Subtelomeres constitute a safeguard for gene expression and chromosome homeostasis.
ABSTRACT: The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.
Project description:Genome sequences have been determined for many model organisms; however, repetitive regions such as centromeres, telomeres, and subtelomeres have not yet been sequenced completely. Here, we report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe. We overcame technical difficulties to obtain subtelomeric repetitive sequences by constructing strains that possess single SH regions of a standard laboratory strain. In addition, some natural isolates of S. pombe were analyzed using previous sequencing data. Whole sequences of SH regions revealed that each SH region consists of two distinct parts with mosaics of multiple common segments or blocks showing high variation among subtelomeres and strains. Subtelomere regions show relatively high frequency of nucleotide variations among strains compared with the other chromosomal regions. Furthermore, we identified subtelomeric RecQ-type helicase genes, tlh3 and tlh4, which add to the already known tlh1 and tlh2, and found that the tlh1-4 genes show high sequence variation with missense mutations, insertions, and deletions but no severe effects on their RNA expression. Our results indicate that SH sequences are highly polymorphic and hot spots for genome variation. These features of subtelomeres may have contributed to genome diversity and, conversely, various diseases.
Project description:Subtelomeres are patchworks of evolutionary conserved sequence blocks and harbor the transcriptional start sites for telomere repeat containing RNAs (TERRA). Recent studies suggest that the interplay between telomeres and subtelomeric chromatin is required for maintaining telomere function. To further characterize chromatin remodeling of subtelomeres in relation to telomere shortening and cellular senescence, we systematically quantified histone modifications and DNA methylation at the subtelomeres of chromosomes 7q and 11q in primary human WI-38 fibroblasts. Upon senescence, both subtelomeres were characterized by a decrease in markers of constitutive heterochromatin, suggesting relative chromatin relaxation. However, we did not find increased levels of markers of euchromatin or derepression of the 7q VIPR2 gene. The repressed state of the subtelomeres was maintained upon senescence, which could be attributed to a rise in levels of facultative heterochromatin markers at both subtelomeres. While senescence-induced subtelomeric chromatin remodeling was similar for both chromosomes, chromatin remodeling at TERRA promoters displayed chromosome-specific patterns. At the 7q TERRA promoter, chromatin structure was co-regulated with the more proximal subtelomere. In contrast, the 11q TERRA promoter, which was previously shown to be bound by CCCTC-binding factor CTCF, displayed lower levels of markers of constitutive heterochromatin that did not change upon senescence, whereas levels of markers of facultative heterochromatin decreased upon senescence. In line with the chromatin state data, transcription of 11q TERRA but not 7q TERRA was detected. Our study provides a detailed description of human subtelomeric chromatin dynamics and shows distinct regulation of the TERRA promoters of 7q and 11q upon cellular senescence.
Project description:It is generally believed that telomeric repeats are a necessary and sufficient cis-element for telomere function. Here we show that telomere structure and meiotic function are stably inherited in fission yeast circular chromosomes that have lost all telomeric repeats. We found that the telomeric repeat binding protein, Taz1, and the heterochromatin protein, Swi6, remain associated with subtelomeres in the absence of telomeric repeats. We also found that the fusion point of circular chromosomes that lack telomeric repeats associates with SPB (the yeast counterpart of the centrosome) in the premeiotic horsetail stage, similarly to wild-type telomeres. However, a taz1+ deletion/reintroduction experiment revealed that the maintenance of Taz1 binding and premeiotic function is achieved via different strategies. Taz1 is recruited to subtelomeres by an autonomous element present in subtelomeric DNA, thus in a genetic mechanism. In contrast, the premeiotic subtelomere-SPB association is maintained in an epigenetic manner. These results shed light on the previously unrecognized role played by the subtelomere and underscore the robust nature of the functional telomere complex that is maintained by both genetic and epigenetic mechanisms. Furthermore, we suggest that the establishment and the maintenance of the functional telomere complex are mechanistically distinguishable.
Project description:Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major variant surface antigen, variant surface glycoprotein (VSG), to evade the host immune response, and VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double-strand breaks (DSBs) in both active and silent subtelomeric bloodstream form expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion induced much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES.
Project description:Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription.
Project description:In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyse their structure and infer how they evolved. Here, we use recent genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is a heterochromatic array of Sultan elements adjacent to the telomere, followed by a transcribed Spacer sequence, a G-rich microsatellite and transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Analysis of other green algae reveals species-specific repeated elements that are shared across subtelomeres, with an overall organization similar to C. reinhardtii. This work uncovers the complexity and evolution of subtelomere architecture in green algae.
Project description:Mapping genome-wide data to human subtelomeres has been problematic due to the incomplete assembly and challenges of low-copy repetitive DNA elements. Here, we provide updated human subtelomere sequence assemblies that were extended by filling telomere-adjacent gaps using clone-based resources. A bioinformatic pipeline incorporating multiread mapping for annotation of the updated assemblies using short-read data sets was developed and implemented. Annotation of subtelomeric sequence features as well as mapping of CTCF and cohesin binding sites using ChIP-seq data sets from multiple human cell types confirmed that CTCF and cohesin bind within 3 kb of the start of terminal repeat tracts at many, but not all, subtelomeres. CTCF and cohesin co-occupancy were also enriched near internal telomere-like sequence (ITS) islands and the nonterminal boundaries of subtelomere repeat elements (SREs) in transformed lymphoblastoid cell lines (LCLs) and human embryonic stem cell (ES) lines, but were not significantly enriched in the primary fibroblast IMR90 cell line. Subtelomeric CTCF and cohesin sites predicted by ChIP-seq using our bioinformatics pipeline (but not predicted when only uniquely mapping reads were considered) were consistently validated by ChIP-qPCR. The colocalized CTCF and cohesin sites in SRE regions are candidates for mediating long-range chromatin interactions in the transcript-rich SRE region. A public browser for the integrated display of short-read sequence-based annotations relative to key subtelomere features such as the start of each terminal repeat tract, SRE identity and organization, and subtelomeric gene models was established.
Project description:Copy number variation is a defining characteristic of human subtelomeres. Human subtelomeric segmental duplication regions ('Subtelomeric Repeats') comprise about 25% of the most distal 500 kb and 80% of the most distal 100 kb in human DNA. Huge allelic disparities seen in subtelomeric DNA sequence content and organization are postulated to have an impact on the dosage of transcripts embedded within the duplicated sequences, on the transcription of genes in adjacent single copy DNA regions, and on the chromatin structures mediating telomere functions including chromosome stability. In addition to the complex duplicon substructure and huge allelic variations in extended subtelomere regions, both copy number variation and alternative sequence organizations for DNA characterize the sequences immediately adjacent to terminal (TTAGGG)n tracts ('subterminal DNA'). The structural variation in subterminal DNA is likely to have important consequences for expression of subterminal transcripts such as a newly-discovered gene family encoding actin-interacting proteins and a non-coding telomeric repeat containing RNA (TERRA) transcript family critical for telomere integrity. Major immediate challenges include discovering the full extent and nature of subtelomeric structural and copy number variation in humans, and developing methods for tracking individual allelic variants in the context of total genomic DNA.
Project description:Detailed comprehensive knowledge of the structures of individual long-range telomere-terminal haplotypes are needed to understand their impact on telomere function, and to delineate the population structure and evolution of subtelomere regions. However, the abundance of large evolutionarily recent segmental duplications and high levels of large structural variations have complicated both the mapping and sequence characterization of human subtelomere regions. Here, we use high throughput optical mapping of large single DNA molecules in nanochannel arrays for 154 human genomes from 26 populations to present a comprehensive look at human subtelomere structure and variation. The results catalog many novel long-range subtelomere haplotypes and determine the frequencies and contexts of specific subtelomeric duplicons on each chromosome arm, helping to clarify the currently ambiguous nature of many specific subtelomere structures as represented in the current reference sequence (HG38). The organization and content of some duplicons in subtelomeres appear to show both chromosome arm and population-specific trends. Based upon these trends we estimate a timeline for the spread of these duplication blocks.
Project description:The contribution of human subtelomeric DNA and chromatin organization to telomere integrity and chromosome end protection is not yet understood in molecular detail. Here, we show by ChIP-Seq that most human subtelomeres contain a CTCF- and cohesin-binding site within ?1-2?kb of the TTAGGG repeat tract and adjacent to a CpG-islands implicated in TERRA transcription control. ChIP-Seq also revealed that RNA polymerase II (RNAPII) was enriched at sites adjacent to the CTCF sites and extending towards the telomere repeat tracts. Mutation of CTCF-binding sites in plasmid-borne promoters reduced transcriptional activity in an orientation-dependent manner. Depletion of CTCF by shRNA led to a decrease in TERRA transcription, and a loss of cohesin and RNAPII binding to the subtelomeres. Depletion of either CTCF or cohesin subunit Rad21 caused telomere-induced DNA damage foci (TIF) formation, and destabilized TRF1 and TRF2 binding to the TTAGGG proximal subtelomere DNA. These findings indicate that CTCF and cohesin are integral components of most human subtelomeres, and important for the regulation of TERRA transcription and telomere end protection.