טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYehezkel-Rekhes Shiran
SubjectThe Role of Human Subtelomeric DNA Methylation in Regulation
of Telomere Length and Function
DepartmentDepartment of Medicine
Supervisors Professor Karl Skorecki
Professor Sara Selig
Full Thesis textFull thesis text - English Version


Abstract

Telomeres and adjacent subtelomeric regions are packaged as heterochromatin facilitating the stabilization and capping of chromosome ends. Subtelomeric DNA is subjected to de novo methylation during early embryonic development. In this thesis the role of subtelomeric DNA methylation in human cells is addressed utilizing two experimental platforms.

              The first section of this thesis explores the effect of hypomorphic mutations in human DNMT3B on telomeric and subtelomeric regions. Such mutations lead to ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) syndrome. This study demonstrates that subtelomeric regions in lymphoblastoid and fibroblast cells of ICF patients are hypomethylated and the telomeres are abnormally short in both telomerase positive- and negative-cells. Fibroblasts derived from two ICF patients reached replicative senescence at an abnormally early population doubling (PD). Ectopic expression of wild-type (WT) human telomerase (hTERT) rescued this phenotype.

              Subtelomeric hypomethylation in ICF cells was also associated with advancement of telomere replication timing and with elevated levels of transcripts emanating from telomeric regions, known as TERRA (telomeric- repeat-containing RNA).

In order to determine whether the abnormal methylation state may be rescued in somatic cells, we introduced WT DNMT3B into immortalized ICF fibroblasts, either with or without DNMT3L, an additional developmental factor involved in the de novo methylation process. Subsequent Southern analyses demonstrated that both telomeric and non-telomeric repetitive sequences remained hypomethylated in both cases.

              The second section of this thesis studies the epigenetic reprogramming of telomeric regions in human induced pluripotent stem (hiPS) cells which serve as a potent in vitro model for studying early embryonic development and subsequent differentiation.

            The study of several hiPS cell clones derived from normal foreskin fibroblasts revealed that telomeres lengthen significantly in hiPS cells in comparison to the parental fibroblast source, and progressively shorten after differentiation, 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 examined subtelomeres, methylation levels differed among subclones and that both de novo methylation and demethylation processes occurred during telomere reprogramming. Notably, whereas overall subtelomeres were very highly methylated in hiPS cells, TERRA levels were elevated, albeit to different degrees in different clones

The findings in this thesis highlight the role of subtelomeric DNA methylation in regulation of telomere length and function during normal development and provide a basis for future studies on hiPS cells derived from ICF patients.