[Copy number variations of CCND1 gene and chromosome 11 centromere in acral melanoma].

Objective: To study the correlation between the copy number variations of CCND1 gene and chromosome 11 and their associations with clinicopathologic features in acral melanoma. Methods: Thirty-three acral melanoma cases diagnosed at the Department of Pathology of Peking University Third Hospital, Beijing, China from January 2018 to August 2021 were collected. Fluorescence in situ hybridization (FISH) was used to detect the copy number of CCND1 gene and centromere of chromosome 11. The relationship between the copy numbers of CCND1 and chromosome 11 centromere, and the correlation between CCND1 copy number and clinicopathologic characteristics were analyzed. Results: There were 15 male and 18 female patients, with an age ranging from 22-86 years. 63.6% (21/33) of the patients had an increased CCND1 gene copy number. 21.2% (7/33) of patients with increased CCND1 copy number had an accompanying chromosome 11 centromere copy number increase. 27.3% (9/33) of the cases had a low copy number of CCND1 gene, and 4 of them (4/33, 12.1%) were accompanied by chromosome 11 centromere copy number increase. 36.4% (12/33) of the cases had a high copy number of CCND1 gene, and 3 (3/33, 9.1%) of them were accompanied by chromosome 11 centromere copy number increase. No cases with CCND1 low copy number increase showed CCND1/CEP11 ratio greater than 2.00. The 11 cases with CCND1 high copy number increase showed CCND1/CEP11 ratio greater than or equal to 2.00. However, there was no significant correlation between CCND1 copy number increase and any of the examined clinicopathologic features such as age, sex, histological type, Breslow thickness, ulcer and Clark level. Conclusions: CCND1 copy number increase is a significant molecular alteration in acral melanoma. In some cases, CCND1 copy number increase may be accompanied by the copy number increase of chromosome 11. For these cases the copy number increase in CCND1 gene may be a result of the copy number change of chromosome 11.

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.

Bipartite binding interface recruiting HP1 to chromosomal passenger complex at inner centromeres.

Maintenance of ploidy depends on the mitotic kinase Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC) whose proficient activity is supported by HP1 enriched at inner centromeres. HP1 is known to associate with INCENP of the CPC in a manner that depends on the PVI motif conserved across HP1 interactors. Here, we found that the interaction of INCENP with HP1 requires not only the PVI motif but also its C-terminally juxtaposed domain. Remarkably, these domains conditionally fold the ß-strand (PVI motif) and the α-helix from a disordered sequence upon HP1 binding and render INCENP with high affinity to HP1. This bipartite binding domain termed SSH domain (Structure composed of Strand and Helix) is necessary and sufficient to attain a predominant interaction of HP1 with INCENP. These results identify a unique HP1-binding module in INCENP that ensures enrichment of HP1 at inner centromeres, Aurora B activity, and thereby mitotic fidelity.

The holocentricity in the dioecious nutmeg (Myristica fragrans) is not based on major satellite repeats.

Holocentric species are characterized by the presence of centromeres throughout the length of the chromosomes. We confirmed the holocentricity of the dioecious, small chromosome-size species Myristica fragrans based on the chromosome-wide distribution of the centromere-specific protein KNL1, α-tubulin fibers, and the cell cycle-dependent histone H3 serine 28 phosphorylation (H3S28ph) mark. Each holocentromere is likely composed of, on average, ten centromere units, but none of the identified and in situ hybridized high-copy satellite repeats is centromere-specific. No sex-specific major repeats are present in the high-copy repeat composition of male or female plants, or a significant difference in genome size was detected. Therefore, it is unlikely that M. fragrans possesses heteromorphic sex chromosomes.

The role of RNA in the maintenance of chromatin domains as revealed by antibody-mediated proximity labelling coupled to mass spectrometry.

Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity biotinylation method targeting the RNA and proteins constituents. The method that we termed antibody-mediated proximity labelling coupled to mass spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X chromosome in Drosophila. This analysis identified a number of known RNA-binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

The Aurora B-controlled PP1/RepoMan complex determines the spatial and temporal distribution of mitotic H2B S6 phosphorylation.

The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.

Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.

Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis.

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

Near telomere-to-telomere genome assemblies of two Chlorella species unveil the composition and evolution of centromeres in green algae.

BACKGROUND: Centromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown. RESULTS: We constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and Chlorella pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated their centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromeric CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements, although they differed significantly in their composition. Species within the Chlorella genus display a more diverse centromere composition, with major constituents including members of the LTR/Copia, LINE/L1, and LINE/RTEX families. This is in contrast to green algae including Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, in which centromere composition instead has a pronounced single-element composition. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeric sequence evolves more rapidly than sequence in non-centromeric regions. CONCLUSIONS: This study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.

Mathematical model for the role of multiple pericentromeric repeats on heterochromatin assembly.

Although the length and constituting sequences for pericentromeric repeats are highly variable across eukaryotes, the presence of multiple pericentromeric repeats is one of the conserved features of the eukaryotic chromosomes. Pericentromeric heterochromatin is often misregulated in human diseases, with the expansion of pericentromeric repeats in human solid cancers. In this article, we have developed a mathematical model of the RNAi-dependent methylation of H3K9 in the pericentromeric region of fission yeast. Our model, which takes copy number as an explicit parameter, predicts that the pericentromere is silenced only if there are many copies of repeats. It becomes bistable or desilenced if the copy number of repeats is reduced. This suggests that the copy number of pericentromeric repeats alone can determine the fate of heterochromatin silencing in fission yeast. Through sensitivity analysis, we identified parameters that favor bistability and desilencing. Stochastic simulation shows that faster cell division and noise favor the desilenced state. These results show the unexpected role of pericentromeric repeat copy number in gene silencing and provide a quantitative basis for how the copy number allows or protects repetitive and unique parts of the genome from heterochromatin silencing, respectively.

Long-read sequencing and optical mapping generates near T2T assemblies that resolves a centromeric translocation.

Long-read genome sequencing (lrGS) is a promising method in genetic diagnostics. Here we investigate the potential of lrGS to detect a disease-associated chromosomal translocation between 17p13 and the 19 centromere. We constructed two sets of phased and non-phased de novo assemblies; (i) based on lrGS only and (ii) hybrid assemblies combining lrGS with optical mapping using lrGS reads with a median coverage of 34X. Variant calling detected both structural variants (SVs) and small variants and the accuracy of the small variant calling was compared with those called with short-read genome sequencing (srGS). The de novo and hybrid assemblies had high quality and contiguity with N50 of 62.85 Mb, enabling a near telomere to telomere assembly with less than a 100 contigs per haplotype. Notably, we successfully identified the centromeric breakpoint of the translocation. A concordance of 92% was observed when comparing small variant calling between srGS and lrGS. In summary, our findings underscore the remarkable potential of lrGS as a comprehensive and accurate solution for the analysis of SVs and small variants. Thus, lrGS could replace a large battery of genetic tests that were used for the diagnosis of a single symptomatic translocation carrier, highlighting the potential of lrGS in the realm of digital karyotyping.

A validation strategy to assess the role of phase separation as a determinant of macromolecular localization.

Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.

Chromodomain mutation in S. pombe Kat5/Mst1 affects centromere dynamics and DNA repair.

KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that is involved in multiple cellular activities. This family is characterized in part by containing a chromodomain, a motif associated with binding methylated histones. We show that a chromodomain mutation in the S. pombe Kat5, mst1-W66R, has defects in pericentromere silencing. mst1-W66R is sensitive to camptothecin (CPT) but only at an increased temperature of 36°C, although it is proficient for growth at this temperature. We also describe a de-silencing effect at the pericentromere by CPT that is independent of RNAi and methylation machinery. We also show that mst1-W66R disrupts recruitment of proteins to repair foci in response to camptothecin-induced DNA damage. Our data suggest a function of Mst1 chromodomain in centromere heterochromatin formation and a separate role in genome-wide damage repair in CPT.

A working model for the formation of Robertsonian chromosomes.

Robertsonian chromosomes form by fusion of two chromosomes that have centromeres located near their ends, known as acrocentric or telocentric chromosomes. This fusion creates a new metacentric chromosome and is a major mechanism of karyotype evolution and speciation. Robertsonian chromosomes are common in nature and were first described in grasshoppers by the zoologist W. R. B. Robertson more than 100 years ago. They have since been observed in many species, including catfish, sheep, butterflies, bats, bovids, rodents and humans, and are the most common chromosomal change in mammals. Robertsonian translocations are particularly rampant in the house mouse, Mus musculus domesticus, where they exhibit meiotic drive and create reproductive isolation. Recent progress has been made in understanding how Robertsonian chromosomes form in the human genome, highlighting some of the fundamental principles of how and why these types of fusion events occur so frequently. Consequences of these fusions include infertility and Down's syndrome. In this Hypothesis, I postulate that the conditions that allow these fusions to form are threefold: (1) sequence homology on non-homologous chromosomes, often in the form of repetitive DNA; (2) recombination initiation during meiosis; and (3) physical proximity of the homologous sequences in three-dimensional space. This Hypothesis highlights the latest progress in understanding human Robertsonian translocations within the context of the broader literature on Robertsonian chromosomes.

Three factors ParA, TipN, and DnaA-mediated chromosome replication initiation are contributors of centromere segregation in Caulobacter crescentus.

The ability of bacteria to maintain chromosomal integrity throughout their life cycle is crucial for survival. In Caulobacter crescentus, the polar factor TipN has been proposed to be involved with the partitioning system ParABS. Cells with tipN knocked out display subtle segregation defects of the centromere-like region parS. We hypothesized that TipN's role with parS segregation is obscured by other forces that are ParABS-independent. To test our hypothesis, we removed one of those forces - chromosome replication - and analyzed the role of TipN with ParA. We first confirm that ParA retains its ability to transport the centromeric region parS from the stalked pole to the opposite pole in the absence of chromosome replication. Our data revealed that in the absence of chromosome replication, TipN becomes essential for ParA's ability to transport parS. Furthermore, we identify a potential connection between the replication initiator DnaA and TipN. Although TipN is not essential for viability, tipN knockout cells lose viability when the regulation of DnaA levels is altered. Our data suggest that the DnaA-dependent susceptibility of tipN knockout cells is connected to parS segregation. Collectively, this work provides insights into the complex regulation involved in the coordination of chromosome replication and segregation in bacteria.

Linear interaction between replication and transcription shapes DNA break dynamics at recurrent DNA break Clusters.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unravel the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA reveals co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity uncovers that head-to-head interactions between replication and transcription machinery result in 60% DSB contribution to the head-on compared to 40% for co-directional. Taken together we reveal TTR as a fragile class and show how the linear interaction between transcription and replication impacts genome stability.

A survey of chromosomal instability measures across mechanistic models.

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

Cytogenetic Analysis of Satellitome of Madagascar Leaf-Tailed Geckos.

Satellite DNA (satDNA) consists of sequences of DNA that form tandem repetitions across the genome, and it is notorious for its diversity and fast evolutionary rate. Despite its importance, satDNA has been only sporadically studied in reptile lineages. Here, we sequenced genomic DNA and PCR-amplified microdissected W chromosomes on the Illumina platform in order to characterize the monomers of satDNA from the Henkel's leaf-tailed gecko U. henkeli and to compare their topology by in situ hybridization in the karyotypes of the closely related Günther's flat-tail gecko U. guentheri and gold dust day gecko P. laticauda. We identified seventeen different satDNAs; twelve of them seem to accumulate in centromeres, telomeres and/or the W chromosome. Notably, centromeric and telomeric regions seem to share similar types of satDNAs, and we found two that seem to accumulate at both edges of all chromosomes in all three species. We speculate that the long-term stability of all-acrocentric karyotypes in geckos might be explained from the presence of specific satDNAs at the centromeric regions that are strong meiotic drivers, a hypothesis that should be further tested.

A pan-genome of 69 Arabidopsis thaliana accessions reveals a conserved genome structure throughout the global species range.

Although originally primarily a system for functional biology, Arabidopsis thaliana has, owing to its broad geographical distribution and adaptation to diverse environments, developed into a powerful model in population genomics. Here we present chromosome-level genome assemblies of 69 accessions from a global species range. We found that genomic colinearity is very conserved, even among geographically and genetically distant accessions. Along chromosome arms, megabase-scale rearrangements are rare and typically present only in a single accession. This indicates that the karyotype is quasi-fixed and that rearrangements in chromosome arms are counter-selected. Centromeric regions display higher structural dynamics, and divergences in core centromeres account for most of the genome size variations. Pan-genome analyses uncovered 32,986 distinct gene families, 60% being present in all accessions and 40% appearing to be dispensable, including 18% private to a single accession, indicating unexplored genic diversity. These 69 new Arabidopsis thaliana genome assemblies will empower future genetic research.