Repeat-based holocentromeres influence genome architecture and karyotype evolution.

The centromere represents a single region in most eukaryotic chromosomes. However, several plant and animal lineages assemble holocentromeres along the entire chromosome length. Here, we compare genome organization and evolution as a function of centromere type by assembling chromosome-scale holocentric genomes with repeat-based holocentromeres from three beak-sedge (Rhynchospora pubera, R. breviuscula, and R. tenuis) and their closest monocentric relative, Juncus effusus. We demonstrate that transition to holocentricity affected 3D genome architecture by redefining genomic compartments, while distributing centromere function to thousands of repeat-based centromere units genome-wide. We uncover a complex genome organization in R. pubera that hides its unexpected octoploidy and describe a marked reduction in chromosome number for R. tenuis, which has only two chromosomes. We show that chromosome fusions, facilitated by repeat-based holocentromeres, promoted karyotype evolution and diploidization. Our study thus sheds light on several important aspects of genome architecture and evolution influenced by centromere organization.

Adaptation to spindle assembly checkpoint inhibition through the selection of specific aneuploidies.

Both the presence of an abnormal complement of chromosomes (aneuploidy) and an increased frequency of chromosome missegregation (chromosomal instability) are hallmarks of cancer. Analyses of cancer genome data have identified certain aneuploidy patterns in tumors; however, the bases behind their selection are largely unexplored. By establishing time-resolved long-term adaptation protocols, we found that human cells adapt to persistent spindle assembly checkpoint (SAC) inhibition by acquiring specific chromosome arm gains and losses. Independently adapted populations converge on complex karyotypes, which over time are refined to contain ever smaller chromosomal changes. Of note, the frequencies of chromosome arm gains in adapted cells correlate with those detected in cancers, suggesting that our cellular adaptation approach recapitulates selective traits that dictate the selection of aneuploidies frequently observed across many cancer types. We further engineered specific aneuploidies to determine the genetic basis behind the observed karyotype patterns. These experiments demonstrated that the adapted and engineered aneuploid cell lines limit CIN by extending mitotic duration. Heterozygous deletions of key SAC and APC/C genes recapitulated the rescue phenotypes of the monosomic chromosomes. We conclude that aneuploidy-induced gene dosage imbalances of individual mitotic regulators are sufficient for altering mitotic timing to reduce CIN.

Clonal selection of stable aneuploidies in progenitor cells drives high-prevalence tumorigenesis.

Chromosome gains and losses are a frequent feature of human cancers. However, how these aberrations can outweigh the detrimental effects of aneuploidy remains unclear. An initial comparison of existing chromosomal instability (CIN) mouse models suggests that aneuploidy accumulates to low levels in these animals. We therefore developed a novel mouse model that enables unprecedented levels of chromosome missegregation in the adult animal. At the earliest stages of T-cell development, cells with random chromosome gains and/or losses are selected against, but CIN eventually results in the expansion of progenitors with clonal chromosomal imbalances. Clonal selection leads to the development of T-cell lymphomas with stereotypic karyotypes in which chromosome 15, containing the Myc oncogene, is gained with high prevalence. Expressing human MYC from chromosome 6 (MYCChr6) is sufficient to change the karyotype of these lymphomas to include universal chromosome 6 gains. Interestingly, while chromosome 15 is still gained in MYCChr6 tumors after genetic ablation of the endogenous Myc locus, this chromosome is not efficiently gained after deletion of one copy of Rad21, suggesting a synergistic effect of both MYC and RAD21 in driving chromosome 15 gains. Our results show that the initial detrimental effects of random missegregation are outbalanced by clonal selection, which is dictated by the chromosomal location and nature of certain genes and is sufficient to drive cancer with high prevalence.

Solving the chromosome puzzle of aneuploidy in cancer.

Chromosome instability (CIN) and aneuploidy are hallmarks of cancer cells, typically associated with aggressiveness and poor outcomes. Historically, the causative link between aneuploidy and cancer has been difficult to study due to its intrinsic complexity and the poor fitness of aneuploid cells. In this issue of Genes & Development, two companion papers (Trakala and colleagues [pp. 1079-1092] and Shoshani and colleagues [pp. 1093-1108]) exploited sophisticated mouse models to study the progression of aneuploidy from early phases to established tumors. Both groups observed that, while in the early nontumoral cells aneuploidy is characterized by random chromosomal gains, established tumors display a stereotypic karyotype with recurrent gains of only a few chromosomes. Thus, aneuploidy in tumors is not random but shows reproducible patterns of chromosomal changes induced by mechanisms that these two studies are beginning to unveil.

Genetic instability from a single S phase after whole-genome duplication.

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.

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.

Condensin-Mediated Chromosome Folding and Internal Telomeres Drive Dicentric Severing by Cytokinesis.

In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.

Subgenome-aware analyses reveal the genomic consequences of ancient allopolyploid hybridizations throughout the cotton family.

Malvaceae comprise some 4,225 species in 243 genera and nine subfamilies and include economically important species, such as cacao, cotton, durian, and jute, with cotton an important model system for studying the domestication of polyploids. Here, we use chromosome-level genome assemblies from representatives of five or six subfamilies (depending on the placement of Ochroma) to differentiate coexisting subgenomes and their evolution during the family's deep history. The results reveal that the allohexaploid Helicteroideae partially derive from an allotetraploid Sterculioideae and also form a component of the allodecaploid Bombacoideae and Malvoideae. The ancestral Malvaceae karyotype consists of 11 protochromosomes. Four subfamilies share a unique reciprocal chromosome translocation, and two other subfamilies share a chromosome fusion. DNA alignments of single-copy nuclear genes do not yield the same relationships as inferred from chromosome structural traits, probably because of genes originating from different ancestral subgenomes. These results illustrate how chromosome-structural data can unravel the evolutionary history of groups with ancient hybrid genomes.

Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization.

Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum, an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Nascent evolution of recombination rate differences as a consequence of chromosomal rearrangements.

Reshuffling of genetic variation occurs both by independent assortment of chromosomes and by homologous recombination. Such reshuffling can generate novel allele combinations and break linkage between advantageous and deleterious variants which increases both the potential and the efficacy of natural selection. Here we used high-density linkage maps to characterize global and regional recombination rate variation in two populations of the wood white butterfly (Leptidea sinapis) that differ considerably in their karyotype as a consequence of at least 27 chromosome fissions and fusions. The recombination data were compared to estimates of genetic diversity and measures of selection to assess the relationship between chromosomal rearrangements, crossing over, maintenance of genetic diversity and adaptation. Our data show that the recombination rate is influenced by both chromosome size and number, but that the difference in the number of crossovers between karyotypes is reduced as a consequence of a higher frequency of double crossovers in larger chromosomes. As expected from effects of selection on linked sites, we observed an overall positive association between recombination rate and genetic diversity in both populations. Our results also revealed a significant effect of chromosomal rearrangements on the rate of intergenic diversity change between populations, but limited effects on polymorphisms in coding sequence. We conclude that chromosomal rearrangements can have considerable effects on the recombination landscape and consequently influence both maintenance of genetic diversity and efficiency of selection in natural populations.

Genetic interactions between specific chromosome copy number alterations dictate complex aneuploidy patterns.

Cells that contain an abnormal number of chromosomes are called aneuploid. High rates of aneuploidy in cancer are correlated with an increased frequency of chromosome missegregation, termed chromosomal instability (CIN). Both high levels of aneuploidy and CIN are associated with cancers that are resistant to treatment. Although aneuploidy and CIN are typically detrimental to cell growth, they can aid in adaptation to selective pressures. Here, we induced extremely high rates of chromosome missegregation in yeast to determine how cells adapt to CIN over time. We found that adaptation to CIN occurs initially through many different individual chromosomal aneuploidies. Interestingly, the adapted yeast strains acquire complex karyotypes with specific subsets of the beneficial aneuploid chromosomes. These complex aneuploidy patterns are governed by synthetic genetic interactions between individual chromosomal abnormalities, which we refer to as chromosome copy number interactions (CCNIs). Given enough time, distinct karyotypic patterns in separate yeast populations converge on a refined complex aneuploid state. Surprisingly, some chromosomal aneuploidies that provided an advantage early on in adaptation are eventually lost due to negative CCNIs with even more beneficial aneuploid chromosome combinations. Together, our results show how cells adapt by obtaining specific complex aneuploid karyotypes in the presence of CIN.

A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

A method for stabilising the XX karyotype in female mESC cultures.

Female mouse embryonic stem cells (mESCs) present differently from male mESCs in several fundamental ways; however, complications with their in vitro culture have resulted in an under-representation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female and male mESCs apart. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state that is near indistinguishable from male mESCs. If female pluripotency is to be studied effectively in this system, it is crucial that high-quality cultures of XX mESCs are available. Here, we report a method for better maintaining XX female mESCs in culture that also stabilises the male karyotype and makes study of female-specific pluripotency more feasible.

Implications of the Evolutionary Trajectory of Centromeres in the Fungal Kingdom.

Chromosome segregation during the cell cycle is an evolutionarily conserved, fundamental biological process. Dynamic interaction between spindle microtubules and the kinetochore complex that assembles on centromere DNA is required for faithful chromosome segregation. The first artificial minichromosome was constructed by cloning the centromere DNA of the budding yeast Saccharomyces cerevisiae. Since then, centromeres have been identified in >60 fungal species. The DNA sequence and organization of the sequence elements are highly diverse across these fungal centromeres. In this article, we provide a comprehensive view of the evolution of fungal centromeres. Studies of this process facilitated the identification of factors influencing centromere specification, maintenance, and propagation through many generations. Additionally, we discuss the unique features and plasticity of centromeric chromatin and the involvement of centromeres in karyotype evolution. Finally, we discuss the implications of recurrent loss of RNA interference (RNAi) and/or heterochromatin components on the trajectory of the evolution of fungal centromeres and propose the centromere structure of the last common ancestor of three major fungal phyla-Ascomycota, Basidiomycota, and Mucoromycota.

High karyotypic complexity is an independent prognostic factor in patients with CLL treated with venetoclax combinations.

Complex karyotypes have been associated with inferior outcomes in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy (CIT), whereas their prognostic impact in the context of venetoclax-based treatments is still debated. In this prospective analysis on karyotype complexity in CLL, we evaluated the impact of complex (≥3 chromosomal aberrations [CAs], CKTs) and highly complex karyotypes (≥5 CAs; hCKTs) as well as specific aberrations in previously untreated patients without TP53 aberrations undergoing either CIT or time-limited venetoclax-based therapies in the phase 3 GAIA/CLL13 trial. Karyotype analyses were available for 895 of 926 patients (96.7%), of whom 153 (17%) had a CKT and 43 (5%) hCKT. In the CIT arm, CKT was associated with shorter progression-free survival (PFS) (hazard ratio [HR] 2.58; 95% confidence interval [95% CI], 1.54-4.32; P < .001) and overall survival (HR, 3.25; 95% CI, 1.03-10.26; P = .044). In the pooled venetoclax arms, a multivariable analysis identified hCKTs (HR, 1.96; 95% CI, 1.03-3.72; P = .041), but not CKTs, as independent adverse prognosticators for PFS. The presence of translocations (unbalanced and/or balanced) was also independently associated with shorter PFSs in the venetoclax arms. CIT led to the acquisition of additional CAs (mean CAs, 2.0-3.4; from baseline to CLL progression), whereas karyotype complexity remained stable after venetoclax-based treatments (2.0, both time points). This analysis establishes highly complex karyotypes and translocations as adverse prognostic factors in the context of venetoclax-based combination treatments. The findings of this study support the incorporation of karyotyping into the standard diagnostic workup of CLL, because it identifies patients at high risk of poor treatment outcomes and thereby improves prognostication. This trial was registered at www.clinicaltrials.gov as #NCT02950051.

Celebrating Mendel, McClintock, and Darlington: On end-to-end chromosome fusions and nested chromosome fusions.

The evolution of eukaryotic genomes is accompanied by fluctuations in chromosome number, reflecting cycles of chromosome number increase (polyploidy and centric fissions) and decrease (chromosome fusions). Although all chromosome fusions result from DNA recombination between two or more nonhomologous chromosomes, several mechanisms of descending dysploidy are exploited by eukaryotes to reduce their chromosome number. Genome sequencing and comparative genomics have accelerated the identification of inter-genome chromosome collinearity and gross chromosomal rearrangements and have shown that end-to-end chromosome fusions (EEFs) and nested chromosome fusions (NCFs) may have played a more important role in the evolution of eukaryotic karyotypes than previously thought. The present review aims to summarize the limited knowledge on the origin, frequency, and evolutionary implications of EEF and NCF events in eukaryotes and especially in land plants. The interactions between nonhomologous chromosomes in interphase nuclei and chromosome (mis)pairing during meiosis are examined for their potential importance in the origin of EEFs and NCFs. The remaining open questions that need to be addressed are discussed.

Meiotic drive against chromosome fusions in butterfly hybrids.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

Hypo-osmotic-like stress underlies general cellular defects of aneuploidy.

Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1-4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis.

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.