Live chromosome identifying and tracking reveals size-based spatial pathway of meiotic errors in oocytes.

Meiotic errors of relatively small chromosomes in oocytes result in egg aneuploidies that cause miscarriages and congenital diseases. Unlike somatic cells, which preferentially mis-segregate larger chromosomes, aged oocytes preferentially mis-segregate smaller chromosomes through unclear processes. Here, we provide a comprehensive three-dimensional chromosome identifying-and-tracking dataset throughout meiosis I in live mouse oocytes. This analysis reveals a prometaphase pathway that actively moves smaller chromosomes to the inner region of the metaphase plate. In the inner region, chromosomes are pulled by stronger bipolar microtubule forces, which facilitates premature chromosome separation, a major cause of segregation errors in aged oocytes. This study reveals a spatial pathway that facilitates aneuploidy of small chromosomes preferentially in aged eggs and implicates the role of the M phase in creating a chromosome size-based spatial arrangement.

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.

Stunning 3D chromosomes preserved in thawed mammoths.

"New type of fossil" may boost efforts to bring beasts back.

Mapping of SINEs in the genome of Proechimys (Mammalia: Rodentia).

This study aimed to analyze the distribution of short interspersed elements (SINEs) in the chromosomes of five species of rodents of the genus Proechimys and in a variant karyotype of P. guyannensis. Molecular cytogenetic techniques were used to characterize the sequences of the B1, B4, MAR and THER SINEs, which were used as probes for hybridization in metaphase chromosomes. A wide distribution of SINEs was observed in the chromosomes of the Proechimys species examined, thus indicating differentiation of these retroelements. The signal of the B4 SINE was more evident than that of the B1 SINE, especially in P. echinothrix, P. longicaudatus, and P. cuvieri. Although the signal of the MAR SINE was more explosive than that of the THER SINE, in the species P. echinothrix, P. guyannensis (2n = 46) and P. longicaudatus, its distribution in the karyotypes was similar. The signals of these retroelements occurred at specific heterochromatic sites and were centromeric/pericentromeric and at the terminal regions in most chromosomes. This appears to be a typical distribution pattern of the SINEs and may indicate involvement with rearrangements during karyotypic diversification in Proechimys. The variation of the SINEs in the genome of Proechimys species demonstrates that these elements are distributed in a specific way in this genus and the preference for some sites, considered hotspots for chromosomal breakage, allows us to propose that these elements are related to the karyotypic evolution of Proechimys.

PP2A-B55 phosphatase counteracts Ki-67-dependent chromosome individualization during mitosis.

Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.

Tachol1 QTL on mouse chromosome 1 is responsible for hypercholesterolemia and diet-induced obesity.

Hypercholesterolemia raises the risk for cardiovascular complications and overall health. Hypercholesterolemia is common, affecting 10% of the general population of the US, and heritable. Most individuals with hypercholesterolemia have a polygenic predisposition to the condition. Previously we identified a quantitative trait locus, Tachol1, linked to hypercholesterolemia on mouse chromosome 1 (Chr1) in a cross between C57BL/6J (B6) and TALLYHO/JngJ (TH) mice, a polygenic model for human obesity, type 2 diabetes and hyperlipidemia. Subsequently, using congenic mice that carry a TH-derived genomic segment of Chr1 on a B6 background, we demonstrated that the distal segment of Chr1, where Tachol1 maps, is necessary to cause hypercholesterolemia, as well as diet-induced obesity. In this study, we generated overlapping subcongenic lines to the distal segment of congenic region and characterized subcongenic mice carrying the smallest TH region of Tachol1, ~ 16.2 Mb in size (B6.TH-Chr1-16.2 Mb). Both male and female B6.TH-Chr1-16.2 Mb mice showed a significantly increased plasma total cholesterol levels compared to B6 on both chow and high fat (HF) diet. B6.TH-Chr1-16.2 Mb mice also had greater fat mass than B6 on HF diet, without increasing food intake. The gene and protein expression levels of absent in melanoma 2 (Aim2) gene were significantly upregulated in B6.TH-Chr1-16.2 Mb mice compared to B6. In summary, we confirmed the effect of Tachol1 on hypercholesterolemia and diet-induced obesity using subcongenic analysis.

Fine mapping and candidate gene analysis of Dravet syndrome modifier loci on mouse chromosomes 7 and 8.

Dravet syndrome is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, comorbidities related to developmental, cognitive, and motor delays, and a high mortality burden due to sudden unexpected death in epilepsy (SUDEP). Most Dravet syndrome cases are attributed to SCN1A haploinsufficiency, with genetic modifiers and environmental factors influencing disease severity. Mouse models with heterozygous deletion of Scn1a recapitulate key features of Dravet syndrome, including seizures and premature mortality; however, severity varies depending on genetic background. Here, we refined two Dravet survival modifier (Dsm) loci, Dsm2 on chromosome 7 and Dsm3 on chromosome 8, using interval-specific congenic (ISC) mapping. Dsm2 was complex and encompassed at least two separate loci, while Dsm3 was refined to a single locus. Candidate modifier genes within these refined loci were prioritized based on brain expression, strain-dependent differences, and biological relevance to seizures or epilepsy. High priority candidate genes for Dsm2 include Nav2, Ptpn5, Ldha, Dbx1, Prmt3 and Slc6a5, while Dsm3 has a single high priority candidate, Psd3. This study underscores the complex genetic architecture underlying Dravet syndrome and provides insights into potential modifier genes that could influence disease severity and serve as novel therapeutic targets.

Chromosome size-dependent polar ejection force impairs mammalian mitotic error correction.

Accurate chromosome segregation requires sister kinetochores to biorient, attaching to opposite spindle poles. To this end, the mammalian kinetochore destabilizes incorrect attachments and stabilizes correct ones, but how it discriminates between these is not yet clear. Here, we test the model that kinetochore tension is the stabilizing cue and ask how chromosome size impacts that model. We live image PtK2 cells, with just 14 chromosomes, widely ranging in size, and find that long chromosomes align at the metaphase plate later than short chromosomes. Enriching for errors and imaging error correction live, we show that long chromosomes exhibit a specific delay in correcting attachments. Using chromokinesin overexpression and laser ablation to perturb polar ejection forces, we find that chromosome size and force on arms determine alignment order. Thus, we propose a model where increased force on long chromosomes can falsely stabilize incorrect attachments, delaying their biorientation. As such, long chromosomes may require compensatory mechanisms for correcting errors to avoid chromosomal instability.

New Karyotype Information for Ctenomys (Rodentia: Ctenomyidae) from Midwest and Northern Brazil.

INTRODUCTION: Its wide karyotypic variation characterizes the genus Ctenomys, and in Brazil, the genus is distributed in the country's southern, Midwest, and northern regions. Recently, populations of Ctenomys have been found in the Midwest and northern Brazil, with two new lineages named C. sp. "xingu" and C. sp. "central." METHODS: This work combines classical cytogenetic and molecular analyses to provide new chromosomal information on the boliviensis group distributed in northern and Midwestern Brazil. This includes the validation of the karyotype of C. bicolor and C. nattereri and the description of the karyotype of C. sp. "xingu" and C. sp. "central." RESULTS: We found three different karyotypes: 2n = 40 for C. bicolor; 2n = 36 for C. nattereri, and specimens from a locality belonging to C. sp. "central"; 2n = 34 for the lineage C. sp. "xingu" and specimens from a locality belonging to C. sp. "central." Furthermore, GTG banding revealed homologous chromosomes between species/lineages and allowed the identification of the rearrangements that occurred, which proved the occurrence of fissions. CONCLUSION: Considering our results on the variation of 2n in the boliviensis group, we found two possibilities: the first, deduced by parsimony, is that 2n = 36 appeared initially, and two fissions produced gave rise to 2n = 40, and an independent fusion gave rise to 2n = 34 from 2n = 36; moreover, the second explanation is that all karyotypes arose independently.

Condensin dysfunction is a reproductive isolating barrier in mice.

Reproductive isolation occurs when the genomes of two populations accumulate genetic incompatibilities that prevent interbreeding1,2. Understanding of hybrid incompatibility at the cell biology level is limited, particularly in the case of hybrid female sterility3. Here we find that species divergence in condensin regulation and centromere organization between two mouse species, Mus musculus domesticus and Mus spretus, drives chromosome decondensation and mis-segregation in their F1 hybrid oocytes, reducing female fertility. The decondensation in hybrid oocytes was especially prominent at pericentromeric major satellites, which are highly abundant at M. m. domesticus centromeres4-6, leading to species-specific chromosome mis-segregation and egg aneuploidy. Consistent with the condensation defects, a chromosome structure protein complex, condensin II7,8, was reduced on hybrid oocyte chromosomes. We find that the condensin II subunit NCAPG2 was specifically reduced in the nucleus in prophase and that overexpressing NCAPG2 rescued both the decondensation and egg aneuploidy phenotypes. In addition to the overall reduction in condensin II on chromosomes, major satellites further reduced condensin II levels locally, explaining why this region is particularly prone to decondensation. Together, this study provides cell biological insights into hybrid incompatibility in female meiosis and demonstrates that condensin misregulation and pericentromeric satellite expansion can establish a reproductive isolating barrier in mammals.

Abandoning the Isochore Theory Can Help Explain Genome Compositional Organization in Fish.

The organization of the genome nucleotide (AT/GC) composition in vertebrates remains poorly understood despite the numerous genome assemblies available. Particularly, the origin of the AT/GC heterogeneity in amniotes, in comparison to the homogeneity in anamniotes, is controversial. Recently, several exceptions to this dichotomy were confirmed in an ancient fish lineage with mammalian AT/GC heterogeneity. Hence, our current knowledge necessitates a reevaluation considering this fact and utilizing newly available data and tools. We analyzed fish genomes in silico with as low user input as possible to compare previous approaches to assessing genome composition. Our results revealed a disparity between previously used plots of GC% and histograms representing the authentic distribution of GC% values in genomes. Previous plots heavily reduced the range of GC% values in fish to comply with the alleged AT/GC homogeneity and AT-richness of their genomes. We illustrate how the selected sequence size influences the clustering of GC% values. Previous approaches that disregarded chromosome and genome sizes, which are about three times smaller in fish than in mammals, distorted their results and contributed to the persisting confusion about fish genome composition. Chromosome size and their transposons may drive the AT/GC heterogeneity apparent on mammalian chromosomes, whereas far less in fishes.

Structural basis of telomeric nucleosome recognition by shelterin factor TRF1.

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1. Our structures reveal that TRF1 binds unwrapped nucleosomal DNA ends by engaging both the nucleosomal DNA and the histone octamer. Unexpectedly, TRF1 binding shifts the register of the nucleosomal DNA by 1 bp. We discovered that phosphorylation of the TRF1 C terminus and a noncanomical DNA binding surface on TRF1 are critical for its association with telomeric nucleosomes. These insights into shelterin-chromatin interactions have crucial implications for understanding telomeric chromatin organization and other roles of shelterin at telomeres including replication and transcription.

TADs: Dynamic structures to create stable regulatory functions.

Mammalian chromosomes are organized at different length scales within the cell nucleus. Topologically Associating Domains (TADs) are structural units of 3D genome organization with functions in gene regulation, DNA replication, recombination and repair. Whereas TADs were initially interpreted as insulated domains, recent studies are revealing that these domains should be interpreted as dynamic collections of actively extruding loops. This process of loop extrusion is subsequently blocked at dedicated TAD boundaries, thereby promoting intra-domain interactions over their surroundings. In this review, we discuss how mammalian TAD structure can emerge from this dynamic process and we discuss recent evidence that TAD boundaries can have regulatory functions.

Mammalian Chromosome Analysis and Sorting by Flow Cytometry.

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO4 low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.

Corruption of DNA end-joining in mammalian chromosomes by progerin expression.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging and a life expectancy of about 14 years. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, an essential component of the nuclear lamina. The HGPS mutation alters splicing of the LMNA transcript, leading to a truncated, farnesylated form of lamin A termed "progerin." Progerin is also produced in small amounts in healthy individuals by alternative splicing of RNA and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting alteration of DNA repair. DSB repair normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by nonhomologous end-joining (NHEJ), a non-templated rejoining of DNA ends that can be error-prone; however a good portion of NHEJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin correlated with increased NHEJ relative to HR. We now report on progerin's impact on the nature of DNA end-joining. We used a model system involving a DNA end-joining reporter substrate integrated into the genome of cultured thymidine kinase-deficient mouse fibroblasts. Some cells were engineered to express progerin. Two closely spaced DSBs were induced in the integrated substrate through expression of endonuclease I-SceI, and DSB repair events were recovered through selection for thymidine kinase function. DNA sequencing revealed that progerin expression correlated with a significant shift away from precise end-joining between the two I-SceI sites and toward imprecise end-joining. Additional experiments revealed that progerin did not reduce HR fidelity. Our work suggests that progerin suppresses interactions between complementary sequences at DNA termini, thereby shifting DSB repair toward low-fidelity DNA end-joining and perhaps contributing to accelerated and normal aging through compromised genome stability.

Preimplantation Genetic Testing for Aneuploidy (PGT-A) Reveals High Levels of Chromosomal Errors in In Vivo-Derived Pig Embryos, with an Increased Incidence When Produced In Vitro.

Preimplantation genetic testing for aneuploidy (PGT-A) is widespread, but controversial, in humans and improves pregnancy and live birth rates in cattle. In pigs, it presents a possible solution to improve in vitro embryo production (IVP), however, the incidence and origin of chromosomal errors remains under-explored. To address this, we used single nucleotide polymorphism (SNP)-based PGT-A algorithms in 101 in vivo-derived (IVD) and 64 IVP porcine embryos. More errors were observed in IVP vs. IVD blastocysts (79.7% vs. 13.6% p < 0.001). In IVD embryos, fewer errors were found at blastocyst stage compared to cleavage (4-cell) stage (13.6% vs. 40%, p = 0.056). One androgenetic and two parthenogenetic embryos were also identified. Triploidy was the most common error in IVD embryos (15.8%), but only observed at cleavage, not blastocyst stage, followed by whole chromosome aneuploidy (9.9%). In IVP blastocysts, 32.8% were parthenogenetic, 25.0% (hypo-)triploid, 12.5% aneuploid, and 9.4% haploid. Parthenogenetic blastocysts arose from just three out of ten sows, suggesting a possible donor effect. The high incidence of chromosomal abnormalities in general, but in IVP embryos in particular, suggests an explanation for the low success of porcine IVP. The approaches described provide a means of monitoring technical improvements and suggest future application of PGT-A might improve embryo transfer success.

Transcription shapes 3D chromatin organization by interacting with loop extrusion.

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.

Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.

Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse.

We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870-61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.