Characterization and rescue of telomeric abnormalities in ICF syndrome type I fibroblasts.

Mutations in the human DNA methyltransferase 3B (DNMT3B) gene lead to ICF (immunodeficiency, centromeric region instability, and facial anomalies) syndrome type I. We have previously described a telomere-related phenotype in cells from these patients, involving severe hypomethylation of subtelomeric regions, abnormally short telomeres and high levels of telomeric-repeat-containing RNA (TERRA). Here we demonstrate that ICF-patient fibroblasts carry abnormally short telomeres at a low population doubling (PD) and enter senescence prematurely. Accordingly, we attempted to rescue the senescence phenotype by ectopic expression of human telomerase, which led to elongated telomeres with hypomethylated subtelomeres. The senescence phenotype was overcome under these conditions, thus dissociating subtelomeric-DNA hypomethylation per se from the senescence phenotype. In addition, we examined whether the subtelomeric methylation could be restored by expression of a normal copy of full length DNMT3B1 in ICF fibroblasts. Ectopic expression of DNMT3B1 failed to rescue the abnormal hypomethylation at subtelomeres. However, partial rescue of subtelomeric-hypomethylation was achieved by co-expression of DNMT3B1 together with DNA methyltransferase 3-like (DNMT3L), encoding a protein that functions as a stimulator of DNMT3A and DNMT3B. DNMT3B1 and DNMT3L are predominantly expressed during early embryonic development, suggesting that de novo subtelomeric DNA methylation during crucial stages of human embryonic development may be necessary for setting and maintaining normal telomere length.

Telomere elongation followed by telomere length reduction, in leukocytes from divers exposed to intense oxidative stress--implications for tissue and organismal aging.

Many cross-sectional studies have tried to assess the in vivo effect of oxidative stress on organismal aging in general and on telomere length dynamics specifically. Here we followed telomere length dynamics over a 12-month interval, in divers exposed to intense hyperbaric oxygen in comparison with an age-matched control group. Both groups were exposed to extreme physical activity, as well. Among the divers following the oxidative stress, significant telomere elongation was observed in granulocytes and naïve T cells, but not in memory T cells and B cells. Telomere length in granulocytes was mildly elongated in the control group as well, a finding that may relate to the extreme physical activity to which they were exposed. While telomere elongation in naïve T cells may be attributed to telomerase activation, we suggest that in granulocytes the elongation results from undifferentiated hematopoietic cells carrying longer telomeres that repopulate the peripheral hematopoietic compartment. This event might be accompanied by enhanced cell division within the repopulating pool. Since the aging of mammalian tissues can be attributed in part to the reduction in the replicative potential of self renewing cells, enhanced cell turnover under conditions of hyperbaric oxidative stress might be directly relevant to tissue and organismal aging.

Reprogramming of telomeric regions during the generation of human induced pluripotent stem cells and subsequent differentiation into fibroblast-like derivatives.

Human induced pluripotent stem (hiPS) cells provide therapeutic promises, as well as a potent in vitro model for studying biological processes which take place during human embryonic development and subsequent differentiation in normal and disease states. The epigenetic characteristics of iPS cells are reprogrammed to the embryonic state at which they acquire pluripotency. In addition, telomeres in hiPS cell must elongate sufficiently to provide the necessary replicative potential. Recent studies have demonstrated that the epigenetic characteristics of telomeric and subtelomeric regions are pivotal in regulating telomere length. Here we study telomere length, subtelomeric DNA methylation and telomeric-repeat-containing RNA (TERRA) expression in several hiPS cell clones derived from normal neonatal foreskin fibroblasts. We find that telomeres lengthen significantly in hiPS cells in comparison to the parental fibroblast source, and progressively shorten after differentiation back into fibroblast-like cells, concomitantly with telomerase activation and down-regulation, respectively. Subtelomeres in hiPS cells were found to be generally hypermethylated in comparison to the parental source. However bisulfite analysis revealed that at several subtelomeres examined, methylation levels differed between hiPS clones and that both de novo methylation and demethylation processes occurred during telomere reprogramming. Notably, although subtelomeres were in general very highly methylated, TERRA levels were elevated in hiPS cells, albeit to different degrees in the various clones. TERRA elevation may reflect enhanced stability or impaired degradation in hiPS cells, and/or alternatively, increased transcription from the hypomethylated subtelomeres. We suggest that TERRA may play a role in regulation of appropriate telomere function and length in hiPS cells.

Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions.

Telomeres and adjacent subtelomeric regions are packaged as heterochromatin in many organisms. The heterochromatic features include DNA methylation, histones H3-Lys9 (Lysine 9) and H4-Lys20 (Lysine 20) methylation and heterochromatin protein1 alpha binding. Subtelomeric DNA is hypomethylated in human sperm and ova, and these regions are subjected to de novo methylation during development. In mice this activity is carried out by DNA methyltransferase 3b (Dnmt3b). Mutations in DNMT3B in humans lead to the autosomal-recessive ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome. Here we show that, in addition to several satellite and non-satellite repeats, the subtelomeric regions in lymphoblastoid and fibroblast cells of ICF patients are also hypomethylated to similar levels as in sperm. Furthermore, the telomeres are abnormally short in both the telomerase-positive and -negative cells, and many chromosome ends lack detectable telomere fluorescence in situ hybridization signals from either one or both sister-chromatids. In contrast to Dnmt3a/b(-/-) mouse embryonic stem cells, increased telomere sister-chromatid exchange was not observed in ICF cells. Hypomethylation of subtelomeric regions was associated in the ICF cells with advanced telomere replication timing and elevated levels of transcripts emanating from telomeric regions, known as TERRA (telomeric-repeat-containing RNA) or TelRNA. The current findings provide a mechanistic explanation for the abnormal telomeric phenotype observed in ICF syndrome and highlights the link between TERRA/TelRNA and structural telomeric integrity.

Mitochondrial hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain hypomyelination and leukodystrophy.

Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration.

Sister chromatid separation at human telomeric regions.

Telomeres are nucleoprotein complexes located at chromosome ends, vital for preserving chromosomal integrity. Telomeric DNA shortens with progressive rounds of cell division, culminating in replicative senescence. Previously we have reported, on the basis of fluorescent in situ hybridization, that several human telomeric regions display solitary signals (singlets) in metaphase cells of presenescent fibroblasts, in comparison to other genomic regions that hybridize as twin signals (doublets). In the current study, we show that an additional 12 out of 12 telomeric regions examined also display metaphase singlet signals in pre-senescent cells, and that excess telomere-metaphase singlets also occur in earlier passage cells harvested from elderly individuals. In cancer cell lines expressing telomerase and in pre-senescent fibroblasts ectopically expressing hTERT, this phenomenon is abrogated. Confocal microscope image analysis showed that the telomere metaphase singlets represent regions that have replicated but not separated; this is presumably because of persistent cohesion. The introduction of mutations that interfere with the normal dissolution of cohesion at the metaphase to anaphase transition induced the cut (chromosomes untimely torn) phenotype in early passage fibroblasts, with predominantly telomeric rather than centromeric DNA, present on the chromatin bridges between the daughter nuclei. These results suggest that telomeric regions in animal cells may potentially be sites of persistent cohesion, and that this cohesion may be the basis for an observed excess of fluorescent in situ hybridization metaphase singlets at telomeres. Persistent cohesion at telomeres may be associated with attempted DNA repair or chromosomal abnormalities, which have been described in pre-senescent cells.

Replication and/or separation of some human telomeres is delayed beyond S-phase in pre-senescent cells.

Cultured primary human cells, which lack telomerase, enter a state of replicative senescence after a characteristic number of population doublings. During this process telomeres shorten to a critical length of approximately 5-7 kb. The mechanistic relationship between advanced cell passage, cellular senescence and telomeric function has yet to be fully elucidated. In the study described here, we investigated the relationship between changes in telomeric replication timing and/or sister chromatid separation at telomeric regions and advanced cell passage. Using fluorescence in situ hybridization, we analyzed the appearance of double hybridization signals (doublets), which indicate that the region of interest has replicated and the replicated products have separated sufficiently to be resolved as two distinct signals. The results showed that the replication and separation of several telomeric regions occurs during the second half of S-phase and that a delay in replication and/or separation of sister chromatids at these regions occurs in pre-senescent human fibroblasts. Surprisingly, in a significant percentage of pre-senescent cells, several telomeric regions did not hybridize as doublets even in metaphase chromosomes. This delay was not associated with extensive changes in methylation levels at subtelomeric regions and was circumvented in human fibroblasts expressing ectopic telomerase. We propose that incomplete replication and/or separation of telomeric regions in metaphase may be associated with proliferative arrest of senescent cells. This cell growth arrest may result from the activation of a mitotic checkpoint, or from chromosomal instability consequent to progression in the cell cycle despite failure to replicate and/or separate these regions completely.

High glucose-induced replicative senescence: point of no return and effect of telomerase.

Primary human cells enter senescence after a characteristic number of population doublings (PDs). In the current study, human skin fibroblasts were propagated in culture under 5.5mM glucose (normoglycemia); addition of 16.5mM D-glucose to a concentration of 22 mM (hyperglycemia); and addition of 16.5mM L-glucose (osmotic control). Hyperglycemia induced premature replicative senescence after 44.42+/-1.5 PDs compared to 57.9+/-3.83 PDs under normoglycemia (p<0.0001). L-Glucose had no effect, suggesting that the effect of hyperglycemia was not attributed to hyperosmolarity. Activated caspase-3 measurement showed a significantly higher percentage of apoptotic cells in high glucose medium. Telomerase overexpression circumvented the effects of hyperglycemia on replicative capacity and apoptosis. The "point of no return," beyond which hyperglycemia resulted in irreversible progression to premature replicative senescence, occurred after exposure to hyperglycemia for as few as 20 PDs. These results may provide a biochemical basis for the relationship between hyperglycemia and those complications of diabetes, which are reminiscent of accelerated senescence.