Super-Resolution Fluorescence Imaging of Telomeres Reveals TRF2-Dependent T-loop Formation

We have applied a super-resolution fluorescence imaging method, stochastic optical reconstruction microscopy (STORM), to visualize the structure of functional telomeres and telomeres rendered dysfunctional through removal of shelterin proteins. The STORM images showed that functional telomeres frequently exhibit a t-loop configuration. Conditional deletion of individual components of shelterin showed that TRF2 was required for the formation and/or maintenance of t-loops, whereas deletion of TRF1, Rap1, or the POT1 proteins (POT1a and POT1b) had no effect on the frequency of t-loop occurrence. Within the shelterin complex, TRF2 uniquely serves to protect telomeres from two pathways that are initiated on free DNA ends: classical nonhomologous end-joining (NHEJ) and ATM-dependent DNA damage signaling. The TRF2-dependent remodeling of telomeres into t-loop structures, which sequester the ends of chromosomes, can explain why NHEJ and the ATM signaling pathway are repressed when TRF2 is present. [Display omitted] •STORM imaging of telomeres reveals t-loops in relaxed mouse chromatin•Deletion of the shelterin component TRF2 converts t-loops into linear telomeres•Other components of shelterin are not required to form/maintain t-loops•TRF2-dependent t-loop formation is proposed to block c-NHEJ and ATM kinase signaling STORM images reveal telomeres in a lariat configuration, called t-loops, in which telomere ends are sequestered. Deletion of components of the telomeric shelterin complex established that TRF2 is the main factor required for t-loop formation.