Human subtelomeric duplicon structure and organization.
ABSTRACT: Human subtelomeric segmental duplications ('subtelomeric repeats') comprise about 25% of the most distal 500 kb and 80% of the most distal 100 kb in human DNA. A systematic analysis of the duplication substructure of human subtelomeric regions was done in order to develop a detailed understanding of subtelomeric sequence organization and a nucleotide sequence-level characterization of subtelomeric duplicon families.The extent of nucleotide sequence divergence within subtelomeric duplicon families varies considerably, as does the organization of duplicon blocks at subtelomere alleles. Subtelomeric internal (TTAGGG)n-like tracts occur at duplicon boundaries, suggesting their involvement in the generation of the complex sequence organization. Most duplicons have copies at both subtelomere and non-subtelomere locations, but a class of duplicon blocks is identified that are subtelomere-specific. In addition, a group of six subterminal duplicon families are identified that, together with six single-copy telomere-adjacent segments, include all of the (TTAGGG)n-adjacent sequence identified so far in the human genome.Identification of a class of duplicon blocks that is subtelomere-specific will facilitate high-resolution analysis of subtelomere repeat copy number variation as well as studies involving somatic subtelomere rearrangements. The significant levels of nucleotide sequence divergence within many duplicon families as well as the differential organization of duplicon blocks on subtelomere alleles may provide opportunities for allele-specific subtelomere marker development; this is especially true for subterminal regions, where divergence and organizational differences are the greatest. These subterminal sequence families comprise the immediate cis-elements for (TTAGGG)n tracts, and are prime candidates for subtelomeric sequences regulating telomere-specific (TTAGGG)n tract length in humans.
Project description:Accurate maps and DNA sequences for human subtelomere regions, along with detailed knowledge of subtelomere variation and long-range telomere-terminal haplotypes in individuals, are critical for understanding telomere function and its roles in human biology. Here, we use a highly automated whole genome mapping technology in nano-channel arrays to analyze large terminal human chromosome segments extending from chromosome-specific subtelomere sequences through subtelomeric repeat regions to terminal (TTAGGG)n repeat tracts. We establish detailed maps for subtelomere gap regions in the human reference sequence, detect many new large subtelomeric variants and demonstrate the feasibility of long-range haplotyping through segmentally duplicated subtelomere regions. These features make the method a uniquely valuable new tool for improving the quality of genome assemblies in complex DNA regions. Based on single molecule mapping of telomere-terminal DNA fragments, we provide proof of principle for a novel method to estimate telomere lengths linked to distinguishable telomeric haplotypes; this single-telomere genotyping method may ultimately enable delineation of human cis elements involved in telomere length regulation.
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.
Project description:Chimpanzee and gorilla chromosomes differ from human chromosomes by the presence of large blocks of subterminal heterochromatin thought to be composed primarily of arrays of tandem satellite sequence. We explore their sequence composition and organization and show a complex organization composed of specific sets of segmental duplications that have hyperexpanded in concert with the formation of subterminal satellites. These regions are highly copy number polymorphic between and within species, and copy number differences involving hundreds of copies can be accurately estimated by assaying read-depth of next-generation sequencing data sets. Phylogenetic and comparative genomic analyses suggest that the structures have arisen largely independently in the two lineages with the exception of a few seed sequences present in the common ancestor of humans and African apes. We propose a model where an ancestral human-chimpanzee pericentric inversion and the ancestral chromosome 2 fusion both predisposed and protected the chimpanzee and human genomes, respectively, to the formation of subtelomeric heterochromatin. Our findings highlight the complex interplay between duplicated sequences and chromosomal rearrangements that rapidly alter the cytogenetic landscape in a short period of evolutionary time.
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:BACKGROUND:DNA packaging into chromatin regulates all DNA-related processes and at chromosomal ends could affect both essential functions of telomeres: protection against DNA damage response and telomere replication. Despite this primordial role of chromatin, little is known about chromatin organization, and in particular about nucleosome positioning on unmodified subtelomere-telomere junctions in Saccharomyces cerevisiae. RESULTS:By ChEC experiments and indirect end-labeling, we characterized nucleosome positioning as well as specialized protein-DNA associations on most subtelomere-telomere junctions present in budding yeast. The results show that there is a relatively large nucleosome-free region at chromosome ends. Despite the absence of sequence homologies between the two major classes of subtelomere-telomere junctions (i.e.: Y'-telomeres and X-telomeres), all analyzed subtelomere-telomere junctions show a terminal nucleosome-free region just distally from the known Rap1-covered telomeric repeats. Moreover, previous evidence suggested a telomeric chromatin fold-back structure onto subtelomeric areas that supposedly was implicated in chromosome end protection. The in vivo ChEC method used herein in conjunction with several proteins in a natural context revealed no evidence for such structures in bulk chromatin. CONCLUSIONS:Our study allows a structural definition of the chromatin found at chromosome ends in budding yeast. This definition, derived with direct in vivo approaches, includes a terminal area that is free of nucleosomes, certain positioned nucleosomes and conserved DNA-bound protein complexes. This organization of subtelomeric and telomeric areas however does not include a telomeric cis-loopback conformation. We propose that the observations on such fold-back structures may report rare and/or transient associations and not stable or constitutive structures.
Project description:We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines.
Project description: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:DNA sequence surveys in yeast and humans suggest that the forces shaping telomeric polymorphism and divergence are distinctly more dynamic than those in the euchromatic, gene-rich regions of the chromosomes. However, the generality of this pattern across outbreeding, multicellular eukaryotes has not been determined. To characterize the structure and evolution of Drosophila telomeres, we collected and analyzed molecular population genetics data from the X chromosome subtelomere in 58 lines of North American Drosophila melanogaster and 29 lines of African D. melanogaster. We found that Drosophila subtelomeres exhibit high levels of both structural and substitutional polymorphism relative to linked euchromatic regions. We also observed strikingly different patterns of variation in the North American and African samples. Moreover, our analyses of the polymorphism data identify a localized hotspot of recombination in the most-distal portion of the X subtelomere. While the levels of polymorphism decline sharply and in parallel with rates of crossing over per physical length over the distal first euchromatic megabase pairs of the X chromosome, our data suggest that they rise again sharply in the subtelomeric region (approximately 80 kbp). These patterns of historical recombination and geographic differentiation indicate that, similar to yeast and humans, Drosophila subtelomeric DNA is evolving very differently from euchromatic DNA.
Project description:Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks that activate DNA damage checkpoints. In budding yeast, the Mre11-Rad50-Xrs2 (MRX) complex associates with DNA ends and promotes checkpoint activation. Rap1 binds to double-stranded telomeric regions and recruits Rif1 and Rif2 to telomeres. Rap1 collaborates with Rif1 and Rif2 and inhibits MRX localization to DNA ends. This Rap1-Rif1-Rif2 function becomes attenuated at shortened telomeres. Here we show that Rap1 acts together with the subtelomere-binding protein Tbf1 and inhibits MRX localization to DNA ends. The placement of a subtelomeric sequence or TTAGGG repeats together with a short telomeric TG repeat sequence inhibits MRX accumulation at nearby DNA ends in a Tbf1-dependent manner. Moreover, tethering of both Tbf1 and Rap1 proteins decreases MRX and Tel1 accumulation at nearby DNA ends. This Tbf1- and Rap1-dependent pathway operates independently of Rif1 or Rif2 function. Depletion of Tbf1 protein stimulates checkpoint activation in cells containing short telomeres but not in cells containing normal-length telomeres. These data support a model in which Tbf1 and Rap1 collaborate to maintain genomic stability of short telomeres.