Analysis of the Role of TRF1 and SMG6 in Telomere Length Maintenance

Abstract

<p>TRF1, a shelterin protein, is a negative mediator of telomere length maintenance. Phosphorylation has been shown to play an important role in modulating TRF1 function. T137 and S249 of TRF1 have been indentified to be candidate phosphorylation sites in vivo, and one of my thesis objectives was to examine their role in regulating TRF1 function. Both T137 and S249 have each been changed to either alanine (nonphosphorylatable) or phosphomimic mutation. The TRF1 mutants were introduced into a TRF1-depleted cell line. Southern analysis revealed that neither T137 nor S249 of TRF1 is involved in telomere length maintenance. Immunoprecipitation studies showed that T137 and S249 are not required for TRF1 interaction with TIN2. In vitro gel-shift assays indicated that T137 and S249 are not important for TRF1 binding to telomeric DNA. Taken together, these results suggest that T137 and S249 may not be required for TRF1 function in telomere length maintenance. Human Est1A has been suggested to play a role in telomere length maintenance. To identify the domain of hEst1A involved in telomere length maintenance, a number of deletion constructs were generated and retrovirally introduced into HT1080 cells. Southern analysis revealed that the RID domain may positively regulate telomere length maintenance whereas the first 220 amino acids at the N-terminus may be a negative mediator of telomere length maintenance. In S. cerevisiae, Est1 recruits telomerase to telomeres in a Tel1- (homolog of ATM) and MRX-dependent manner. To assess whether atm-1 and smg-6 may function in the same genetic pathway of regulation of telomere length in C.elegans, the single mutant strain atm-1(gk186) was crossed with three of smg-6 mutant strains (tm1308, ok1794 and r896) to generate double mutants. Southern analysis revealed that deletion of ATM-1 or SMG-6 (tm1308) results in telomere shortening, suggesting that atm-1 and smg-6 may function in the same genetic pathway to regulate telomere length maintenance.</p>