The effect of centromere protein Fta2 phosphorylation during meiosis.

During meiosis, defects in cohesin localization within the centromere region can result in various diseases. Accurate cohesin localization depends on the Mis4-Ssl3 loading complex. Although it is known that cohesin completes the loading process with the help of the loading complex, the mechanisms underlying its localization in the centromere region remain unclear. Previous studies suggest cohesin localization in the centromere is mediated by phosphorylation of centromeric proteins. In this study, we focused on the Fta2 protein, a component of the Sim4 centromere protein complex. Using bioinformatics methods, potential phosphorylation sites were identified, and fta2-9A and fta2-9D mutants were constructed in Schizosaccharomyces pombe. The phenotypes of these mutants were characterized through testing thiabendazole (TBZ) sensitivity and fluorescent microscopy localization. Results indicated that Fta2 phosphorylation did not impact mitosis but affected chromosome segregation during meiosis. This study suggests that Fta2 phosphorylation is vital for meiosis and may be related to the specific localization of cohesin during this process.

Immunodetection of tubulin and centromeric histone H3 (CENH3) proteins in Glycine species.

BACKGROUND: The centromeres appear as primary constrictions on monocentric metaphase chromosomes; where sister chromatids are held together and assemble the proteinaceous kitechore complex at which microtubule proteins attach during nuclear divisions for pulling sister chromatids to opposite cell poles. The movement of chromosomes is usually governed by structural proteins that are either species-specific or highly conserved, such as the centromere-specific histone H3 (CENH3) and tubulin proteins, respectively. METHODS AND RESULTS: We aimed to detect these proteins across eight different Glycine species by an immunofluorescence assay using specific antibodies. Furthermore, with the α-tubulin antibody we traced the dynamics of microtubules during the mitotic cell cycle in Glycine max. With two-color immunofluorescence staining, we showed that both proteins interact during nuclear division. CONCLUSIONS: Finally, we proved that in different diploid and tetraploid Glycine species CENH3 can be detected in functional centromeres with spatial proximity of microtubule proteins.

Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus.

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.

Pericentromeric satellite RNAs as flexible protein partners in the regulation of nuclear structure.

Pericentromeric heterochromatin is mainly composed of satellite DNA sequences. Although being historically associated with transcriptional repression, some pericentromeric satellite DNA sequences are transcribed. The transcription events of pericentromeric satellite sequences occur in highly flexible biological contexts. Hence, the apparent randomness of pericentromeric satellite transcription incites the discussion about the attribution of biological functions. However, pericentromeric satellite RNAs have clear roles in the organization of nuclear structure. Silencing pericentromeric heterochromatin depends on pericentromeric satellite RNAs, that, in a feedback mechanism, contribute to the repression of pericentromeric heterochromatin. Moreover, pericentromeric satellite RNAs can also act as scaffolding molecules in condensate subnuclear structures (e.g., nuclear stress bodies). Since the formation/dissociation of nuclear condensates provides cell adaptability, pericentromeric satellite RNAs can be an epigenetic platform for regulating (sub)nuclear structure. We review current knowledge about pericentromeric satellite RNAs that, irrespective of the meaning of biological function, should be functionally addressed in regular and disease settings. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA in Disease and Development > RNA in Disease.

JRK binds satellite III DNA and is necessary for the heat shock response.

JRK is a DNA-binding protein of the pogo superfamily of transposons, which includes the well-known centromere binding protein B (CENP-B). Jrk null mice exhibit epilepsy, and growth and reproductive disorders, consistent with its relatively high expression in the brain and reproductive tissues. Human JRK DNA variants and gene expression levels are implicated in cancers and neuropsychiatric disorders. JRK protein modulates ß-catenin-TCF activity but little is known of its cellular functions. Based on its homology to CENP-B, we determined whether JRK binds centromeric or other satellite DNAs. We show that human JRK binds satellite III DNA, which is abundant at the chromosome 9q12 juxtacentromeric region and on Yq12, both sites of nuclear stress body assembly. Human JRK-GFP overexpressed in HeLa cells strongly localises to 9q12. Using an anti-JRK antiserum we show that endogenous JRK co-localises with a subset of centromeres in non-stressed cells, and with heat shock factor 1 following heat shock. Knockdown of JRK in HeLa cells proportionately reduces heat shock protein gene expression in heat-shocked cells. A role for JRK in regulating the heat shock response is consistent with the mouse Jrk null phenotype and suggests that human JRK may act as a modifier of diseases with a cellular stress component.

Targeting pericentric non-consecutive motifs for heterochromatin initiation.

Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation1-3. However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear4, as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences2,5. Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates.

Rapid meiotic prophase chromosome movements in Arabidopsis thaliana are linked to essential reorganization at the nuclear envelope.

Meiotic rapid prophase chromosome movements (RPMs) require connections between the chromosomes and the cytoskeleton, involving SUN (Sad1/UNC-84)-domain-containing proteins at the inner nuclear envelope (NE). RPMs remain significantly understudied in plants, with respect to their importance in the regulation of meiosis. Here, we demonstrate that Arabidopsis thaliana meiotic centromeres undergo rapid (up to 500 nm/s) and uncoordinated movements during the zygotene and pachytene stages. These centromere movements are not affected by altered chromosome organization and recombination but are abolished in the double mutant sun1 sun2. We also document the changes in chromosome dynamics and nucleus organization during the transition from leptotene to zygotene, including telomere attachment to SUN-enriched NE domains, bouquet formation, and nucleolus displacement, all of which were defective in sun1 sun2. These results establish A. thaliana as a model species for studying the functional implications of meiotic RPMs and demonstrate the mechanistic conservation of telomere-led RPMs in plants.

Precise identification of cascading alpha satellite higher order repeats in T2T-CHM13 assembly of human chromosome 3.

AIM: To precisely identify and analyze alpha-satellite higher-order repeats (HORs) in T2T-CHM13 assembly of human chromosome 3. METHODS: From the recently sequenced complete T2T-CHM13 assembly of human chromosome 3, the precise alpha satellite HOR structure was computed by using the novel high-precision GRM2023 algorithm with global repeat map (GRM) and monomer distance (MD) diagrams. RESULTS: The major alpha satellite HOR array in chromosome 3 revealed a novel cascading HOR, housing 17mer HOR copies with subfragments of periods 15 and 2. Within each row in the cascading HOR, the monomers were of different types, but different rows within the same cascading 17mer HOR contained more than one monomer of the same type. Each canonical 17mer HOR copy comprised 17 monomers belonging to 16 different monomer types. Another pronounced 10mer HOR array was of the regular Willard's type. CONCLUSION: Our findings emphasize the complexity within the chromosome 3 centromere as well as deviations from expected highly regular patterns.

Comparative genomics of the closely related fungal genera Cryptococcus and Kwoniella reveals karyotype dynamics and suggests evolutionary mechanisms of pathogenesis.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

The Interaction of NF-κB Transcription Factor with Centromeric Chromatin.

Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. The centromere is composed of both CENP-A nucleosomes (CENP-Anuc) and H3 nucleosomes (H3nuc) and is enriched with alpha-satellite (α-sat) DNA repeats. These CENP-Anuc have a different structure than H3nuc, decreasing the base pairs (bp) of wrapped DNA from 147 bp for H3nuc to 121 bp for CENP-Anuc. All these factors can contribute to centromere function. We investigated the interaction of H3nuc and CENP-Anuc with NF-κB, a crucial transcription factor in regulating immune response and inflammation. We utilized atomic force microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-κB. We found that NF-κB unravels H3nuc, removing more than 20 bp of DNA, and that NF-κB binds to the nucleosomal core. Similar results were obtained for the truncated variant of NF-κB comprised only of the Rel homology domain and missing the transcription activation domain (TAD), suggesting that RelATAD is not critical in unraveling H3nuc. By contrast, NF-κB did not bind to or unravel CENP-Anuc. These findings with different affinities for two types of nucleosomes to NF-κB may have implications for understanding the mechanisms of gene expression in bulk and centromere chromatin.

Neotelomeres and telomere-spanning chromosomal arm fusions in cancer genomes revealed by long-read sequencing.

Alterations in the structure and location of telomeres are pivotal in cancer genome evolution. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeats, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. These results provide a framework for the systematic study of telomeric repeats in cancer genomes, which could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

Meiosis-specific functions of kinetochore protein SPC105R required for chromosome segregation in Drosophila oocytes.

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and coorientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and biorientation of homologues, which are critical for accurate chromosome segregation in meiosis I.

Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription.

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

[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.

Chromosome segregation: Brushing up on centromeres.

Turning centromere DNA into a mechanical spring is central to the fidelity of chromosome segregation. A recent study shows how centromere DNA loops and partitioning cohesin and condensin convert centromeres and pericentromeres into bipartite bottlebrushes.

Centromere sequence-independent but biased loading of subgenome-specific CENH3 variants in allopolyploid Arabidopsis suecica.

Centromeric nucleosomes are determined by the replacement of the canonical histone H3 with the centromere-specific histone H3 (CENH3) variant. Little is known about the centromere organization in allopolyploid species where different subgenome-specific CENH3s and subgenome-specific centromeric sequences coexist. Here, we analyzed the transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species Arabidopsis suecica. Synthetic A. thaliana x A. arenosa hybrids were generated and analyzed to mimic the early evolution of A. suecica. Our expression analyses indicated that CENH3 has generally higher expression levels in A. arenosa compared to A. thaliana, and this pattern persists in the hybrids. We also demonstrated that despite a different centromere DNA composition, the centromeres of both subgenomes incorporate CENH3 encoded by both subgenomes, but with a positive bias towards the A. arenosa-type CENH3. The intermingled arrangement of both CENH3 variants demonstrates centromere plasticity and may be an evolutionary adaption to handle more than one CENH3 variant in the process of allopolyploidization.

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 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.

Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin.

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.

Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms.

Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a⋅H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a⋅H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.