Search

Previous studies in human cells indicate that sister telomeres have distinct requirements for their separation at mitosis. In cells depleted for tankyrase 1, a telomeric poly(ADP-ribose) polymerase, sister chromatid arms and centromeres separate normally, but telomeres remain associated and cells arrest in mitosis. Here, we use biochemical and genetic approaches to identify proteins that might mediate the persistent association at sister telomeres. We use immunoprecipitation analysis to show that the telomeric proteins, TRF1 (an acceptor of PARsylation by tankyrase 1) and TIN2 (a TRF1 binding partner) each bind to the SA1 ortholog of the cohesin Scc3 subunit. Sucrose gradient sedimentation shows that TRF1 cosediments with the SA1-cohesin complex. Depletion of the SA1 cohesin subunit or the telomeric proteins (TRF1 and TIN2) restores the normal resolution of sister telomeres in mitosis in tankyrase 1-depleted cells. Moreover, depletion of TRF1 and TIN2 or SA1 abrogates the requirement for tankyrase 1 in mitotic progression. Our studies indicate that sister telomere association in human cells is mediated by a novel association between a cohesin subunit and components of telomeric chromatin.

Cohesins keep sister chromatids associated from the time of their replication in S phase until the onset of anaphase. In vertebrate cells, two distinct pathways dissociate cohesins, one acts on chromosome arms and the other on centromeres. Here, we describe a third pathway that acts on telomeres. Knockdown of tankyrase 1, a telomeric poly(ADP-ribose) polymerase caused mitotic arrest. Chromosomes aligned normally on the metaphase plate but were unable to segregate. Sister chromatids separated at centromeres and arms but remained associated at telomeres, apparently through proteinaceous bridges. Thus, telomeres may require a unique tankyrase 1–dependent mechanism for sister chromatid resolution before anaphase.

Tankyrase 1 is a PARP [poly(ADP-ribose) polymerase] that localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Previous studies demonstrated that cells deficient in tankyrase 1 suffered a block in resolution of sister telomeres and arrested in early anaphase [Dynek and Smith (2004) Science 304, 97–100]. This phenotype was dependent on the catalytic PARP activity of tankyrase 1. To identify critical acceptors of PARsylation [poly(ADP-ribosyl)ation] by tankyrase 1 in mitosis, tankyrase 1 immunoprecipitates were analysed for associated PARsylated proteins. We identified NuMA (nuclear mitotic apparatus protein) as a major acceptor of poly(ADP-ribose) from tankyrase 1 in mitosis. We showed by immunofluorescence and immunoprecipitation that association between tankyrase 1 and NuMA increases dramatically at the onset of mitosis, concomitant with PARsylation of NuMA. Knockdown of tankyrase 1 by siRNA (small interfering RNA) eliminates PARsylation of NuMA in mitosis, confirming tankyrase 1 as the PARP responsible for this modification. However, even in the absence of tankyrase 1 and PARsylation, NuMA localizes to spindle poles. By contrast, siRNA knockdown of NuMA results in complete loss of tankyrase 1 from spindle poles. We discuss our result in terms of a model where PARsylation of NuMA by tankyrase 1 in mitosis could play a role in sister telomere separation and/or mitotic progression.

Mammalian telomeres are coated by the sequence-specific, DNA-binding protein, TRF1, a negative regulator of telomere length. Previous results showed that ADP-ribosylation of TRF1 by tankyrase 1 released TRF1 from telomeres and promoted telomere elongation. We now show that loss of TRF1 from telomeres results in ubiquitination and degradation of TRF1 by the proteasome and that degradation is required to keep TRF1 off telomeres. Ubiquitination of TRF1 is regulated by its telomere-binding status; only the telomere-unbound form of TRF1 is ubiquitinated. Our findings suggest a novel mechanism of sequential post translational modification of TRF1 (ADP-ribosylation and ubiquitination) for regulating access of telomerase to telomeres.

Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.