Compartmentalization of telomeres through DNA-scaffolded phase separation.

Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

Ccq1-Raf2 interaction mediates CLRC recruitment to establish heterochromatin at telomeres.

Telomeres, highly ordered DNA-protein complexes at eukaryotic linear chromosome ends, are specialized heterochromatin loci conserved among eukaryotes. In Schizosaccharomyces pombe, the shelterin complex is important for subtelomeric heterochromatin establishment. Despite shelterin has been demonstrated to mediate the recruitment of the Snf2/histone deacetylase-containing repressor complex (SHREC) and the Clr4 methyltransferase complex (CLRC) to telomeres, the mechanism involved in telomeric heterochromatin assembly remains elusive due to the multiple functions of the shelterin complex. Here, we found that CLRC plays a dominant role in heterochromatin establishment at telomeres. In addition, we identified a series of amino acids in the shelterin subunit Ccq1 that are important for the specific interaction between Ccq1 and the CLRC subunit Raf2. Finally, we demonstrated that the Ccq1-Raf2 interaction is essential for the recruitment of CLRC to telomeres, that contributes to histone H3 lysine 9 methylation, nucleosome stability and the shelterin-chromatin association, promoting a positive feedback mechanism for the nucleation and spreading of heterochromatin at subtelomeres. Together, our findings provide a mechanistic understanding of subtelomeric heterochromatin assembly by shelterin-dependent CLRC recruitment to chromosomal ends.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.