Sumoylation of TRF1 Is Essential for Its Recruitment to ALT-Associated PML Bodies.

To achieve unlimited proliferative potential, most cancer cells activate telomerase to maintain telomeres. However, some cancer cells elongate telomeres through a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). These ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB), which comprises telomeric DNA and a number of proteins, including PML protein, the telomere binding proteins TRF1 and TRF2, replication factor A, and recombination factors Rad51, Rad52, and the Rad50/Mre11/NBS1 complex. TRF1, as the first identified telomeric binding protein, binds the duplex telomeric repeats at telomere ends. It plays an important role in telomere length control, telomeric ends shelter and cell cycle regulation. In ALT cell lines, TRF1 co-localized with PML protein at APBs, but the exact mechanism of its recruitment to APBs is not clear. Here we show that TRF1 localizes to PML bodies in about 5% of an asynchronously growing culture of U2OS cells and the percentage of cells with colocalization of TRF1 and PML bodies increases to about 40% in cells enriched in G2/M, which is consistent with the previous studies. Furthermore, our results show that TRF1 is modified by the small ubiquitin-like protein SUMO-1 in vivo and in vitro. Firstly, 293T cells were transfected with Flag-TRF1, HA-Ubc9, GFP-SUMO1 and then immunoprecipitated by using FLAG-M2 gel under denaturing conditions. Immunoblotting with GFP and Flag antibodies demonstrated that TRF1 is modified by SUMO-1 in vivo. Next, in vitro SUMO-1 conjugation assay of TRF1 was employed. The results showed that TRF1 is conjugated with SUMO-1 in the presence of purified recombinant protein SAE1/SAE2, Ubc9, SUMO1, His-TRF1 and ATP. Either SAE1/SAE2, Ubc9, SUMO-1 or ATP was omitted from the reaction abolished the sumoylation of TRF1. Previous studies have shown that sumoylation controls the recruitment of several proteins to PML bodies, so we examined whether sumoylation of TRF1 is required for its recruitment to APBs. We mutated the potential sumoylation sites of TRF1 according to the computational prediction and then transfected it into U2OS cells to examine its localization. The results showed that TRF1 mutant does not localize to PML bodies. Taken together, all these results suggest that TRF is modified by SUMO-1 and sumoylation of TRF1 is essential for its recruitment to APBs in ALT cells.

Sudden Telomere Lengthening Triggers a Rad53-dependent Checkpoint inSaccharomyces cerevisiae

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.