Eukaryotic Chromosomes Research Articles

Telomere Length Variation in Model Bryophytes.

The ends of linear chromosomes of most eukaryotes consist of protein-bound DNA arrays called telomeres, which play essential roles in protecting genome integrity. Despite general evolutionary conservation in function, telomeric DNA is known to drastically vary in length and sequence between different eukaryotic lineages. Bryophytes are a group of early diverging land plants that include mosses, liverworts, and hornworts. This group of ancient land plants recently emerged as a new model for important discoveries in genomics and evolutionary biology, as well as for understanding plant adaptations to a terrestrial lifestyle. We measured telomere length in different ecotypes of model bryophyte species, including Physcomitrium patens, Marchantia polymorpha, Ceratodon purpureus, and in Sphagnum isolates. Our data indicate that all analyzed moss and liverwort genotypes have relatively short telomeres. Furthermore, all analyzed ecotypes and isolates of model mosses and liverworts display evidence of substantial natural variation in telomere length. Interestingly, telomere length also differs between male and female strains of the dioecious liverwort M. polymorpha and dioecious moss C. purpureus. Given that bryophytes are extraordinarily well adapted to different ecological niches from polar to tropical environments, our data will contribute to understanding the impact of natural telomere length variation on evolutionary adaptations in this ancient land plant lineage.

A designer synthetic chromosome fragment functions in moss.

Rapid advances in DNA synthesis techniques have enabled the assembly and engineering of viral and microbial genomes, presenting new opportunities for synthetic genomics in multicellular eukaryotic organisms. These organisms, characterized by larger genomes, abundant transposons and extensive epigenetic regulation, pose unique challenges. Here we report the in vivo assembly of chromosomal fragments in the moss Physcomitrium patens, producing phenotypically virtually wild-type lines in which one-third of the coding region of a chromosomal arm is replaced by redesigned, chemically synthesized fragments. By eliminating 55.8% of a 155 kb endogenous chromosomal region, we substantially simplified the genome without discernible phenotypic effects, implying that many transposable elements may minimally impact growth. We also introduced other sequence modifications, such as PCRTag incorporation, gene locus swapping and stop codon substitution. Despite these substantial changes, the complex epigenetic landscape was normally established, albeit with some three-dimensional conformation alterations. The synthesis of a partial multicellular eukaryotic chromosome arm lays the foundation for the synthetic moss genome project (SynMoss) and paves the way for genome synthesis in multicellular organisms.

2.7 Å cryo-EM structure of human telomerase H/ACA ribonucleoprotein

Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase is comprised of two distinct functional lobes tethered by telomerase RNA (hTR): a catalytic core, responsible for DNA extension; and a Hinge and ACA (H/ACA) box RNP, responsible for telomerase biogenesis. H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs, which is critical for the biogenesis of the spliceosome and ribosome. Much of our structural understanding of eukaryotic H/ACA RNPs comes from structures of the human telomerase H/ACA RNP. Here we report a 2.7 Å cryo-electron microscopy structure of the telomerase H/ACA RNP. The significant improvement in resolution over previous 3.3 Å to 8.2 Å structures allows us to uncover new molecular interactions within the H/ACA RNP. Many disease mutations are mapped to these interaction sites. The structure also reveals unprecedented insights into a region critical for pseudouridylation in canonical H/ACA RNPs. Together, our work advances understanding of telomerase-related disease mutations and the mechanism of pseudouridylation by eukaryotic H/ACA RNPs.

Structural effects of inosine substitution in telomeric DNA quadruplex.

The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)n telomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)4, (IGG)4, (GIG)4, (GGI)4, (IGI)4, (IIG)4, (GII)4, and (III)4, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na+ and K+ containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)4 and the terminal modified DNAs (IGG)4 and (GGI)4 in both Na+ and K+ containing buffers. Specifically, quadruplex hybrid was observed for (GGG)4 in K+ buffer. Among the other modified sequences, (GIG)4, (IGI)4 and (GII)4 show parallel features, while (IIG)4 and (III)4 show no detectable conformation in the presence of either Na+ or K+. Our studies indicate that terminal lesions (IGG)4 and (GGI)4 may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)4 in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K+ cation confers more thermodynamic stability compared to Na+. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions.

A Nested PCR Telomere Fusion Assay Highlights the Widespread End-Capping Protection of Arabidopsis CTC1.

Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-strand breaks. Two major protein complexes are involved in the protection of telomeres: shelterin and CST. The dysfunction of these complexes can challenge the function of telomeres and lead to telomere fusions, breakage-fusion-bridge cycles, and cell death. Therefore, monitoring telomere fusions helps to understand telomeres biology. Telomere fusions are often analyzed by Fluorescent In Situ Hybridization (FISH) or PCR. Usually, both methods involve hybridization with a telomeric probe, which allows the detection of fusions containing telomeric sequences, but not of those lacking them. With the aim of detecting both types of fusion events, we have developed a nested PCR method to analyze telomere fusions in Arabidopsis thaliana. This method is simple, accurate, and does not require hybridization. We have used it to analyze telomere fusions in wild-type and mutant plants altered in CTC1, one of the three components of the Arabidopsis CST telomere capping complex. Our results show that null ctc1-2 mutant plants display fusions between all telomeric regions present in Arabidopsis chromosomes 1, 3 and 5, thus highlighting the widespread end-capping protection achieved by CTC1.

Molecular Dynamics Simulation of a Feather-Boa Model of a Bacterial Chromosome.

The chromosome of a bacterium consists of a mega-base pair-long circular DNA, which self-organizes within the micron-sized bacterial cell volume, compacting itself by three orders of magnitude. Unlike eukaryotic chromosomes, it lacks a nuclear membrane and freely floats in the cytosol confined by the cell membrane. It is believed that strong confinement, cross-linking by associated proteins, and molecular crowding all contribute to determine chromosome size and morphology. Modelling the chromosome simply as a circular polymer decorated with closed side loops in a cylindrical confining volume has been shown to already recapture some of the salient properties observed experimentally. Here we describe how a computer simulation can be set up to study structure and dynamics of bacterial chromosomes using this model.

Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.

Building a eukaryotic chromosome arm by de novo design and synthesis

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a “one-amino-acid-one-codon” strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

The Altered Functions of Shelterin Components in ALT Cells

Telomeres are nucleoprotein complexes that cap the ends of eukaryotic linear chromosomes. Telomeric DNA is bound by shelterin protein complex to prevent telomeric chromosome ends from being recognized as damaged sites for abnormal repair. To overcome the end replication problem, cancer cells mostly preserve their telomeres by reactivating telomerase, but a minority (10–15%) of cancer cells use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). Recent studies have found that shelterin components play an important role in the ALT mechanism. The binding of TRF1, TRF2, and RAP1 to telomeres attenuates ALT activation, while the maintenance of ALT telomere requires TRF1 and TRF2. POT1 and TPP1 can also influence the occurrence of ALT. The elucidation of how shelterin regulates the initiation of ALT remains elusive. This review presents a comprehensive overview of the current findings on the regulation of ALT by shelterin components, aiming to enhance the insight into the altered functions of shelterin components in ALT cells and to identify potential targets for the treatment of ALT tumor cells.

Bridging condensins mediate compaction of mitotic chromosomes.

Eukaryotic chromosomes compact during mitosis into elongated cylinders-and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamic simulations involving two types of such proteins with different activities. One, which we refer to as looping condensins, anchors long-lived chromatin loops to create bottlebrush structures. The second, referred to as bridging condensins, forms multivalent bridges between distant parts of these loops. We show that binding of bridging condensins leads to the formation of shorter and stiffer mitotic-like cylinders without requiring any additional energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes seen experimentally and points to a mechanistic basis for the structure of common fragile sites in mitotic chromosomes.

IMMU-06. THE ROLE OF BRAIN TUMOR MICROENVIRONMENT ON T CELL TELOMERE SHORTENING

Abstract INTRODUCTION Glioblastoma (GBM) stands as the most frequent and the most aggressive of the primary brain tumors. GBM-driven immunomodulatory mechanisms induce T-cell exhaustion or senescence leading to impaired anti-tumor responses. Telomeres are nucleoprotein complexes located at the ending regions of eukaryotic chromosomes that become shorter during each cell cycle or due to cellular stress leading to DNA damage response and cell cycle arrest. Tumor-associated T-cell senescence associated with telomere loss on different tumor models still represents a barrier to the success of T-cell-based tumor immunotherapies. However, the mechanisms through which the GBM microenvironment promotes T-cell telomere loss remain poorly understood. METHODS We evaluated the expression profile of telomere maintenance-related gene sets of single-cell RNA sequencing from public datasets of GBM patients matched with the peripheral blood. We determined the telomere length, telomere-associated DNA secondary structures (G-quadruplex), β-galactosidase activity, and reactive oxygen species (ROS) production of CD8+ T-cells after 72 h exposure to the conditioned media of malignant glioma murine cell cultures CT2A and GL261. We analyzed the telomere length and G-quadruplex abundance in peripheral and tumor-infiltrated T-cells at different time points of tumor-bearing mice injected with CT2A and GL261 cells. RESULTS CD8+ T-cells exposed to the malignant cell conditioned media showed a 20 % reduction of telomere length compared to controls (p< 0.05), a 1.5-fold increase of β-galactosidase activity (p< 0.05), and a 2-fold increase of ROS production (p< 0.05). Both tumor-infiltrated CD4+ and CD8+ T-cells presented a relative telomere shortening of 22 % compared to spleen CD4+ and CD8+ T-cells (p< 0.05). Besides, G-quadruplex abundance increased by 5-fold in tumor-infiltrated CD4+ and 3-fold in tumor-infiltrated CD8+ T-cells compared to peripheral T-cells (p< 0.05). CONCLUSIONS T-cell telomere maintenance is affected by malignant cell conditioned media. In vivo tumor microenvironment induced telomere shortening of tumor-infiltrated T-cells in a time-dependent fashion.

Centromere structure and function: lessons from Drosophila.

The fruit fly Drosophila melanogaster serves as a powerful model organism for advancing our understanding of biological processes, not just by studying its similarities with other organisms including ourselves but also by investigating its differences to unravel the underlying strategies that evolved to achieve a common goal. This is particularly true for centromeres, specialized genomic regions present on all eukaryotic chromosomes that function as the platform for the assembly of kinetochores. These multiprotein structures play an essential role during cell division by connecting chromosomes to spindle microtubules in mitosis and meiosis to mediate accurate chromosome segregation. Here, we will take a historical perspective on the study of fly centromeres, aiming to highlight not only the important similarities but also the differences identified that contributed to advancing centromere biology. We will discuss the current knowledge on the sequence and chromatin organization of fly centromeres together with advances for identification of centromeric proteins. Then, we will describe both the factors and processes involved in centromere organization and how they work together to provide an epigenetic identity to the centromeric locus. Lastly, we will take an evolutionary point of view of centromeres and briefly discuss current views on centromere drive.

Telomouse—a mouse model with human-length telomeres generated by a single amino acid change in RTEL1

Telomeres, the ends of eukaryotic chromosomes, protect genome integrity and enable cell proliferation. Maintaining optimal telomere length in the germline and throughout life limits the risk of cancer and enables healthy aging. Telomeres in the house mouse, Mus musculus, are about five times longer than human telomeres, limiting the use of this common laboratory animal for studying the contribution of telomere biology to aging and cancer. We identified a key amino acid variation in the helicase RTEL1, naturally occurring in the short-telomere mouse species M. spretus. Introducing this variation into M. musculus is sufficient to reduce the telomere length set point in the germline and generate mice with human-length telomeres. While these mice are fertile and appear healthy, the regenerative capacity of their colonic epithelium is compromised. The engineered Telomouse reported here demonstrates a dominant role of RTEL1 in telomere length regulation and provides a unique model for aging and cancer.

3D models of fungal chromosomes to enhance visual integration of omics data.

The functions of eukaryotic chromosomes and their spatial architecture in the nucleus are reciprocally dependent. Hi-C experiments are routinely used to study chromosome 3D organization by probing chromatin interactions. Standard representation of the data has relied on contact maps that show the frequency of interactions between parts of the genome. In parallel, it has become easier to build 3D models of the entire genome based on the same Hi-C data, and thus benefit from the methodology and visualization tools developed for structural biology. 3D modeling of entire genomes leverages the understanding of their spatial organization. However, this opportunity for original and insightful modeling is underexploited. In this paper, we show how seeing the spatial organization of chromosomes can bring new perspectives to omics data integration. We assembled state-of-the-art tools into a workflow that goes from Hi-C raw data to fully annotated 3D models and we re-analysed public omics datasets available for three fungal species. Besides the well-described properties of the spatial organization of their chromosomes (Rabl conformation, hypercoiling and chromosome territories), our results highlighted(i) in Saccharomyces cerevisiae, the backbones of the cohesin anchor regions, which were aligned all along the chromosomes, (ii) in Schizosaccharomyces pombe, the oscillations of the coiling of chromosome arms throughout the cell cycle and (iii) in Neurospora crassa, the massive relocalization of histone marks in mutants of heterochromatin regulators. 3D modeling of the chromosomes brings new opportunities for visual integration of omics data. This holistic perspective supports intuition and lays the foundation for building new concepts.

Telomere sequence variability in genotypes from natural plant populations: unusual block-organized double-monomer terminal telomeric arrays

BackgroundTelomeres are the nucleoprotein complexes that physically cap the ends of eukaryotic chromosomes. Most plants possess Arabidopsis-type telomere sequences (TSs). In addition to terminal TSs, more diverse interstitial TSs exists in plants. Although telomeres have been sufficiently studied, the actual diversity of TSs in land plants is underestimated.ResultsWe investigate genotypes from seven natural populations with contrasting environments of four Chenopodium species to reveal the variability in TSs by analyzing Oxford Nanopore reads. Fluorescent in situ hybridization was used to localize telomeric repeats on chromosomes. We identified a number of derivative monomers that arise in part of both terminal and interstitial telomeric arrays of a single genotype. The former presents a case of block-organized double-monomer telomers, where blocks of Arabidopsis-type TTTAGGG motifs were interspersed with blocks of derivative TTTAAAA motifs. The latter is an integral part of the satellitome with transformations specific to the inactive genome fraction.ConclusionsWe suggested two alternative models for the possible formation of derivative monomers from telomeric heptamer motifs of Arabidopsis-type. It was assumed that derivatization of TSs is a ubiquitous process in the plant genome but occurrence and frequencies of derivatives may be genotype-specific. We also propose that the formation of non-canonical arrays of TSs, especially at chromosomal termini, may be a source for genomic variability in nature.

The Shortening of Leukocyte Telomere Length Contributes to Alzheimer's Disease: Further Evidence from Late-Onset Familial and Sporadic Cases.

Telomeres are structures at the ends of eukaryotic chromosomes that help maintain genomic stability. During aging, telomere length gradually shortens, producing short telomeres, which are markers of premature cellular senescence. This may contribute to age-related diseases, including Alzheimer's disease (AD), and based on this, several studies have hypothesized that telomere shortening may characterize AD. Current research, however, has been inconclusive regarding the direction of the association between leukocyte telomere length (LTL) and disease risk. We assessed the association between LTL and AD in a retrospective case-control study of a sample of 255 unrelated patients with late-onset AD (LOAD), including 120 sporadic cases and 135 with positive family history for LOAD, and a group of 279 cognitively healthy unrelated controls, who were all from Calabria, a southern Italian region. Following regression analysis, telomeres were found significantly shorter in LOAD cases than in controls (48% and 41% decrease for sporadic and familial cases, respectively; p < 0.001 for both). Interestingly, LTL was associated with disease risk independently of the presence of conventional risk factors (e.g., age, sex, MMSE scores, and the presence of the APOE-ε4 allele). Altogether, our findings lend support to the notion that LTL shortening may be an indicator of the pathogenesis of LOAD.

Role of Telomeres and Telomerase in Parkinson's Disease-A New Theranostics?

Parkinson's disease (PD) is a complex condition that is significantly influenced by oxidative stress and inflammation. It is also suggested that telomere shortening (TS) is regulated by oxidative stress which leads to various diseases including age-related neurodegenerative diseases like PD. Thus, it is anticipated that PD would result in TS of peripheral blood mononuclear cells (PBMCs). Telomeres protect the ends of eukaryotic chromosomes preserving them against fusion and destruction. The TS is a normal process because DNA polymerase is unable to replicate the linear ends of the DNA due to end replication complications and telomerase activity in various cell types counteracts this process. PD is usually observed in the aged population and progresses over time therefore, disparities among telomere length in PBMCs of PD patients are recorded and it is still a question whether it has any useful role. Here, the likelihood of telomere attrition in PD and its implications concerning microglia activation, ageing, oxidative stress, and the significance of telomerase activators are addressed. Also, the possibility of telomeres and telomerase as a diagnostic and therapeutic biomarker in PD is discussed.

Causes of DNA Damage and Genomic Instability: A Review

Genomic instability defines all genetic alterations resulting from excessive or high frequency of mutation; base pairs sequence alterations known as microsatellite instability (MSI) and aneuploidy also called chromosome instability (CIN), chromosomal re- arrangements within the genome or the susceptibility of genome to alterations that occur during cell division cycle. The genome of most cancer cells is highly unstable, mostly due to damage to tumour suppressor genes example tp53 which encodes p53 or other genes coordinating cell division. Telomere is a nucleoprotein complex which extends the physical ends of eukaryotic chromosomes, protecting it from degradation, counteracting sequence loss, protecting genes closer to the chromosomes end and inhibiting cell cycle arrest. Studies have implicated the dysfunction of telomere to the formation of sub-tetraploid aneuploid cells that exhibit tumourigenic capacity and formation of anueploid cells that can elude programmed cell death (apoptosis) and form tumour cells, thus initiating genomic instability. In this review, we presented an insight to the causes of DNA damage and its associated genomic instability. We found that many factors such as fragile sites, end replication problem, DNA double strand breaks, DNA replication defects, deletion, insertion and translocation all are responsible to the fragile nature of the genome.

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader.

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of ~500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex and addressed the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We discuss these results with respect to how chromosomal replication and telomere elongation are coordinated.

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of ~500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex and addressed the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We discuss these results with respect to how chromosomal replication and telomere elongation are coordinated.