Novel Cascade Alpha Satellite HORs in Orangutan Chromosome 13 Assembly: Discovery of the 59mer HOR-The largest Unit in Primates-And the Missing Triplet 45/27/18 HOR in Human T2T-CHM13v2.0 Assembly.

From the recent genome assembly NHGRI_mPonAbe1-v2.0_NCBI (GCF_028885655.2) of orangutan chromosome 13, we computed the precise alpha satellite higher-order repeat (HOR) structure using the novel high-precision GRM2023 algorithm with Global Repeat Map (GRM) and Monomer Distance (MD) diagrams. This study rigorously identified alpha satellite HORs in the centromere of orangutan chromosome 13, discovering a novel 59mer HOR-the longest HOR unit identified in any primate to date. Additionally, it revealed the first intertwined sequence of three HORs, 18mer/27mer/45mer HORs, with a common aligned "backbone" across all HOR copies. The major 7mer HOR exhibits a Willard's-type canonical copy, although some segments of the array display significant irregularities. In contrast, the 14mer HOR forms a regular Willard's-type HOR array. Surprisingly, the GRM2023 high-precision analysis of chromosome 13 of human genome assembly T2T-CHM13v2.0 reveals the presence of only a 7mer HOR, despite both the orangutan and human genome assemblies being derived from whole genome shotgun sequences.

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.

Canonical and noncanonical regulators of centromere assembly and maintenance.

Centromeres are specialized chromosomal domains where the kinetochores assemble during cell division to ensure accurate transmission of the genetic information to the two daughter cells. The centromeric function is evolutionary conserved and, in most organisms, centromeres are epigenetically defined by a unique chromatin containing the histone H3 variant CENP-A. The canonical regulators of CENP-A assembly and maintenance are well-known, yet some of the molecular mechanisms regulating this complex process have only recently been unveiled. We review the most recent advances on the topic, including the emergence of new and unexpected factors that favor and regulate CENP-A assembly and/or maintenance.

macroH2A1 drives nucleosome dephasing and genome instability in histone humanized yeast.

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects.

The Structure of Simple Satellite Variation in the Human Genome and Its Correlation With Centromere Ancestry.

Although repetitive DNA forms much of the human genome, its study is challenging due to limitations in assembly and alignment of repetitive short-reads. We have deployed k-Seek, software that detects tandem repeats embedded in single reads, on 2,504 human genomes from the 1,000 Genomes Project to quantify the variation and abundance of simple satellites (repeat units <20 bp). We find that the ancestral monomer of Human Satellite 3 makes up the largest portion of simple satellite content in humans (mean of ∼8 Mb). We discovered ∼50,000 rare tandem repeats that are not detected in the T2T-CHM13v2.0 assembly, including undescribed variants of telomericand pericentromeric repeats. We find broad homogeneity of the most abundant repeats across populations, except for AG-rich repeats which are more abundant in African individuals. We also find cliques of highly similar AG- and AT-rich satellites that are interspersed and form higher-order structures that covary in copy number across individuals, likely through concerted amplification via unequal exchange. Finally, we use pericentromeric polymorphisms to estimate centromeric genetic relatedness between individuals and find a strong predictive relationship between centromeric lineages and pericentromeric simple satellite abundances. In particular, ancestral monomers of Human Satellite 2 and Human Satellite 3 abundances correlate with clusters of centromeric ancestry on chromosome 16 and chromosome 9, with some clusters structured by population. These results provide new descriptions of the population dynamics that underlie the evolution of simple satellites in humans.

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.

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes.

Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.

Structural basis for Mis18 complex assembly and its implications for centromere maintenance.

The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

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.

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.

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.

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.

Vertebrate centromere architecture: from chromatin threads to functional structures.

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.

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.

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.