Following this, the cells were incubated at normal growth conditions (37?C, 5% CO2) overnight. utilizes the telomeric C-rich leading strand as its template7 (reviewed in ref. 13). TERRA has been implicated in numerous telomeric roles, such as regulation of telomere length, replication and heterochromatinization14,15,16,17,18,19 (reviewed in refs 2, 13). Evidence is emerging that the function and regulation of TERRA are telomere state dependent such that telomere length, telomerase expression and ALT pathway activity can influence the role that TERRA has at telomeres (reviewed in ref. 20). R-loops, three-stranded nucleic acid structures that consist of a DNA:RNA hybrid and a displaced single-stranded DNA loop21, are predisposed by strand asymmetry in the distribution of guanines and cytosines, termed GC-skewing. These structures form mainly co-transcriptionally when positive GC skew is present such that DNA:RNA hybrids form between the G-rich RNA strand and the C-rich complementary DNA strand22. Although various studies indicate that DNA:RNA hybrids have a positive effect on gene transcription and are beneficial to the cell22,23,24,25, these structures have also been shown to mediate genome instability and replication stress26. R-loops have been implicated in human diseases, including trinucleotide expansion diseases, neurological diseases and cancer (reviewed in ref. 27). Telomeric DNA and TERRA transcripts are predicted to form hybrids, with the G-rich (UUAGGG)TERRA transcript annealing to the C-rich (CCCTAA)DNA template. Indeed, recent studies support the existence of such hybrids at telomeres in (whose telomeres are comprised of a different G-rich repeat)14,28,29 and suggest that, in the absence of a telomere-maintenance mechanism, TERRA-telomeric DNA hybrids may promote accelerated BNS-22 telomere loss in gene31,32, the major DNA methyltransferase involved in methylation of repetitive sequences in mammalian cells during development32. Subtelomeres, as other repetitive sequences, are severely hypomethylated in ICF type I syndrome cells33,34,35. We detected accelerated telomere shortening and significant telomere loss, premature replicative senescence and significantly elevated levels of TERRA transcripts in both ICF fibroblast and lymphoblastoid cells (LCLs)33,35. Although it was proposed that TERRA has a causative role in the generation of telomeric abnormalities in ICF syndrome14,17,33,34,35,36,37, the underlying mechanism by which this occurs is as yet unclear. Here we further investigate the occurrence of human telomeric hybrids in various cell types. Furthermore, we address the question of whether all telomeres are equally competent in generating these hybrids and whether the subtelomeric regions may affect this capacity. Our findings establish that telomeric DNA:RNA hybrids LEPREL2 antibody occur also in primary human cells and that subtelomeric sequences have an effect on generation of telomeric hybrids. We demonstrate that elevated TERRA levels are associated with higher levels of telomeric hybrids in ICF syndrome and suggest a role for these DNA:RNA hybrids in promoting damage and instability at telomeric regions in this disease. Results Human subtelomeres are predicted to form DNA:RNA hybrids Human telomere-hexameric (TTAGGG)repeats are predicted to form DNA:RNA hybrids, with the C-rich template annealing to the G-rich TERRA transcript. We validated this capacity and BNS-22 demonstrated, as in a previous study30, that these hybrids are formed only in a specific direction and are sensitive to RNase H, an enzyme that specifically degrades RNA strands within DNA:RNA hybrids (Supplementary Fig. 1). BNS-22 The majority of TERRA transcripts initiate at the last few hundred base-pairs (bps) of the subtelomeric region7, although some TERRA species may start 5C10? kb upstream of the telomere tract38. As most DNA:RNA hybrids are assumed to form co-transcriptionally22,39, we speculated that subtelomeric sequences might facilitate the formation of telomeric hybrids. To test this hypothesis, we first analysed the sequence of the distal 2?kb region adjacent to the telomere tract at both chromosome ends for CpG density, GC content and GC skew23. Regions with a strong GC skew downstream of the BNS-22 TERRA promoter may be prone to DNA:RNA hybrid formation. For this analysis, we utilized the previously described subtelomeric sequences8,10, focussing on high-confidence subtelomeric regions whose sequence is available in the UCSC GRCh38/hg38 release with a clearly defined telomeric region or at least three consecutive TTAGGG repeats at the 3 end. These subtelomeric regions were overlaid with the predicted TERRA promoters and transcription start sites (TSSs), as determined by the Genomatix software40. Most human subtelomeric regions exhibit high CpG density and GC content in regions corresponding to the predicted promoters for TERRA (Fig. 1a), thus closely resembling CpG island promoters. This is consistent with a similar analysis of a subgroup of TERRA promoters7 and reinforced by the findings that TERRA transcribing telomeres show higher GC content in comparison to the non-transcribing ones38. Examination of GC skew revealed that.