosome segregation defects in Sf9 cells. All kinetochore genes except cenp-S were more strongly expressed in gonadal than in somatic tissues. Immunofluorescence microscopy and RT-qPCR demonstrated the effective silencing of the target genes by transfection with dsRNA. In Sf9 cells depleted of CENP-L and NDC80, immunofluorescence microscopy revealed increases in mitotic index and in the proportion of cells with unaligned chromosomes or multipolar spindles. The depletion of CENP-S and CENP-X had no effect on mitotic index and no division defects were observed. This suggests that CENP-L and NDC80 play key roles in chromosome segregation, whereas the functions of CENP-S and CENP-X remain unknown. We have begun to characterize the kinetochore proteins (CENP-L, CENP-S, CENP-X, NDC80), a prerequisite for holocentromere identification in S. frugiperda. This study also provides the first information about the role, in Lepidoptera, of CENP-L, a protein essential to the structure of the constitutive centromere-associated network in species with monocentric chromosomes.
Satellite DNAs are highly repetitive, tandemly arranged sequences, typically making up large portions (> 20
Therian female mammals compensate for the dosage of X-linked gene expression by inactivating one of the X-chromosomes. X-inactivation is facilitated by the master regulator Xist long non-coding RNA, which coats the inactive-X and facilitates heterochromatinization through recruiting different chromatin modifiers and changing the X-chromosome 3D conformation. However, many mechanistic aspects behind the X-inactivation process remain poorly understood. Among the many contributing players, CTCF has emerged as one of the key players in orchestrating various aspects related to X-chromosome inactivation by interacting with several other protein and RNA partners. In general, CTCF is a well-known architectural protein, which plays an important role in chromatin organization and transcriptional regulation. Here, we provide significant insight into the role of CTCF in orchestrating X-chromosome inactivation and highlight future perspectives.
Polyploidy is a common feature in eukaryotes with one of paramount consequences leading to better environmental adaptation. Heterochromatin is often located at telomeres and centromeres and contains repetitive DNA sequences. Sainfoin (Onobrychis viciifolia) is an important perennial forage legume for sustainable agriculture. However, there are only a few studies on the sainfoin genome and chromosomes. In this study, novel tandem repetitive DNA sequences of the sainfoin genome (OnVi180, OnVi169, OnVi176 and OnVidimer) were characterized using bioinformatics, molecular and cytogenetic approaches. The OnVi180 and OnVi169 elements colocalized within functional centromeres. The OnVi176 and OnVidimer elements were localized in centromeric, subtelomeric and interstitial regions. We constructed a sainfoin karyotype that distinguishes the seven basic chromosome groups. Our study provides the first detailed description of heterochromatin and chromosome structure of sainfoin and proposes an origin of heterozygous ancestral genomes, possibly from the same ancestral diploid species, not necessarily from different species, or for chromosome rearrangements after polyploidy. Overall, we discuss our novel and complementary findings in a polyploid crop with unique and complex chromosomal features.
Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.
Among the repetitive elements, satellite DNA (SatDNA) emerges as extensive arrays of highly similar tandemly repeated units, spanning megabases in length. Given that the satDNA PboSat01-176, previously characterized in P. boiei, prompted our interest for having a high abundance in P. boiei and potential for centromeric satellite, here, we employed various approaches, including low coverage genome sequencing, followed by computational analysis and chromosomal localization techniques in four Proceratophrys species and, investigating the genomic presence and sharing, as well as its potential for chromosomal centromere marker in Proceratophrys frog species. Our findings demonstrate that PboSat01-176 exhibits high abundance across all four Proceratophrys species, displaying distinct characteristics that establish it as the predominant repetitive DNA element in these species. The satellite DNA is prominently clustered in the peri/centromeric region of the chromosomes, particularly in the heterochromatic regions. The widespread presence of PboSat01-176 in closely related Proceratophrys species reinforces the validity of the library hypothesis for repetitive sequences. Thus, this study highlighted the utility of the satDNA family PboSat01-176 as a reliable centromeric marker in Proceratophrys species, with potential to be applied in other species of anuran amphibians. The observed sharing and maintenance of this sequence within the genus suggest possibilities for future research, particularly through expanded sampling to elucidate parameters that underlie the library hypothesis and the evolutionary dynamics of satDNA sequences.
Centromeres are chromatin structures specialized in sister chromatid cohesion, kinetochore assembly, and microtubule attachment during chromosome segregation. The regional centromere of vertebrates consists of long regions of highly repetitive sequences occupied by the Histone H3 variant CENP-A, and which are flanked by pericentromeres. The three-dimensional organization of centromeric chromatin is paramount for its functionality and its ability to withstand spindle forces. Alongside CENP-A, key contributors to the folding of this structure include components of the Constitutive Centromere-Associated Network (CCAN), the protein CENP-B, and condensin and cohesin complexes. Despite its importance, the intricate architecture of the regional centromere of vertebrates remains largely unknown. Recent advancements in long-read sequencing, super-resolution and cryo-electron microscopy, and chromosome conformation capture techniques have significantly improved our understanding of this structure at various levels, from the linear arrangement of centromeric sequences and their epigenetic landscape to their higher-order compaction. In this review, we discuss the latest insights on centromere organization and place them in the context of recent findings describing a bipartite higher-order organization of the centromere.
Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.
