The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin

Abstract

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.

The nucleolar protein GNL3 prevents resection of stalled replication forks

“…Loss of ORC2 increases inter-origin distance (Shibata et al, 2016), therefore GNL3 level in the nucleolus may directly controls ORC2 amount in the nucleoplasm to regulates the number of fired origins. Alternatively, GNL3 may sequester ORC2 in the nucleolus to regulate some of its functions that are not directly linked with the ORC complex such as its role at centromeres (Bauwens et al, 2021; Huang et al, 2016; Prasanth et al, 2004) or in sister chromatid cohesion (MacAlpine et al, 2010; Shimada and Gasser, 2007). In support to this, we found that GNL3 depletion decreases ORC2 recruitment to centromeres and deregulates heterochromatin formation.…”

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress

“…However, overexpression of TRF2 ΔB also induces cell cycle arrest, senescence, and rapid loss of telomeres ( 37 , 40 , 62 , 67 , 68 ). These phenotypes may be a consequence of the multiple roles the TRF2 basic domain has been shown to play in preventing homologous recombination-mediated telomere deletion ( 67–70 ), promoting the replication of heterochromatin ( 71 , 72 ), and mediating ORC recruitment at telomeres.…”

“…These results suggest that ORC1 (244–511) overexpression selectively impairs the telomere binding activity of ORC. However, it remains possible that the overexpression of ORC1 (244–511) affects particular replication origins in other genomic regions such as pericentromeric heterochromatin, where TRF2 has been suggested to promote ORC recruitment ( 72 ). This issue should be addressed in future studies.…”

Multifunctionality of the Telomere-Capping Shelterin Complex Explained by Variations in Its Protein Composition

“…Besides replication elongation, TRF2 also influences the initiation step. Indeed, it regulates origin activation on telomeres and pericentromeres by binding and recruiting ORC2 [ 96 , 97 , 98 , 99 , 100 ]. Finally, and perhaps more surprising, is the transcriptional role of TRF2.…”