Chromosome Research最新文献

While the centromeric function is conserved and epigenetically specified by CENP-A, centromeric DNA, typically composed of satellite repeats, is highly divergent and rapidly evolving. In the species of the genus Equus (horses, asses and zebras), also known as equids, the numerous centromeres devoid of satellite repeats enabled us to carry out molecular analysis of centromeric chromatin establishing a unique model system for mammalian centromere biology. In this review, after a brief description of the rapid evolution of equids, we outline one of our most relevant initial discoveries: the position of CENP-A binding domains is variable among individuals giving rise to epialleles which are inherited as Mendelian traits. This positional variability was recently confirmed in human centromeres whose repetitive DNA organization could be analyzed thanks to telomere-to-telomere (T2T) genome assemblies. Another unexpected observation was that, in equids, CENP-B does not bind the centromeric core and is uncoupled from CENP-A and CENP-C. CENP-B is absent from the majority of chromosomes while the CENP-B binding DNA sequence (CENP-B box) is comprised within a satellite that is localized at pericentromeric or terminal positions. Finally, comparative molecular and cytogenetic analyses of satellite-free centromeres revealed that the birth of neocentromeres during the evolution of this genus occurred through two alternative mechanisms: centromere repositioning and Robertsonian fusion. These events played a key role in karyotype reshuffling and speciation. Investigating centromere organization in equids provided new insights into the complexity of centromere organization across the vast biodiversity of the mammalian world, where the majority of species remain understudied.

Objectives: This study aimed to analyze chromosomal arm-level copy number variations (CNVs) in benign diseases, cervical intraepithelial neoplasia (CIN), and cervical cancer (CC) using low-coverage whole genome sequencing (LC-WGS) and evaluate the efficacy of the ultrasensitive chromosomal aneuploidy detector (UCAD) model in distinguishing CC from CIN and benign diseases.

Methods: Cervical exfoliated cell specimens from 50 patients were collected for high-risk human papillomavirus(hr-HPV) testing, ThinPrep Cytologic Test (TCT), and CNV detection via LC-WGS. UCAD was employed to analyze chromosomal changes, with validation using WGS data from the National Center for Biotechnology Information(NCBI) database.

Results: Among 50 patients, 8 had benign disease, 3 CIN1, 15 CIN2, 6 CIN2-3, 13 CIN3, and 5 CC. Chromosomal instability was detected in 9 patients (18%): all 5 CC cases, 3 CIN3 cases, and 1 CIN1 case. Gains in 3q were observed in all CC and CIN3 cases with CNVs. UCAD achieved 100% sensitivity and 91.11% specificity in differentiating CC from CIN and benign diseases, outperforming hr-HPV and TCT. The UCAD model was also applied to 5 CC and 1 high-grade squamous intraepithelial lesion (HSIL) cases obtained from the NCBI database, and the findings validated its ability to detect chromosomal aberrations in all cases.

Conclusions: CNV analysis of cervical exfoliated cells shows promise for CC detection, with UCAD demonstrating high accuracy. Further validation in larger cohorts is needed to confirm its clinical utility.

Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural α-satellite DNA from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

Glyphosate resistance in crop weeds is commonly attributed to rapid evolution through the amplification of the target gene, EPSPS (5-enolpyruvylshikimate-3-phosphate synthase). This amplification typically occurs through mechanisms such as unequal recombination, segmental duplications within the target chromosome, or the formation of ring chromosomes and extrachromosomal circular (ecc) DNA elements containing EPSPS. However, structural abnormalities in chromosomes not directly associated with EPSPS amplification have not been documented in the glyphosate-resistant weed population. Here, we describe the presence of a large chromosome found exclusively in the glyphosate-resistant Amaranthus tuberculatus (waterhemp) population but absent in susceptible counterparts. This large chromosome (~ 6 μm) is approximately twice the size of normal chromosomes (~ 2-3 μm) and is present in both male and female euploid plants (2n = 32) in a heteromorphic state. It aroses through pericentromeric heterochromatin expansion and duplications of the 5S rDNA locus but notably lacks the EPSPS gene. The large chromosome pairs with its normal homolog but was not transmitted to progeny in controlled greenhouse matings, suggesting a fitness cost in the absence of glyphosate selection pressure. This large chromosome offers a potential resource for the investigation of chromosome evolution of adaptive traits for glyphosate resistance in A. tuberculatus.

During spermiogenesis, histones are removed from most genomic loci and are replaced by protamines in mature sperm nuclei. Yet, centromeres appear resistant to this process. We review the experimental evidence that the centromeric histone CENP-A is maintained in mature sperm nuclei, comparing human, bovine, mouse and fly species. We also recall how the detection of centromeres in mature sperm nuclei in the 1990's contributed to the isolation of the CENP-A protein and the eventual cloning of the human CENP-A gene. Further, based on more recent genetic studies carried out in flies and in mice, we discuss the inheritance and functional importance of paternal CENP-A and how it is complemented by maternal CENP-A to give rise to a healthy embryo. Finally, we raise some unanswered questions regarding the exclusive maintenance of CENP-A on sperm, the organisation of sperm centromeric chromatin and its importance for fertility and early embryo development.

In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.

Transposable elements (TEs) are widely present in eukaryotic genomes, where they can contribute to genome size and functional modifications. As new genomes are sequenced and annotated, more studies can be conducted regarding TE content, distribution, and genome evolution. TEs are extensively diversified in fish genomes resulting in an important role in genome and chromosome evolution. However, curated TE libraries are still scarce in non-model organisms, making it difficult to evaluate TE's impact on genomic modifications thoroughly. Here, we aimed to obtain a curated TE library from the neotropical fish Apareiodon sp. genome. The prospection and curation of the TE library resulted in 244 families from 18 superfamilies of DNA transposons and retrotransposons, which comprise about 10% of the genome, with most insertions fitting in one or a few families. A greater diversity of retrotransposon families is present, especially for Ty3 superfamily. Despite the greater number of retrotransposon families, DNA transposons are the most abundant in the genome, with 37% of all TE insertions belonging to the Tc1-Mariner superfamily. Complete TE copies were observed for almost all superfamilies, with most of the sequences on the Tc1-Mariner group. DNA transposons and SINEs presented older insertions in the genome, followed by LINEs and LTR retrotransposons. TE genome density is highest in the cs25 scaffold, and enriched for Helitron elements. With these data, allied to previous studies on chromosome evolution, we suggest that cs25 bears the W chromosome specific region of the Apareiodon sp. genome, with the presence of significant amount of Helitron insertions.

DNA topoisomerase II beta (TOP2B) is required for correct execution of certain developmental transcriptional programs and for signal-induced transcriptional activation, including transcriptional activation by nuclear hormone ligands such as retinoic acid. In addition, TOP2B is enriched at genomic locations occupied by CCCTC-Binding factor (CTCF) and cohesin (RAD21). suggesting a role in chromosome looping and/or establishing or maintaining aspects of chromosome 3D structure. This led us to investigate the effect of TOP2B inactivation on patterns of intra- and inter- chromosomal interaction that reflect the 3D architecture of the genome. Using the retinoic acid responsive SH-SY5Y neuroblastoma cell line model, we had previously demonstrated many gene expression changes upon retinoic acid treatment and upon deletion of TOP2B. We report here that these expression changes in TOP2B null versus WT cells are accompanied by surprisingly subtle changes in local chromosome organization. However, we do observe quantitative changes in chromosome organization on a megabase scale. First, lack of TOP2B did affect compartment strength changes that occur upon ATRA treatment. Second, we observe an excess of very long-range interactions, reminiscent of interactions seen in mitotic cells, suggesting the possibility that in the absence of TOP2B some mitotic interactions are retained. Third, we see quantitative changes in centromere-telomere interactions, again indicating global changes at the megabase and chromosome level. These data support the surprising conclusion that TOP2B has only a minor role in chromosome dynamics and organization.

Jumping translocations and jumping-like translocations constitute a rare category of complex chromosomal rearrangements, which are primarily observed in hematologic disorders and solid tumors. This study outlines a complex structural mosaic rearrangement involving a single recipient chromosome and three distinct donor chromosomes, with varying patterns of mosaicism observed across different cell lines. The rearrangement was confirmed by karyotyping, FISH, and array-CGH. These analyses revealed significant chromosomal duplications and deletions, which may contribute to the observed phenotypic abnormalities. Following characterization via various cytogenetic techniques, this rearrangement appears to be the first reported instance of a jumping-like translocation in prenatal constitutional genetics. This finding enables the formulation of hypotheses regarding the mechanisms underlying such intricate structural variants and their detection via contemporary genetic methods.

Danio rerio, commonly known as zebrafish, is an established model organism for the developmental and cell biology studies. Although significant progress has been made in the analysis of the D. rerio genome, cytogenetic studies face challenges due to the unclear identification of chromosomes. Here, we present a novel approach to the study of the D. rerio karyotype, focusing on the analysis of lampbrush chromosomes isolated from growing oocytes. Lampbrush chromosomes, existing during diplotene, serve as a powerful tool for high-resolution mapping and transcription analysis due to their profound decondensation and remarkable lateral loops decorated by RNA polymerases and ribonucleoprotein (RNP) matrix. In D. rerio, lampbrush chromosomes are about 20 times longer than corresponding metaphase chromosomes. We found that the lampbrush chromosome stage karyotype of D. rerio is generally undifferentiated, except for several bivalents bearing distinct marker structures, including loops with complex RNP matrix and locus-associated nuclear bodies. Locus-associated nuclear bodies were enriched for coilin and snRNAs; the loci where they formed presumably correspond to the histone gene clusters. Further, we observed the accumulation of splicing factors in giant terminal RNP aggregates on one bivalent. DAPI staining of Danio rerio lampbrush chromosomes revealed large and small chromomeres non-uniformly distributed along the axis. For example, D. rerio lampbrush chromosome 4, comprising the sex-determining region, is divided into two halves-with small chromomeres bearing long lateral loops and with large dense chromomeres bearing no or very tiny lateral loops. As centromeres were not distinguishable, we identified centromeric regions in all bivalents by FISH mapping of pericentromeric RFAL1, RFAL2, and RFAM tandem repeats. Through a combination of morphological analysis, immunostaining of marker structures, and centromere mapping, we developed cytological maps of D. rerio lampbrush chromosomes. Finally, by RNA FISH we revealed transcripts of pericentromeric and telomeric tandem repeats at the lampbrush chromosome stage.