Age-Related Phenotypes Linked to Aberrant Expression and Localization of a Telomeric Protein

Telomere attrition is associated with telomere biology disorders and age-related diseases. In telomere biology disorders, telomere uncapping induces a DNA damage response that evokes cell death or senescence. However, a causal mechanism for telomere attrition in age-related diseases remains elusive. Telomere capping and integrity are maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not required for telomere capping and is expressed at non-telomeric genomic and cytosolic regions. The objective of this study was to determine aberrant phenotypes attributed to non-telomeric Rap1. To test this, we generated a Rap1 mutant knock-in (KI) mouse model using CRISPR/Cas9 editing, in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Cell fractionation/western blotting of primary fibroblasts from Rap1 KI mice demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, with an altered cellular distribution. This same difference in Rap1 is also observed in human cells with telomere erosion, indicating that aberrant Rap1 in our model may recapitulate Rap1 in aging and human telomere biology disorders. Compared to wild-type control mice, Rap1 KI mice exhibited increased body weight, altered cytokine levels, behavioral deficits, and decreased lifespan. In conclusion, our results reveal a novel mechanism by which telomere shortening may contribute to age-related pathologies by disrupting Rap1 expression and cell localization.

Taming active transposons at Drosophila telomeres: The interconnection between HipHop’s roles in capping and transcriptional silencing

Drosophila chromosomes are elongated by retrotransposon attachment, a process poorly understood. Here we characterized a mutation affecting the HipHop telomere-capping protein. In mutant ovaries and the embryos that they produce, telomere retrotransposons are activated and transposon RNP accumulates. Genetic results are consistent with that this hiphop mutation weakens the efficacy of HP1-mediated silencing while leaving piRNA-based mechanisms largely intact. Remarkably, mutant females display normal fecundity suggesting that telomere de-silencing is compatible with germline development. Moreover, unlike prior mutants with overactive telomeres, the hiphop stock does not over-accumulate transposons for hundreds of generations. This is likely due to the loss of HipHop’s abilities both to silence transcription and to recruit transposons to telomeres in the mutant. Furthermore, embryos produced by mutant mothers experience a checkpoint activation, and a further loss of maternal HipHop leads to end-to-end fusion and embryonic arrest. Telomeric retroelements fulfill an essential function yet maintain a potentially conflicting relationship with their Drosophila host. Our study thus showcases a possible intermediate in this arm race in which the host is adapting to over-activated transposons while maintaining genome stability. Our results suggest that the collapse of such a relationship might only occur when the selfish element acquires the ability to target non-telomeric regions of the genome. HipHop is likely part of this machinery restricting the elements to the gene-poor region of telomeres. Lastly, our hiphop mutation behaves as a recessive suppressor of PEV that is mediated by centric heterochromatin, suggesting its broader effect on chromatin not limited to telomeres.

Suppression of telomere capping defects of Saccharomyces cerevisiae yku70 and yku80 mutants by telomerase

The Ku complex performs multiple functions inside eukaryotic cells, including protection of chromosomal DNA ends from degradation and fusion events, recruitment of telomerase, and repair of double-strand breaks (DSBs). Inactivation of Ku complex genes YKU70 or YKU80 in cells of the yeast S. cerevisiae gives rise to mutants that exhibit shortened telomeres and temperature-sensitive growth. In this study we have investigated the mechanism by which overexpression of telomerase suppresses the temperature sensitivity of yku mutants. Viability of yku cells was restored by overexpression of the Est2 reverse transcriptase and TLC1 RNA template subunits of telomerase, but not the Est1 or Est3 proteins. Overexpression of other telomerase- and telomere-associated proteins (Cdc13, Stn1, Ten1, Rif1, Rif2, Sir3, Sir4) did not suppress the growth defects of yku70 cells. Mechanistic features of suppression were assessed using several TLC1 RNA deletion derivatives and Est2 enzyme mutants. Supraphysiological levels of three catalytically inactive reverse transcriptase mutants (Est2-D530A, Est2-D670A and Est2-D671A) suppressed the loss of viability as efficiently as the wildtype Est2 protein, without inducing cell senescence. Roles of proteins regulating telomere length were also determined. The results support a model in which chromosomes in yku mutants are stabilized via a replication-independent mechanism involving structural reinforcement of protective telomere cap structures.

Multifunctionality of the Telomere-Capping Shelterin Complex Explained by Variations in Its Protein Composition

Protecting telomere from the DNA damage response is essential to avoid the entry into cellular senescence and organismal aging. The progressive telomere DNA shortening in dividing somatic cells, programmed during development, leads to critically short telomeres that trigger replicative senescence and thereby contribute to aging. In several organisms, including mammals, telomeres are protected by a protein complex named Shelterin that counteract at various levels the DNA damage response at chromosome ends through the specific function of each of its subunits. The changes in Shelterin structure and function during development and aging is thus an intense area of research. Here, we review our knowledge on the existence of several Shelterin subcomplexes and the functional independence between them. This leads us to discuss the possibility that the multifunctionality of the Shelterin complex is determined by the formation of different subcomplexes whose composition may change during aging.

Telomere transcription in MLL-rearranged acute leukemia: increased levels of TERRA associate with lymphoid lineage and are independent of telomere length and ploidy

Telomere transcription into telomeric repeat-containing RNA (TERRA) is an integral component of all aspects of chromosome end protection consisting of telomerase- or recombination-dependent telomere elongation, telomere capping, and preservation of (sub)telomeric heterochromatin structure. The chromatin modifier and transcriptional regulator MLL associates with telomeres and regulates TERRA transcription in telomere length homeostasis and response to telomere dysfunction. MLL fusion proteins (MLL-FPs), the product of MLL rearrangements in leukemia, also associate with telomeric chromatin. However, an effect on telomere transcription in MLL-rearranged (MLL-r) leukemia has not yet been evaluated. Here, we show increased UUAGGG repeat-containing RNA levels in MLL-r acute lymphoblastic leukemia (ALL). MLL rearrangements do not affect telomere length and increased levels of UUAGGG repeat-containing RNA correlate with mean telomere length and reflect increased levels of TERRA. Also, increased levels of TERRA in MLL-r ALL occur in the presence of telomerase activity and are independent of ploidy, an underestimated source of variation on the overall transcriptome size in a cell. This MLL rearrangement-dependent and lymphoid lineage-associated increase in levels of TERRA supports a sustained telomere transcription by MLL-FPs that correlates with marked genomic stability previously reported in pediatric MLL-r ALL.

Rap1, Rif2, and the Ku complex work in concert to cap chromosome ends

SummaryRap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Rap1 can also bind and promote the formation of G-quadruplexes, which are thought to form at telomeres. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association and G-quadruplex formation by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. Remarkably, tlc1-tm cells grow as well as wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. We find that Rap1, Rif2, and the Ku complex work in parallel to prevent telomere degradation, and absence of all three causes lethality. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.

Getting to grips with circular chromosomes

A strain of budding yeast that contains one large chromosome reveals how the telomere capping complex CST maintains linear but not circular chromosomes.