Cell-cycle Dependent Regulation of Telomere-Associate Proteins
Telomeres are protein-DNA structures at the ends of eukaryotic chromosomes. The DNA portion is comprised of double-stranded and single-stranded G-rich repetitive DNA. The protein portion is anchored by the "shelterin complex" composed of six proteins. Inappropriate DNA repair and telomere length dysregulation result in cell cycle arrest, genome instability, and carcinogenesis. Thus, this DNA/protein structure protects telomere ends and regulates telomere length.
The shelterin component TRF1, a double-stranded telomeric DNA binding protein, was found to bind accessory protein PinX1 at mitosis. Given this, I investigated the effect of reducing PinX1 level on cell cycle progression and apoptosis. I found that reducing PinX1 expression with shRNA, as assessed by immunoblot, led to delayed entry into mitosis and elevated levels of apoptosis in human cells. These results indicated that PinX1 plays an important role in mitosis progression and cell viability.
Intriguingly, binding of PinX1 to TRF1 at mitosis increased the stability of the latter. Moreover, PinX1 binds to the same site on TRF1 as the protein TIN2, which can suppress degradation of TRF1 by inhibiting poly ADP-ribosylation of TRF1 by the enzyme tankyrase. Collectively, these results suggested that TIN2 might be released from TRF1 to promote the binding of PinX1 on TRF1 at mitosis. Given that proteins are often regulated in the cell cycle by phosphorylation, I investigated whether TIN2 was phosphorylated at mitosis. To this end, I performed phospho-proteomic analysis of human TIN2, which revealed two phosphorylated residues, serines 295 and 330. Both sites were phosphorylated specifically during mitosis, as detected by two independent approaches, namely Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2 in vitro and in vivo. The identification of these specifically timed post-translational events during the cell cycle demonstrates the mitotic regulation of TIN2 by phosphorylation. However, as expressing non-phosphorylatable mutants of TIN2 failed to reveal any overt phenotypes, the consequences of these phosphorylation events remain to be determined.
Lastly, the TRF1-related double-stranded telomeric DNA binding protein, TRF2, was shown to associate with another shelterin component, POT1. POT1 forms heterodimer with TPP1 to bind single-stranded telomeric DNA. Previous research found that mutations of POT1 with reduced binding affinity to either TRF2 or to TPP1 cause distinct phenotypes. To determine whether similar separation-of-function mutants could be generated to dissect the function of POT1s in mice, which are encoded by two genes, Pot1a and Pot1b, I screened a panel of substitution mutants of mPOT1a for loss of binding to mTRF2 and mTPP1. These studies revealed that mPOT1a does not bind mTRF2, but the association with mTPP1 could be disrupted.
In summary, the described studies have shed insight into the complexity of shelterin regulation, and in particular, highlighted protein-protein interactions and post-translational modifications.
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.
Rights for Collection: Duke Dissertations