Together, our results reveal that shelterin is not a single static complex but two functional subcomplexes with distinct dynamics. Both subcomplexes are dimeric and bind every other telomeric nucleosome.

Building on this model, we demonstrate that TRF1 and TRF2 bind non-overlapping sites on telomeric chromatin, as they can’t compete each other from telomeres. Consistent with an in vitro observation by Hu et al. from @kellythd-nguyen.bsky.social group.

However, TRF2 and RAP1 are very dynamic, providing an evidence that shelterin exists predominantly at two distinct subcomplexes.

Using single-molecule imaging in living cells, we found that TRF1, TIN2, TPP1 and POT1 subunits display static behavior – tightly bound to telomeres.

We found ~25–30 copies of each shelterin protein at telomeres in HeLa cells. Surprisingly, in HeLa 1.3 cells with telomeres 5× longer, the number rises only modestly (~40), suggesting shelterin is more sparsely distributed on longer telomeres.

7/8 We proposed a model of telomeric architecture in HeLa cells. The shelterin associates with telomeres in a form of two distinct subcomplexes: TRF1-TIN2-TPP1-POT1 and TRF2-RAP1. Both subcomplexes are dimeric and are equally presented at telomeres binding approximately every other nucleosome.

6/8 We also probed the shelterin recruitment to telomeres hierarchy using a selective degradation Halo-PROTAC system. Not only TIN2 and POT1 recruitment to telomeres is dependent solely on their association with TRF1, but we found that TRF1 and TRF2 occupy distinct binding sites on telomeres.

5/8 TRF2-RAP1 dissociates from telomeres more rapidly compared to TRF1-TIN2-TPP1-POT1, which enables TRF2-RAP1 to dynamically engage with multiple telomeres and recruit variety of factors necessary for telomere protection.

4/8 Strikingly, our live-cell single-molecule imaging showed that TRF2 and RAP1 subunits are more dynamic when bound to telomeres compared to TRF1, TIN2, TPP1 and POT1 that are statically associated with telomeres, suggesting that TRF2-RAP1 and TRF1-TIN2-TPP1-POT1 form distinct subcomplexes.

3/8 We determined that all shelterin proteins are equally represented at telomeres in HeLa cells at approx. 30 copies per telomere, strongly suggesting that shelterin adopts dimeric composition at telomeres.