Cdk-dependent phosphorylation regulates TRF1 recruitment to PML bodies and promotes C-circle production in ALT cells

Abstract

TRF1, a duplex telomeric DNA binding protein, is implicated in homologous-recombination-based alternative lengthening of telomeres, known as ALT. However, how TRF1 promotes ALT activity has yet to be fully characterized. Here we report that Cdkdependent TRF1 phosphorylation on T371 acts as a switch to create a pool of TRF1, referred to as ( pT371)TRF1, which is recruited to ALT-associated PML bodies (APBs) in S and G2 phases independently of its binding to telomeric DNA. We find that phosphorylation of T371 is essential for APB formation and C-circle production, both of which are hallmarks of ALT. We show that the interaction of ( pT371)TRF1 with APBs is dependent upon ATM and homologous-recombination-promoting factors Mre11 and BRCA1. In addition, ( pT371)TRF1 interaction with APBs is sensitive to transcription inhibition, which also reduces DNA damage at telomeres. Furthermore, overexpression of RNaseH1 impairs ( pT371)TRF1 recruitment to APBs in the presence of campothecin, an inhibitor that prevents topoisomerase I from resolving RNA-DNA hybrids. These results suggest that transcription-associated DNA damage, perhaps arising from processing RNA-DNA hybrids at telomeres, triggers ( pT371)TRF1 recruitment to APBs to facilitate ALT activity.

TRF1 phosphorylation on T271 modulates telomerase-dependent telomere length maintenance as well as the formation of ALT-associated PML bodies

“…Analysis of C-circle assays revealed that depletion of TRF1 resulted in a significant loss in the level of C-circles in GM847 cells (Fig. 4c,d), in agreement with previous finding14. Like Myc-tagged wild type TRF1, both Myc-tagged TRF1-T271A and Myc-tagged TRF1-T271D were able to rescue the level of C-circles in TRF1-depleted cells (Fig.…”

“…Analysis of indirect immunofluorescence revealed that depletion of TRF1 resulted in a significant reduction in the percentage of GM847 cells exhibiting colocalization of large PML bodies with TRF2 and hRap1 (Fig. 5a–d), in agreement with previous finding that TRF1 is required for the formation of APBs1314. We observed that among cells scored positive for APBs, the average number of APB foci per cell was about 1.5 (Fig.…”

“…In addition to its role in negatively regulating telomerase-dependent telomere maintenance, TRF1 has been implicated in promoting C-circle production and APB formation14, characteristic features associated with alternative lengthening of telomeres. To investigate if TRF1 phosphorylation on T271 might regulate C-circle production, we depleted endogenous TRF1 in GM847 cells (Fig.…”

“…On the other hand, in ALT cells, TRF1 is implicated as a positive regulator of HR-based telomere maintenance13. Depletion of TRF1 impairs the formation of APBs and C-circle production1314. TRF1 undergoes extensive phosphorylation, which has been implicated in the regulation of TRF1 binding to telomeric DNA as well as its stability in telomerease-expressing cells15.…”

“…TRF1 undergoes extensive phosphorylation, which has been implicated in the regulation of TRF1 binding to telomeric DNA as well as its stability in telomerease-expressing cells15. Recent work also suggests that TRF1 phosphorylation modulates APB formation and C-circle production in ALT cells14.…”