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The electrostatics-based dsDNA binding activity in TbRAP1 NLS can be diminished by phosphorylation of NLS-adjacent S residues., Localization of Repressor Activator Protein 1 (RAP1) to the telomere is essential for its telomeric functions. RAP1 homologs either directly bind the duplex telomere DNA or interact with telomere-binding proteins. We find that Trypanosoma brucei RAP1 relies on a unique double-stranded DNA (dsDNA) binding activity to achieve this goal. T. brucei causes human sleeping sickness and regularly switches its major surface antigen, variant surface glycoprotein (VSG), to evade the host immune response. VSGs are monoallelically expressed from subtelomeres, and TbRAP1 is essential for VSG regulation. We identify dsDNA and single-stranded DNA binding activities in TbRAP1, which require positively charged 737RKRRR741 residues that overlap with TbRAP1’s nuclear localization signal in the MybLike domain. Both DNA binding activities are electrostatics-based and sequence nonspecific. The dsDNA binding activity can be substantially diminished by phosphorylation of two 737RKRRR741-adjacent S residues and is essential for TbRAP1’s telomere localization, VSG silencing, telomere integrity, and cell proliferation.

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.

Malaria is caused by infection with Plasmodium parasites and is a major public health concern. The CRISPR/Cas9 system is a promising technology, but still has technical problems, such as low efficiency and unexpected recombination. Here, we solved these problems by transfecting Cas9-expressing parasites with linear donor templates. The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large-scale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the post-genomic era of Plasmodium species., Shinzawa et al. develop a method that improves the transfection efficiency for Plasmodium parasites that cause malaria by transfecting Cas9-expressing parasites with linear donor templates. This method allows efficient integration of the donor template while preventing unexpected recombination.

Background Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887–898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. Results Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. Conclusion Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

Facioscapulohumeral dystrophy (FSHD) is the most frequent muscular disease in adults. FSHD is characterized by a weakness and atrophy of a specific set of muscles located in the face, the shoulder, and the upper arms. FSHD patients may present different genetic defects, but they all present epigenetic alterations of the D4Z4 array located on the subtelomeric part of chromosome 4, leading to chromatin relaxation and, ultimately, to the aberrant expression of one gene called DUX4. Once expressed, DUX4 triggers a cascade of deleterious events, eventually leading to muscle dysfunction and cell death. Here, we review studies on DUX4 expression in skeletal muscle to determine the genetic/epigenetic factors and regulatory proteins governing DUX4 expression, with particular attention to the different transcripts and their very low expression in muscle.

Telomeres are the ends of linear chromosomes comprised of repetitive nucleotide sequences in humans. Telomeres preserve chromosomal stability and genomic integrity. Telomere length shortens with every cell division in somatic cells, eventually resulting in replicative senescence once telomere length becomes critically short. Telomere shortening can be overcome by telomerase enzyme activity that is undetectable in somatic cells, while being active in germline cells, stem cells, and immune cells. Telomeres are bound by a shelterin complex that regulates telomere lengthening as well as protects them from being identified as DNA damage sites. Telomeres are transcribed by RNA polymerase II, and generate a long noncoding RNA called telomeric repeat-containing RNA (TERRA), which plays a key role in regulating subtelomeric gene expression. Replicative immortality and genome instability are hallmarks of cancer and to attain them cancer cells exploit telomere maintenance and telomere protection mechanisms. Thus, understanding the role of telomeres and their associated proteins in cancer initiation, progression and treatment is very important. The present review highlights the critical role of various telomeric components with recently established functions in cancer. Further, current strategies to target various telomeric components including human telomerase reverse transcriptase (hTERT) as a therapeutic approach in human malignancies are discussed.

Significance Meiosis is fundamental to eukaryotic reproduction and shapes patterns of genetic variation. Meiotic recombination is also a vital tool during crop improvement, which allows introgression of wild variation into agricultural strains. Despite this, the levels and distributions of crossovers along chromosomes can limit breeding. For example, many crops show highly skewed crossover distributions toward the telomeres. This can lead to the problem of linkage drag when variation within nonrecombining regions is selected. Our findings demonstrate how gene dosage of key components of the meiotic chromosome axis can be used to remodel the recombination landscape. Therefore, modifying ASY1 and ASY3 gene dosage in crop species may provide a strategy to change recombination patterns or levels in order to accelerate strain improvement., During meiosis, interhomolog recombination produces crossovers and noncrossovers to create genetic diversity. Meiotic recombination frequency varies at multiple scales, with high subtelomeric recombination and suppressed centromeric recombination typical in many eukaryotes. During recombination, sister chromatids are tethered as loops to a polymerized chromosome axis, which, in plants, includes the ASY1 HORMA domain protein and REC8–cohesin complexes. Using chromatin immunoprecipitation, we show an ascending telomere-to-centromere gradient of ASY1 enrichment, which correlates strongly with REC8–cohesin ChIP-seq data. We mapped crossovers genome-wide in the absence of ASY1 and observe that telomere-led recombination becomes dominant. Surprisingly, asy1/+ heterozygotes also remodel crossovers toward subtelomeric regions at the expense of the pericentromeres. Telomeric recombination increases in asy1/+ occur in distal regions where ASY1 and REC8 ChIP enrichment are lowest in wild type. In wild type, the majority of crossovers show interference, meaning that they are more widely spaced along the chromosomes than expected by chance. To measure interference, we analyzed double crossover distances, MLH1 foci, and fluorescent pollen tetrads. Interestingly, while crossover interference is normal in asy1/+, it is undetectable in asy1 mutants, indicating that ASY1 is required to mediate crossover interference. Together, this is consistent with ASY1 antagonizing telomere-led recombination and promoting spaced crossover formation along the chromosomes via interference. These findings provide insight into the role of the meiotic axis in patterning recombination frequency within plant genomes.

International audience; In the budding yeast Saccharomyces cerevisiae, telomerase is constitutively active and is essential for chromosome end protection and illimited proliferation of cell populations. However, upon inactivation of telomerase, alternative mechanims of telomere maintenance allow proliferation of only extremely rare survivors. S. cerevisiae type I and type II survivors differ by the nature of the donor sequences used for repair by homologous recombination of the uncapped terminal TG1-3 telomeric sequences. Type I amplifies the subtelomeric Y' sequences and is more efficient than type II, which amplifies the terminal TG1-3 repeats. However, type II survivors grow faster than type I survivors and can easily outgrow them in liquid cultures. The mechanistic interest of studying S. cerevisiae telomeric recombination is reinforced by the fact that type II recombination is the equivalent of the alternative lengthening of telomeres (ALT) pathway that is used by 5-15 % of cancer types as an alternative to telomerase reactivation. In budding yeast, only around half of the 32 telomeres harbor Y' subtelomeric elements. We report here that in strains harboring Y' elements on all telomeres, type II survivors are not observed, most likely due to an increase in the efficiency of type I recombination. However, in a temperature-sensitive cdc13-1 mutant grown at semi-permissive temperature, the increased amount of telomeric TG1-3 repeats could overcome type II inhibition by the subtelomeric Y' sequences. Strikingly, in the 100 % Y' strain the replicative senescence crisis normally provoked by inactivation of telomerase completely disappeared and the severity of the crisis was proportional to the percentage of chromosome-ends lacking Y' subtelomeric sequences. The present study highlights the fact that the nature of subtelomeric elements can influence the selection of the pathway of telomere maintenance by recombination, as well as the response of the cell to telomeric damage caused by telomerase inactivation.

Telomere length is measured using Southern blotting of the chromosomal terminal restriction fragments (TRFs) released by endonuclease digestion in cells from yeast to human. In the budding yeast Saccharomyces cerevisiae, XhoI or PstI is applied to cut the subtelomere Y' element and release TRFs from the 17 subtelomeres. However, telomeres from other 15 X-element-only subtelomeres are omitted from analysis. Here, we report a method for measuring all 32 telomeres in S. cerevisiae using the endonuclease MmeI. Based on analyses of the endonuclease cleavage sites, we found that the TRFs generated by MmeI displayed two distinguishable bands in the sizes of ~500 and ~700 bp comprising telomeres (300 bp) and subtelomeres (200-400 bp). The modified MmeI-restricted TRF (mTRF) method recapitulated telomere shortening and lengthening caused by deficiencies of YKu and Rif1 respectively in S. cerevisiae. Furthermore, we found that mTRF was also applicable to telomere length analysis in S. paradoxus strains. These results demonstrate a useful tool for simultaneous detection of telomeres from all chromosomal ends with both X-element-only and Y'-element subtelomeres in S. cerevisiae species.

© 2021 Viceconte et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/., Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing., Author summary During aging, gene silencing also decreases and it has been hypothesized that the collapse of epigenetic control networks may in part explain age-related diseases. For example, changes in epigenetic silencing are linked with different stages of tumor formation and progression. Great efforts have been made on investigating the mechanisms of establishment and maintenance silencing at silent mating cassettes in yeast. In this work, by applying a genome-wide silencing screening approach, we identified the conserved subunits of the mismatch repair (MMR) machinery (Pms1, Mlh1 and Msh2) as new components of the epigenetic silencing regulation machinery in yeast. We also found that depletion of mismatch repair subunits (Mlh1 and Msh2) led to impaired telomere-length related expression in mammalian cells. This indicates that these components probably have an evolutionarily conserved role on influencing gene silencing from yeast to humans. Further studies the functional roles of these MMR components on epigenetic silencing in mammalian model systems or relevant cancer patient samples will increase our understanding of MMR-related oncogenesis.

Subtelomere Y' elements get amplified by homologous recombination in sustaining the survival and division of the budding yeast Saccharomyces cerevisiae. However, current method for measurement of the subtelomere structures uses Southern blotting with labelled specific probes, which is laborious and time-consuming. By multiple sequence alignment analysis of all 19 subtelomere Y' elements across the 13 chromosomes of the sequenced S288C strain deposited in the yeast genome SGD database, we identified 12 consensus and relative longer fragments and 14 pairs of unique primers for real-time quantitative PCR analysis. With a PAC2 or ACT1 located near the centromere of chromosome V and VI as internal controls, these primers were applied to real-time quantitative PCR analysis, so the relative Y' element intensity normalised to that of wild type (WT) cells was calculated for subtelomere Y' element copy numbers across all different chromosomes using the formula: 2^[-((CTmutant Y' - CTmutant control ) - (CTWT Y' - CTWT control ))]. This novel quantitative subtelomere amplification assay across chromosomes by real-time PCR proves to be a much simpler and more sensitive way than the traditional Southern blotting method to analyse the Y' element recombination events in survivors derived from telomerase deficiency or recruitment failure.

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. Whole sequences of SH regions revealed that each SH region consists of two distinct parts: the telomere-proximal part with mosaics of multiple common segments showing high variation among subtelomeres and strains, and the telomere-distal part showing high sequence similarity among subtelomeres with some insertions and deletions. The newly sequenced SH regions showed differences in nucleotide sequences and common segment composition compared to those in the S. pombe genome database (PomBase), which is in striking contrast to the regions outside of SH, where mutations are rarely detected. Furthermore, we identified new 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. 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.

The parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host’s immune system in a process known as antigenic variation. One route to change VSG expression is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B-interactors. One of these was the Ribonuclease H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage and recombination-based ES switching events. Surprisingly, a similar pattern of VSG deregulation was observed in Ribonuclease H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly-regulated process of antigenic variation.

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., Sequencing and mapping of long repetitive regions can be challenging due to technical difficulties in sequencing and assembly of the sequence data. Here authors report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe to reveal highly polymorphic and hot spots for genome variation features.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing., RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1’s self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.

Telomere anchoring to nuclear envelope (NE) is a key feature of nuclear genome architecture. Peripheral localization of telomeres is important for chromatin silencing, telomere replication and for the control of inappropriate recombination. Here, we report that fission yeast quiescent cells harbor predominantly a single telomeric cluster anchored to the NE. Telomere cluster association to the NE relies on Rap1–Bqt4 interaction, which is impacted by the length of telomeric sequences. In quiescent cells, reducing telomere length or deleting bqt4, both result in an increase in transcription of the telomeric repeat-containing RNA (TERRA). In the absence of Bqt4, telomere shortening leads to deep increase in TERRA level and the concomitant formation of subtelomeric rearrangements (STEEx) that accumulate massively in quiescent cells. Taken together, our data demonstrate that Rap1–Bqt4-dependent telomere association to NE preserves telomere integrity in post-mitotic cells, preventing telomeric transcription and recombination. This defines the nuclear periphery as an area where recombination is restricted, creating a safe zone for telomeres of post-mitotic cells.

Like in most eukaryotes, the linear chromosomes of Trypanosoma cruzi end in a nucleoprotein structure called the telomere, which is preceded by regions of variable length called subtelomeres. Together telomeres and subtelomeres are dynamic sites where DNA sequence rearrangements can occur without compromising essential interstitial genes or chromosomal synteny. Good examples of subtelomeres involvement are the expansion of human olfactory receptors genes, variant surface antigens in Trypanosoma brucei, and Saccharomyces cerevisiae mating types. T. cruzi telomeres are made of long stretches of the hexameric repeat 5′-TTAGGG-OH-3′, and its subtelomeres are enriched in genes and pseudogenes from the large gene families RHS, TS and DGF1, DEAD/H-RNA helicase and N-acetyltransferase, intermingled with sequences of retrotransposons elements. In particular, members of the Trans-sialidase type II family appear to have played a role in shaping the current T. cruzi telomere structure. Although the structure and function of T. cruzi telomeric and subtelomeric regions have been documented, recent experiments are providing new insights into T. cruzi's telomere-subtelomere dynamics. In this review, I discuss the co-evolution of telomere, subtelomeres and the TS gene family, and the role that these regions may have played in shaping T. cruzi's genome.

BackgroundThe broad host range pathogenSclerotinia sclerotioruminfects over 400 plant species and causes substantial yield losses in crops worldwide. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but little is known about the secondary metabolite repertoire ofS. sclerotiorum. In this study, we predicted secondary metabolite biosynthetic gene clusters in the genome ofS. sclerotiorumand analysed their expression during infection ofBrassica napususing an existing transcriptome data set. We also investigated their sequence diversity among a panel of 25 previously publishedS. sclerotiorumisolate genomes.ResultsWe identified 80 putative secondary metabolite clusters. Over half of the clusters contained at least three transcriptionally coregulated genes. Comparative genomics revealed clusters homologous to clusters in the closely related plant pathogenBotrytis cinereafor production of carotenoids, hydroxamate siderophores, DHN melanin and botcinic acid. We also identified putative phytotoxin clusters that can potentially produce the polyketide sclerin and an epipolythiodioxopiperazine. Secondary metabolite clusters were enriched in subtelomeric genomic regions, and those containing paralogues showed a particularly strong association with repeats. The positional bias we identified was borne out by intraspecific comparisons that revealed putative secondary metabolite genes suffered more presence / absence polymorphisms and exhibited a significantly higher sequence diversity than other genes.ConclusionsThese data suggest thatS. sclerotiorumproduces numerous secondary metabolites during plant infection and that their gene clusters undergo enhanced rates of mutation, duplication and recombination in subtelomeric regions. The microevolutionary regimes leading toS. sclerotiorumsecondary metabolite diversity have yet to be elucidated. Several potential phytotoxins documented in this study provide the basis for future functional analyses.

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While heterochromatin and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1's catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

Retrotransposon Hot Spot (RHS) is the most abundant gene family in Trypanosoma cruzi, with unknown function in this parasite. The aim of this work was to shed light on the organization and expression of RHS in T. cruzi. The diversity of the RHS protein family in T. cruzi was demonstrated by phylogenetic and recombination analyses. Transcribed sequences carrying the RHS domain were classified into ten distinct groups of monophyletic origin. We identified numerous recombination events among the RHS and traced the origins of the donors and target sequences. The transcribed RHS genes have a mosaic structure that may contain fragments of different RHS inserted in the target sequence. About 30% of RHS sequences are located in the subtelomere, a region very susceptible to recombination. The evolution of the RHS family has been marked by many events, including gene duplication by unequal mitotic crossing-over, homologous, as well as ectopic recombination, and gene conversion. The expression of RHS was analyzed by immunofluorescence and immunoblotting using anti-RHS antibodies. RHS proteins are evenly distributed in the nuclear region of T. cruzi replicative forms (amastigote and epimastigote), suggesting that they could be involved in the control of the chromatin structure and gene expression, as has been proposed for T. brucei.

Any theory suggesting an adaptive meaning for aging implicitly postulates the existence of specific mechanisms, genetically determined and modulated, causing progressive decline of an organism. According to the subtelomere–telomere theory, each telomere is covered by a hood formed in the first cell of an organism having a size preserved at each subsequent duplication. Telomere shortening, which is quantitatively different for each cell type according to the telomerase regulation, causes the hood to slide on the subtelomere repressing it by the telomeric position effect. At this point, the theory postulates existence of subtelomeric regulatory sequences, whose progressive transcriptional repression by the hood should cause cellular alterations that would be the likely determinant of aging manifestations. However, sequences with characteristics of these hypothetical sequences have already been described and documented. They are the [sub]TElomeric Repeat-containing RNA (TERRA) sequences. The repression of TERRA sequences causes progressively: (i) down- or up-regulation of many other regulatory sequences; (ii) increase in the probability of activation of cell senescence program (blockage of the ability to replicate and very significant alterations of the cellular functions). When cell senescence program has not been triggered and the repression is partial, there is a partial alteration of the cellular functions that is easily reversible by telomerase activation. Location of the extremely important sequences in chromosomal parts that are most vulnerable to repression by the telomeric hood is evolutionarily unjustifiable if aging is not considered adaptive: this location must be necessarily adaptive with the specific function of determining aging of the cell and consequently of the whole organism.

Telomere length is maintained by either telomerase, a reverse transcriptase, or alternative lengthening of telomeres (ALT), a mechanism that utilizes homologous recombination (HR) proteins. Since access to DNA for HR enzymes is regulated by the chromatin status, it is expected that telomere elongation is linked to epigenetic modifications. The aim of this review is to elucidate the epigenetic features of ALT-positive cells. In order to do this, it is first necessary to understand the telomeric chromatin peculiarities. So far, the epigenetic nature of telomeres is still controversial: some authors describe them as heterochromatic, while for others, they are euchromatic. Similarly, ALT activity should be characterized by the loss (according to most researchers) or formation (as claimed by a minority) of heterochromatin in telomeres. Besides reviewing the main works in this field and the most recent findings, some hypotheses involving the role of telomere non-canonical sequences and the possible spatial heterogeneity of telomeres are given.

Contains fulltext : 218917pub.pdf (Publisher’s version ) (Closed access) A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.

In the fission yeast Schizosaccharomyces pombe, both RNAi machinery and RNAi- independent factors mediate transcriptional and posttranscriptional silencing and heterochromatin formation. Here, we show that the silencing of reporter genes at major native heterochromatic loci (centromeres, telomeres, mating-type locus and rDNA regions) and an artificially induced heterochromatin locus is alleviated in a fission yeast hsp90 mutant, hsp90-G84C. Also, H3K9me2 enrichment at heterochromatin regions, especially at the mating-type locus and subtelomeres, is compromised, suggesting heterochromatin assembly defects. We further discovered that Hsp90 is required for stabilization or assembly of the RNAi effector complexes RITS and ARC, RNAi-independent factor Fft3, shelterin complex subunit Poz1 and SHREC. Our ChIP data suggest that Hsp90 regulates the efficient recruitment of CLRC by shelterin to chromosome ends and targeting of SHREC and Fft3 to mating type locus and/or rDNA region. Finally, our genetic analyses demonstrated that increased heterochromatin spreading restores silencing at subtelomeres in hsp90-G84C mutant. Thus, this work uncovers a conserved factor critical for promoting RNAi-dependent and -independent heterochromatin assembly and gene silencing through stabilizing multiple effectors and effector complexes.

International audience; Specificities associated with chromosomal linearity are not restricted to telomeres. Here, recent results obtained on fission and budding yeast are summarized and an attempt is made to define subtelomeres using chromatin features extending beyond the heterochromatin emanating from telomeres. Subtelomeres, the chromosome domains adjacent to telomeres, differ from the rest of the genome by their gene content, rapid evolution, and chromatin features that together contribute to organism adaptation. However, current definitions of subtelomeres are generally based on synteny and are largely gene‐centered. Taking into consideration both the peculiar gene content and dynamics as well as the chromatin properties of those domains, it is discussed how chromatin features can contribute to subtelomeric properties and functions, and play a pivotal role in the emergence of subtelomeres.

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here we investigate the well conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing complex SIR composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles ofSIR3, we show that the binding of Sir3 directly promotes long range contacts between distant regions, including the rDNA, telomeres, and internal Sir3 bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, and Sir4), Sir2 activity or chromosome context. Altogether these data suggest that Sir3 represents an uncommon example of protein able to bridge directly distant loci.

Mindfulness and meditation techniques have proven successful for the reduction of stress and improvement in general health. In addition, meditation is linked to longevity and longer telomere length, a proposed biomarker of human aging. Interestingly, DNA methylation changes have been described at specific subtelomeric regions in long-term meditators compared to controls. However, the molecular basis underlying these beneficial effects of meditation on human health still remains unclear. Here we show that DNA methylation levels, measured by the Infinium HumanMethylation450 BeadChip (Illumina) array, at specific subtelomeric regions containing GPR31 and SERPINB9 genes were associated with telomere length in long-term meditators with a strong statistical trend when correcting for multiple testing. Notably, age showed no association with telomere length in the group of long-term meditators. These results may suggest that long-term meditation could be related to epigenetic mechanisms, in particular gene-specific DNA methylation changes at distinct subtelomeric regions. The project has received funding from the Network for Prevention and Health Promotion in primary Care (RD12/0005 and RD16/0007/0005) grant and a FIS grant (PI16/00962) from the Instituto de Salud Carlos III of the Spanish Ministry of Economy and Competitiveness, co-financed with European Union ERDF funds. MM has received a grant 'intensificación' from Fundación LaCaixa.

Background: Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran families, structure of chromosome termini is usually conserved within genera. With the aim to assess whether or not the evolutionary distance between genera implies chromosome end diversification, this report exploits two representatives of Sciaridae, Rhynchosciara americana, and Trichomegalosphyspubescens. Methods: Probes and plasmid microlibraries obtained by chromosome end microdissection, in situ hybridization, cloning, and sequencing are among the methodological approaches employed in this work. Results: The data argue for the existence of either specific terminal DNA sequences for each chromosome tip in T. pubescens, or sequences common to all chromosome ends but their extension does not allow detection by in situ hybridization. Both sciarid species share terminal sequences that are significantly underrepresented in chromosome ends of T. pubescens. Conclusions: The data suggest an unusual terminal structure in T. pubescens chromosomes compared to other dipterans investigated. A putative, evolutionary process of repetitive DNA expansion that acted differentially to shape chromosome ends of the two flies is also discussed.

Histone H2B lysine 123 mono-ubiquitination (H2Bub1), catalyzed by Rad6 and Bre1 in Saccharomyces cerevisiae, modulates chromatin structure and affects diverse cellular functions. H2Bub1 plays roles in telomeric silencing and telomere replication. Here, we have explored a novel role of H2Bub1 in telomere protection at uncapped telomeres in yku70Δ and cdc13-1 cells. Deletion of RAD6 or BRE1, or mutation of H2BK123R enhances the temperature sensitivity of both yku70Δ and cdc13-1 telomere capping mutants. Consistently, BRE1 deletion increases accumulation of telomeric single-stranded DNA (ssDNA) in yku70Δ and cdc13-1 cells, and EXO1 deletion improves the growth of yku70Δ bre1Δ and cdc13-1 bre1Δ cells and decreases ssDNA accumulation. Additionally, deletion of BRE1 exacerbates the rate of entry into senescence of yku70Δ mre11Δ cells with telomere defects, and increases the recombination of subtelomeric Y' element that is required for telomere maintenance and survivor generation. Furthermore, Exo1 contributes to the abrupt senescence of yku70Δ mre11Δ bre1Δ cells, and Rad51 is essential for Y' recombination to generate survivors. Finally, deletion of BRE1 or mutation of H2BK123R results in nucleosome instability at subtelomeric regions. Collectively, this study provides a mechanistic link between H2Bub1-mediated chromatin structure and telomere protection after telomere uncapping.

Cell senescence is an artificially reversible condition activated by various factors and characterized by replicative senescence and typical general alteration of cell functions, including extra-cellular secretion. The number of senescent cells increases with age and contributes strongly to the manifestations of aging. For these reasons, research is under way to obtain "senolytic" compounds, defined as drugs that eliminate senescent cells and therefore reduce aging-associated decay, as already shown in some experiments on animal models. This objective is analyzed in the context of the programmed aging paradigm, as described by the mechanisms of the subtelomere-telomere theory. In this regard, positive effects of the elimination of senescent cells and limits of this method are discussed. For comparison, positive effects and limits of telomerase activation are also analyzed, as well of the combined action of the two methods and the possible association of opportune gene modifications. Ethical issues associated with the use of these methods are outlined.

Various stresses increase disease susceptibility and accelerate aging, and increasing evidence suggests that these effects can be transmitted over generation. Epidemiological studies suggest that stressors experienced by parents affect the longevity of their offspring, possibly by regulating telomere dynamics. Telomeres are elongated by telomerase and shortened by certain stresses as well as telomere repeat-containing RNA (TERRA), a telomere transcript. However, the mechanism underlying the transgenerational effects is poorly understood. Here, we show that TNF-α, which is induced by various psychological stresses, induces the p38-dependent phosphorylation of ATF7, a stress-responsive chromatin regulator, in mouse testicular germ cells. This caused a release of ATF7 from the TERRA gene promoter in the subtelomeric region, which disrupted heterochromatin and induced TERRA. TERRA was transgenerationally transmitted to zygotes via sperm and caused telomere shortening. These results suggest that ATF7 and TERRA play key roles in paternal stress-induced telomere shortening in the offspring. papers2://publication/uuid/098E3E6E-869C-4DAB-97AF-BDA0E6DF6B90 PMC6326783

Key Clinical Message Hypopituitarism could have been overlooked so far in the patients with 9q subtelomere deletion syndrome (9qSTDS); thus, further investigations or reevaluation of clinical information, especially hormonal evaluations, are warranted to determine whether hypopituitarism is a rare or relatively common presentation in patients with 9qSTDS.

Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disorder. The disease locus for this condition was mapped some 12 years ago and the mutations for the disease are known, but the exact identity of FSHD gene remains elusive. Keywords: facioscapulohumeral muscular dystrophy; mutations; gene; position effect; somatic mosaicism; subtelomeric repeat exchange

Human telomeres and adjacent subtelomeres are packaged as heterochromatin. Subtelomeric DNA undergoes methylation during development by DNA methyltransferase 3B (DNMT3B), including the CpG-rich promoters of the long non-coding RNA (TERRA) embedded in these regions. The factors that direct DNMT3B methylation to human subtelomeres and maintain this methylation throughout lifetime are yet unknown. The importance of subtelomeric methylation is manifested through the abnormal telomeric phenotype in Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome type 1 patients carrying mutations in DNMT3B. Patient cells demonstrate subtelomeric hypomethylation, accompanied by elevated TERRA transcription, accelerated telomere shortening and premature senescence of fibroblasts. ICF syndrome can arise due to mutations in at least three additional genes, ZBTB24 (ICF2), CDCA7 (ICF3) and HELLS (ICF4). While pericentromeric repeat hypomethylation is evident in all ICF syndrome subtypes, the status of subtelomeric DNA methylation had not been described for patients of subtypes 2-4. Here we explored the telomeric phenotype in cells derived from ICF2-4 patients with the aim to determine whether ZBTB24, CDCA7 and HELLS also play a role in establishing and/or maintaining human subtelomeric methylation. We found normal subtelomeric methylation in ICF2-4 and accordingly low TERRA levels and unperturbed telomere length. Moreover, depleting the ICF2-4-related proteins in normal fibroblasts did not influence subtelomeric methylation. Thus, these gene products are not involved in establishing or maintaining subtelomeric methylation. Our findings indicate that human subtelomeric heterochromatin has specialized methylation regulation and highlight the telomeric phenotype as a characteristic that distinguishes ICF1 from ICF2-4.

In 10–15% of cancers, telomere length is maintained by a telomerase-independent, recombination-mediated pathway called alternative lengthening of telomeres (ALT). ALT mechanisms were first seen, and have been best studied, in telomerase-null Saccharomyces cerevisiae cells called “survivors”. There are two main types of survivors. Type I survivors amplify Y′ subtelomeric elements while type II survivors, similar to the majority of human ALT cells, amplify the terminal telomeric repeats. Both types of survivors require Rad52, a key homologous recombination protein, and Pol32, a non-essential subunit of DNA polymerase δ. A number of additional proteins have been reported to be important for either type I or type II survivor formation, but it is still unclear how these two pathways maintain telomeres. In this study, we performed a genome-wide screen to identify novel genes that are important for the formation of type II ALT-like survivors. We identified 23 genes that disrupt type II survivor formation when deleted. 17 of these genes had not been previously reported to do so. Several of these genes (DUN1, CCR4, and MOT2) are known to be involved in the regulation of dNTP levels. We find that dNTP levels are elevated early after telomerase inactivation and that this increase favors the formation of type II survivors.

Summary Chemically induced pluripotent stem cells (CiPSCs) may provide an alternative and attractive source for stem cell-based therapy. Sufficient telomere lengths are critical for unlimited self-renewal and genomic stability of pluripotent stem cells. Dynamics and mechanisms of telomere reprogramming of CiPSCs remain elusive. We show that CiPSCs acquire telomere lengthening with increasing passages after clonal formation. Both telomerase activity and recombination-based mechanisms are involved in the telomere elongation. Telomere lengths strongly indicate the degree of reprogramming, pluripotency, and differentiation capacity of CiPSCs. Nevertheless, telomere damage and shortening occur at a late stage of lengthy induction, limiting CiPSC formation. We find that histone crotonylation induced by crotonic acid can activate two-cell genes, including Zscan4; maintain telomeres; and promote CiPSC generation. Crotonylation decreases the abundance of heterochromatic H3K9me3 and HP1α at subtelomeres and Zscan4 loci. Taken together, telomere rejuvenation links to reprogramming and pluripotency of CiPSCs. Crotonylation facilitates telomere maintenance and enhances chemically induced reprogramming to pluripotency., Graphical Abstract, Highlights • CiPSCs acquire telomere elongation after clonal formation with increasing passages • Both telomerase and recombination mechanisms are involved in the telomere elongation • Telomere damage and shortening can occur during late stage of lengthy induction • Crotonylation activates Zscan4 and promotes telomere elongation and CiPSC induction, In this article, Liu and colleagues show that telomere rejuvenation links to reprogramming and pluripotency of CiPSCs. Moreover, crotonylation induced by crotonic acid facilitates telomere maintenance and enhances chemically induced reprogramming to pluripotency.

Holoprosencephaly (HPE), a common developmental forebrain malformation, is characterized by failure of the cerebrum to completely divide into left and right hemispheres. The etiology of HPE is heterogeneous and a number of environmental and genetic factors have been identified. Cytogenetically visible alterations occur in 25% to 45% of HPE patients and cytogenetic techniques have long been used to study copy number variants (CNV) in this disorder. The karyotype approach initially demonstrated several recurrent chromosomal anomalies, which led to the identification of HPE-specific loci and, eventually, several major HPE genes. More recently, higher-resolution cytogenetic techniques such as subtelomeric multiplex ligation-dependent probe amplification (MLPA) and chromosomal microarray have been used to analyze chromosomal anomalies. By using chromosomal microarray, we sought to identify submicroscopic chromosomal deletions and duplications in patients with HPE. In an analysis of 222 individuals with HPE, a deletion or duplication was detected in 107 individuals. Of these 107 individuals, 23 (21%) had variants that were classified as pathogenic or likely pathogenic by board-certified medical geneticists. We identified multiple patients with deletions in established HPE loci as well as three patients with deletions encompassed by 6q12–q14.3, a CNV previously reported by Bendavid et al. In addition, we identified a new locus, 16p13.2, that warrants further investigation for HPE association. Incidentally, we also found a case of Potocki-Lupski Syndrome, a case of Phelan-McDermid Syndrome, and multiple cases of 22q11.2 deletion syndrome within our cohort. These data confirm the genetically heterogeneous nature of HPE, and also demonstrate clinical utility of chromosomal microarray in diagnosing patients affected by HPE.

[EN] TERRA is an RNA molecule transcribed from human subtelomeric regions toward chromosome ends potentially involved in regulation of heterochromatin stability, semiconservative replication, and telomerase inhibition, among others. TERRA contains tandem repeats of the sequence GGGUUA, with a strong tendency to fold into a four-stranded arrangement known as a parallel G-quadruplex. Here, we demonstrate by using single-molecule force spectroscopy that this potential is limited by the inherent capacity of RNA to self-associate randomly and further condense into entropically more favorable structures. We stretched RNA constructions with more than four and less than eight hexanucleotide repeats, thus unable to form several G-quadruplexes in tandem, flanked by non-G-rich overhangs of random sequence by optical tweezers on a one by one basis. We found that condensed RNA stochastically blocks G-quadruplex folding pathways with a near 20% probability, a behavior that is not found in DNA analogous molecules., Dedicated to the memory of Alfredo Villasante. The authors thank E. Poyatos-Racionero for auxiliary experiments. I.G. is supported by Fundacion IMDEA Nanociencia and M.G. by a Juan de la Cierva contract (FJCI-2016-28474). This research received grants from the MINECO (MAT2015-71806-R and BFU2017-89707-P). IMDEA Nanociencia acknowledges support from the "Severo Ochoa" Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686).

R-loops (RLs) are three-stranded nucleic acid structures that contain a DNA:RNA hybrid and a displaced DNA strand. Genomic regions with GC skew and a G-rich transcript are particularly prone to form RLs. RLs play important physiological roles in cells; however, when present at abnormally high levels, they may threaten genome stability. The perfect GC skew of telomeric repeats and the discovery of telomeric repeat-containing RNA (TERRA), a long noncoding transcript that consists of the G-rich telomeric sequence, make telomeric sequences the perfect candidates for generating RLs. Indeed, in the past 5 years, telomere R-loops (TRLs) have been demonstrated in Saccharomyces cerevisiae, Trypanosoma brucei, and human cells. The presence of TRLs in normal human cells that transcribe low levels of TERRA, suggests a physiological role for these nucleic structures in telomere maintenance. Abnormally enhanced TERRA transcription, as found in several human pathological conditions, leads to high TRL levels and various cellular outcomes, depending on the recombinogenic capabilities of the cells. Study of TRLs in various organisms highlights the necessity for tight regulation of these structures, which can switch from beneficial to detrimental under different conditions. Here, we review the current state of knowledge on TRLs, describe several means by which TRLs are regulated, and discuss how findings from yeast are relevant to human pathological scenarios in which TRLs are deregulated.

Telomeres serve as protective caps that help the cell differentiate between the naturally occurring ends of chromosomes and double-stranded breaks. When telomere capping function becomes compromised, chromosome ends are subjected to elevated rates of chromosome alterations. These effects can be particularly dramatic in the telomere-adjacent subtelomeric region. While the catastrophic impact of severe telomere dysfunction on genome stability has been well documented, the adaptive telomere failure hypothesis considers an alternative role telomere dysfunction may play in adaptive evolution. This hypothesis suggests that low levels of telomere failure, induced by certain environmental stresses, can lead to elevated subtelomeric recombination. Mutational loss, duplication, or modification of subtelomeric contingency genes could ultimately facilitate adaptation by generating novel mutants better able to survive environmental stress. In this perspective, we discuss recent work that examined mild telomere dysfunction and its role in altering the adaptive potential of subtelomeric genes.

Ephrin B2, one of the ligand of the EphB receptors, is involved in a complex signaling pathway regulating the development of the nervous system, neuronal migration, erythropoiesis and vasculogenesis. We report a patient with a de novo variant in EFNB2 and a family in which segregates a 610-kb deletion at chromosome 13q33 encompassing only ARGLU1 and EFNB2 genes. The de novo variant was observed in a patient with anal stenosis, hypoplastic left ventricle and mild developmental delay. The deletion was identified in 2 sibs with congenital heart defect and mild developmental delay. One of the affected sibs further had myoclonic epilepsy and bilateral sensorineural hearing loss. The carrier mother was apparently asymptomatic. Because EFNB2 is located in the subtelomeric region of 13q chromosome, we reviewed the previous reports of terminal 13q deletion. We suggest that haploinsufficiency of the EFNB2 could be at the origin of several clinical features reported in 13qter deletions, including intellectual disability, seizures, congenital heart defects, anorectal malformation and hearing loss.

International audience; During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100-200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the sub-telomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

Subtelomeric regions have several unusual characteristics, including complex repetitive structures, increased rates of evolution, and enrichment for genes involved in niche adaptation. The adaptive telomere failure hypothesis suggests that certain environmental stresses can induce a low level of telomere failure, potentially leading to elevated subtelomeric recombination that could result in adaptive mutational changes within subtelomeric genes. Here, we tested a key prediction of the adaptive telomere failure hypothesis—that telomere dysfunction mild enough to have little or no overall effect on cell fitness could still lead to substantial increases in the mutation rates of subtelomeric genes. Our results show that a mutant of Kluyveromyces lactis with stably short telomeres produced a large increase in the frequency of mutations affecting the native subtelomeric β-galactosidase (LAC4) gene. All lac4 mutants examined from strains with severe telomere dysfunction underwent terminal deletion/duplication events consistent with being due to break-induced replication. In contrast, although cells with mild telomere dysfunction also exhibited similar terminal deletion and duplication events, up to 50% of lac4 mutants from this background unexpectedly contained base changes within the LAC4 coding region. This mutational bias for producing base changes demonstrates that mild telomere dysfunction can be well suited as a force for altering the adaptive potential of subtelomeric genes.

The genome of Plasmodium falciparum, the causative agent of malaria in Africa, has been extensively studied since it was first fully sequenced in 2002. However, many open questions remain, including understanding the chromosomal context of molecular evolutionary changes (e.g., relationship between chromosome map and phylogenetic conservation, patterns of gene duplication, and patterns of selection). Here, we present PhyloChromoMap, a method that generates a phylogenomic map of chromosomes from a custom-built bioinformatics pipeline. Using P. falciparum 3D7 as a model, we analyze 2,116 genes with homologs in up to 941 diverse eukaryotic, bacterial and archaeal lineages. We estimate the level of conservation along chromosomes based on conservation across clades, and identify “young” regions (i.e., those with recent or fast evolving genes) that are enriched in subtelomeric regions as compared with internal regions. We also demonstrate that patterns of molecular evolution for paralogous genes differ significantly depending on their location as younger paralogs tend to be found in subtelomeric regions whereas older paralogs are enriched in internal regions. Combining these observations with analyses of synteny, we demonstrate that subtelomeric regions are actively shuffled among chromosome ends, which is consistent with the hypothesis that these regions are prone to ectopic recombination. We also assess patterns of selection by comparing dN/dS ratios of gene family members in subtelomeric versus internal regions, and we include the important antigenic gene family var. These analyses illustrate the highly dynamic nature of the karyotype of P. falciparum, and provide a method for exploring genome dynamics in other lineages.

Spinal muscular atrophy (SMA) is caused by dysfunction of the alpha motor neurons of the spinal cord. It is an autosomal recessive disease associated to the SMN1 gene, located in the subtelomeric region of 5q13. A paralog SMN2 gene is located at the centromeric region of the same chromosome, which apparently originated by an ancestral inverted duplication occurring only in humans. The exon sequence differs in two nucleotides in exon 7 and exon 8, which leads to an SMN2 transcript that lacks exon 7 and results in a truncated protein. Part (10%) of the SMN2 transcripts avoids the splicing of exon 7 but most of the copies are dysfunctional. In a disease scenario, the more SMN2 copies the higher possibility to restore at least partly the effects of SMN1 deficiency. Some therapeutic approaches are being developed to increase the expression of SMN2. To determine the number of SMN1 and SMN2 copies, the methodology must distinguish accurately between both genes. In this work, we present the results obtained using multiplex ligation-dependent probe amplification (MLPA) in 60 SMA suspected patients/carriers derived from different regions of Argentina. In 32 of these DNA samples we found alterations in SMN1. Among these, 16 presented a heterozygous deletion (carrier status) and 14 an homozygous deletion (patient status) in exon 7 and 8 of SMN1. In one case, exon 7 was found homozygously deleted but exon 8 presented a single copy, and in another case, exon 7 was found heterozygously deleted while exon 8 was normal. Almost half of the patients (7/15) presented a normal diploid number of SMN2 while the other half (8/15) presented an increased number. In this work we showed how a probe-based methodology such as MLPA was able to distinguish between the paralog genes and determine the amount of copies in DNA samples from suspected patients/carriers of SMA.