The nucleolus constitutes a prominent nuclear compartment, a membraneless organelle that was first documented in the 1830s. The fact that specific chromosomal regions were present in the nucleolus ...was recognized by Barbara McClintock in the 1930s, and these regions were termed nucleolar organizing regions, or NORs. The primary function of ribosomal DNA (rDNA) is to produce RNA components of ribosomes. Yet, ribosomal DNA also plays a pivotal role in nuclear organization by assembling the nucleolus. This review is focused on the rDNA and associated proteins in the context of genome organization. Recent advances in understanding chromatin organization suggest that chromosomes are organized into topological domains by a DNA loop extrusion process. We discuss the perspective that rDNA may also be organized in topological domains constrained by structural maintenance of chromosome protein complexes such as cohesin and condensin. Moreover, biophysical studies indicate that the nucleolar compartment may be formed by active processes as well as phase separation, a perspective that lends further insight into nucleolar organization. The application of the latest perspectives and technologies to this organelle help further elucidate its role in nuclear structure and function.
Aneuploidy and chromosomal instability frequently co-exist, and aneuploidy is recognized as a direct outcome of chromosomal instability. However, chromosomal instability is widely viewed as a ...consequence of mutations in genes involved in DNA replication, chromosome segregation, and cell cycle checkpoints. Telomere attrition and presence of extra centrosomes have also been recognized as causative for errors in genomic transmission. Here, we examine recent studies suggesting that aneuploidy
itself
can be responsible for the procreation of chromosomal instability. Evidence from both yeast and mammalian experimental models suggests that changes in chromosome copy number can cause changes in dosage of the products of many genes located on aneuploid chromosomes. These effects on gene expression can alter the balanced stoichiometry of various protein complexes, causing perturbations of their functions. Therefore, phenotypic consequences of aneuploidy will include chromosomal instability if the balanced stoichiometry of protein machineries responsible for accurate chromosome segregation is affected enough to perturb the function. The degree of chromosomal instability will depend on specific karyotypic changes, which may be due to dosage imbalances of specific genes or lack of scaling between chromosome segregation load and the capacity of the mitotic system. We propose that the relationship between aneuploidy and chromosomal instability can be envisioned as a “vicious cycle,” where aneuploidy potentiates chromosomal instability leading to further karyotype diversity in the affected population.
The complete sequence of a human genome Nurk, Sergey; Koren, Sergey; Rhie, Arang ...
Science (American Association for the Advancement of Science),
04/2022, Volume:
376, Issue:
6588
Journal Article
Peer reviewed
Open access
Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining ...8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.
After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end ...to end, and hundreds of unresolved gaps persist
. Here we present a human genome assembly that surpasses the continuity of GRCh38
, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome
, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
Complete genomic and epigenetic maps of human centromeres Altemose, Nicolas; Logsdon, Glennis A; Bzikadze, Andrey V ...
Science (American Association for the Advancement of Science),
04/2022, Volume:
376, Issue:
6588
Journal Article
Peer reviewed
Open access
Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which ...include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions.
Mistakes during cell division frequently generate changes in chromosome content, producing aneuploid or polyploid progeny cells. Polyploid cells may then undergo abnormal division to generate ...aneuploid cells. Chromosome segregation errors may also involve fragments of whole chromosomes. A major consequence of segregation defects is change in the relative dosage of products from genes located on the missegregated chromosomes. Abnormal expression of transcriptional regulators can also impact genes on the properly segregated chromosomes. The consequences of these perturbations in gene expression depend on the specific chromosomes affected and on the interplay of the aneuploid phenotype with the environment. Most often, these novel chromosome distributions are detrimental to the health and survival of the organism. However, in a changed environment, alterations in gene copy number may generate a more highly adapted phenotype. Chromosome segregation errors also have important implications in human health. They may promote drug resistance in pathogenic microorganisms. In cancer cells, they are a source for genetic and phenotypic variability that may select for populations with increased malignance and resistance to therapy. Lastly, chromosome segregation errors during gamete formation in meiosis are a primary cause of human birth defects and infertility. This review describes the consequences of mitotic and meiotic errors focusing on novel concepts and human health.
Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression ...by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.
Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates ...changes in gene expression evoked in acute and adapted tetraploid cells and their effect on cell-cycle progression. Acute polyploidy was generated by knockdown of the essential regulator of cytokinesis anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology. Transcriptome analysis of these acute tetraploid cells revealed common signatures of activation of the tumor-suppressor protein p53. Suppression of proliferation in these cells was dependent on p53 and its transcriptional target, CDK inhibitor p21. Rare proliferating tetraploid cells can emerge from acute polyploid populations. Gene expression analysis of single cell-derived, adapted tetraploid clones showed up-regulation of several p53 target genes and cyclin D2, the activator of CDK4/6/2. Overexpression of cyclin D2 in diploid cells strongly potentiated the ability to proliferate with increased DNA content despite the presence of functional p53. These results indicate that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as cyclin D2, elucidating a possible route for tetraploidy-mediated genomic instability in carcinogenesis.
Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on ...microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit.
Here we show that during experimentally induced metaphase delay, spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term “cohesion fatigue.” Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the “prophase pathway” of cohesin release from chromosomes, or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids.
In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.
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► Metaphase delays induce “cohesion fatigue” and asynchronous chromatid separation ► Cohesion fatigue does not require Plk1 or separase but requires intact microtubules ► Cohesin remains associated with chromatids separated by cohesion fatigue ► Cohesion fatigue during metaphase delay may contribute to chromosome instability
The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution ...microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization.
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