|Ph.D Student||Sagie Shira|
|Subject||ICF Syndrome as a Model for Studying the Role of Human|
|Department||Department of Medicine||Supervisors||Professor Sara Selig|
|Professor Karl Skorecki|
|Full Thesis text|
Telomeres, the chromosomes termini, function in protection of chromosome ends from digestion and breakage. Telomeres are packaged as constitutive heterochromatin, and the adjacent CpG rich subtelomeres are extensively methylated. The long non-coding RNA TERRA (telomeric repeat-containing RNA) is transcribed from telomeres and is postulated to play a role in telomere maintenance. 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 cells display hypomethylated subtelomeres, abnormally short telomeres, elevated levels of TERRA and premature senescence.
The research described in this thesis aims to study the role of subtelomeric methylation in telomere function and maintenance. We took advantage of the abnormal state of hypomethylated subtelomeres in ICF syndrome in order to understand the molecular mechanism by which the failure to de novo methylate subtelomeres results in accelerated telomere shortening.
In the first part of the thesis, we generated induced pluripotent stem cells (iPSCs) from three ICF type I patients. iPSCs represent the developmental stage at which subtelomeres are normally methylated by DNMT3B. We found that telomeres were elongated in ICF iPSCs during reprogramming, and that 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.
While it was demonstrated that the mean telomere length is significantly shorter in ICF cells, it was unknown whether all telomeres are equally vulnerable to shortening. By combining telomere-FISH and spectral karyotyping we determined the relative length of each individual telomere in multiple ICF syndrome patients and controls. We demonstrated that telomere length variance in the ICF group is much greater than in the control group, suggesting that not all telomeres shorten uniformly. Further, we identified that certain individual telomeres in ICF are consistently shorter than others.
Next, we studied a possible molecular mechanism that drives telomere shortening in ICF cells. DNA:RNA hybrids, three stranded nucleic acid structures, can disrupt genome integrity when dysregulated. Human telomeres were shown to form such hybrids with TERRA. We demonstrated that ICF syndrome cells are enriched for telomeric hybrids throughout the cell cycle. High levels of DNA damage at chromosome ends in ICF cells were significantly reduced with overexpression of RNase H1 that dissolves hybrids. These findings suggest that the abnormally high TERRA levels lead to accumulation of telomeric hybrids that, in turn, can result in DNA damage at telomeres and telomere loss.
In summary, we show that telomeric dysfunction due to subtelomeric hypomethylation is dependent on the lack of telomerase activity, and that telomeres vary in their vulnerability to shorten due to subtelomeric hypomethylation. We demonstrate that telomeric DNA:RNA hybrids are at least one mechanism by which damage is mediated through subtelomeric hypomethylation.