DNA:RNA hybrids, nucleic acid structures with diverse physiological functions, can disrupt genome integrity when dysregulated. Human telomeres were shown to form hybrids with the lncRNA TERRA, yet the formation and distribution of these hybrids among telomeres, their regulation and their cellular effects remain elusive. Here we predict and confirm in several human cell types that DNA:RNA hybrids form at many subtelomeric and telomeric regions. We demonstrate that ICF syndrome cells, which exhibit short telomeres and elevated TERRA levels, are enriched for hybrids at telomeric regions throughout the cell cycle. Telomeric hybrids are associated with high levels of DNA damage at chromosome ends in ICF cells, which are significantly reduced with overexpression of RNase H1. Our findings suggest that abnormally high TERRA levels in ICF syndrome lead to accumulation of telomeric hybrids that, in turn, can result in telomeric dysfunction.
Somech and colleagues identify two new mutations in STN1 that causes Coats plus syndrome and telomere abnormalities in human, recapitulated in a zebra fish model.
Human telomeric regions are packaged as constitutive heterochromatin, characterized by extensive subtelomeric DNA methylation and specific histone modifications. ICF (immunodeficiency, centromeric instability, facial anomalies) type I patients carry mutations in DNA methyltransferase 3B (DNMT3B) that methylates de novo repetitive sequences during early embryonic development. ICF type I patient fibroblasts display hypomethylated subtelomeres, abnormally short telomeres and premature senescence. In order to study the molecular mechanism by which the failure to de novo methylate subtelomeres results in accelerated telomere shortening, we generated induced pluripotent stem cells (iPSCs) from 3 ICF type I patients. Telomeres were elongated in ICF-iPSCs during reprogramming, and the senescence phenotype was abolished despite sustained subtelomeric hypomethylation and high TERRA levels. Fibroblast-like cells (FLs) isolated from differentiated ICF-iPSCs maintained abnormally high TERRA levels, and telomeres in these cells shortened at an accelerated rate, leading to early senescence, thus recapitulating the telomeric phenotype of the parental fibroblasts. These findings demonstrate that the abnormal telomere phenotype associated with subtelomeric hypomethylation is overridden in cells expressing telomerase, therefore excluding telomerase inhibition by TERRA as a central mechanism responsible for telomere shortening in ICF syndrome. The data in the current study lend support to the use of ICF-iPSCs for modeling of phenotypic and molecular defects in ICF syndrome and for unraveling the mechanism whereby subtelomeric hypomethylation is linked to accelerated telomeric loss in this syndrome.
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.
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