Telomere instability in the male germline

Telomeres play a key role in upholding the integrity of the genome, and telomerase expression in spermatogonial stem cells is responsible for the maintenance of telomere length in the human male germline. We have previously described extensive allelic variation in somatic cell telomere length that is set in the zygote, the ultimate source of which may be the germline. This implies that despite telomerase activity, substantial telomere length variation can be generated and tolerated in the germline; in order to investigate this further, we have examined the nature of telomere length variation in the human male germline. Here, we describe an analysis of both genome-wide telomere length and single molecule analysis of specific chromosome ends in human sperm. We observed individual specific differences in genome-wide telomere length. This variation may result from genetic differences within the components that determine the telomere length setting of each individual. Superimposed on the genome wide telomere length setting was a stochastic component of variation that generates germ-cells containing severely truncated telomeres. If not re-lengthened during early embryogenesis, such telomeres may limit the replicative capacity of cells derived from the zygote and have the potential to create fusagenic chromosomes, unbalanced translocations and terminal micro-deletions. These data may have implications for the genetic determination of ageing, genetic disease and fertility

Telomere dysfunction accurately predicts clinical outcome in chronic lymphocytic leukaemia, even in patients with early stage disease

© 2014 John Wiley & Sons Ltd. Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P  <  0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI]  = 11·6-106·4) and this was preserved in early-stage disease patients (P  <  0·0001, HR=19·3, 95% CI = 17·8-802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL

Mre11 modulates the fidelity of fusion between short telomeres in human cells

The loss of telomere function can result in the fusion of telomeres with other telomeric loci, or non-telomeric double-stranded DNA breaks. Sequence analysis of fusion events between short dysfunctional telomeres in human cells has revealed that fusion is characterized by a distinct molecular signature consisting of extensive deletions and micro-homology at the fusion points. This signature is consistent with alternative error-prone end-joining processes. We have examined the role that Mre11 may play in the fusion of short telomeres in human cells; to do this, we have analysed telomere fusion events in cells derived from ataxia-telangiectasia-like disorder (ATLD) patients that exhibit hypomorphic mutations in MRE11. The telomere dynamics of ATLD fibroblasts were indistinguishable from wild-type fibroblasts and they were proficient in the fusion of short telomeres. However, we observed a high frequency of insertion of DNA sequences at the fusion points that created localized sequence duplications. These data indicate that Mre11 plays a role in the fusion of short dysfunctional telomeres in human cells and are consistent with the hypothesis that as part of the MRN complex it serves to stabilize the joining complex, thereby controlling the fidelity of the fusion reaction

Intra-allelic mutation at human telomeres

The maintenance of telomere length is important in upholding the integrity of the genome. However, it is clear from detailed observations of both telomere length and internal repeat structure that human telomeres are extremely dynamic structures and are subjected to multiple processes that create considerable heterogeneity. Genetic evidence suggests that meiotic recombination within telomeres is rare. However, there are various lines of evidence that implicate the involvement of intra-allelic processes in human telomere dynamics. In this paper, we briefly review some of this evidence and the putative mechanisms of intra-allelic telomeric mutation

The Naphthalene Catabolic (nag) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

In Ralstonia sp. strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter. nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes. A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon. The transcriptional start of the nag operon was mapped, and a putative −10, −35 σ(70)-type promoter binding site was identified. Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways. Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate. Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon. However, other mutations in the region increased the effectiveness of salicylate as an inducer

Short telomeres are preferentially elongated by telomerase in human cells

Short telomeres have been shown to be preferentially elongated in both yeast and mouse models. We examined this in human cells, by utilising cells with large allelic telomere length differentials and observing the relative rates of elongation following the expression of hTERT. We observed that short telomeres are gradually elongated in the first 26 PDs of growth, whereas the longer telomeres displayed limited elongation in this period. Telomeres coalesced at similar lengths irrespective of their length prior to the expression of hTERT. These data indicate that short telomeres are marked for gradual elongation to a cell strain specific length threshold

Structural stability and chromosome-specific telomere length is governed by cis-acting determinants in humans

Single telomere length analysis (STELA) of the XpYp telomere has revealed extensive allelic variation and ultra-short telomeres in senescent cells. Superimposed on end-replication losses are additional mutational events that result in large-scale changes in telomere length. In order to establish if the dynamics of the XpYp telomere are typical of human telomeres, here we describe an analysis using STELA of the telomeres of 2p, 11q, 12q, 17p and XpYp. The dynamics of telomere loss (erosion rates and stochastic length changes) was conserved among 2p, 11q, 12q and XpYp within the same cell strains and was dependent on the replicative kinetics of the cells in culture. However, of the telomeres analysed, the telomere of 17p was more stable with a striking paucity of large-scale length changes, and exhibited the shortest recorded allelic distribution (300 bp) in senescent cells and displayed a general, but not absolute, trend towards being the shortest telomere. Ectopic over-expression of hTERT homogenized both allelic and chromosome-specific telomeric distributions. However, telomerase-expressing cancer cells displayed both allelic variation and chromosome-specific telomere length, with 17p displaying the shortest allelic telomere length. Although other telomeres in the genome may share the properties of 17p, these data suggest that physiological levels of telomerase allow differential telomere length regulation and indicate the presence of cis-acting factors that govern both telomeric stability and chromosome-specific telomere length in the presence of telomerase

Telomere dynamics during replicative senescence are not directly modulated by conditions of oxidative stress in IMR90 fibroblast cells

The replicative lifespan of many cell types is determined by the length of telomeres in the initiating cell population. In 20% oxygen, IMR90 cells have a shorter replicative lifespan compared to that achieved in conditions that lower the levels of oxidative stress. We sought to address the role of telomere dynamics in determining the replicative lifespan of IMR90 cells. We analysed clonal populations cultured in parallel in 3 and 20% oxygen. We observed that, at senescence, telomere length was shorter in 3% oxygen and this was proportional to the lifespan extension. We observed no detectable difference in the rate of telomere erosion in the two culture conditions, however as the cells approached senescence the growth rate of the cultures slowed with a commensurate increase in the rate of telomere erosion. We conclude that, in 20% oxygen senescence of IMR90 is telomere-independent, but telomere-dependent in 3% oxygen