Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters

Abstract

The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex-forming DNA sequences. Furthermore, we validated TRF2 occupancy at several promoter G-quadruplex motifs, which did adopt quadruplex forms in solution. TRF2 binding altered expression and the epigenetic state of several target promoters, indicated by histone modifications resulting in transcriptional repression of eight of nine genes investigated here. Furthermore, TRF2 occupancy and target gene expression were also sensitive to the well-known intracellular G-quadruplex-binding ligand 360A. Together, these results reveal an extensive genome-wide association of TRF2 outside telomeres and that it regulates gene expression in a G-quadruplex-dependent fashion.

Keywords: DNA secondary structure; DNA transcription; DNA-binding protein; G-quadruplex; TRF2; epigenetics; extra-telomeric; gene regulation; genome-wide; histone mark; shelterin complex; telomere; telomere repeat-binding factor 2.

Figure 1.

Thousands of extra-telomeric TRF2 binding sites found across the genome. A, extra-telomeric TRF2 peaks found in HT1080 cells following TRF2 ChIP-Seq: 20304 TRF2 peaks were common between two independent experiments. B, distribution of common TRF2 peaks around TSSs. Distance from the TSS is shown in kb. C, replicate consistency plot generated using irreproducible discovery rate analysis; 1956 peaks found at ≤0.01 are marked. D, distribution of 1956 TRF2 peaks with IDR 0.01 (TRF2_HC peaks) around the TSS. Distance from the TSS is shown in kb.

Figure 2.

TRF2 peaks harbor G-quadruplex motifs genome-wide. A, schematic representation of a G4 motif; sequence pattern with loop/stem and PG4 motif formed by a tetrad of guanine trimers interspersed with loops that can vary in length. B and C, PG4 motifs and TRF2 peaks significantly overlap. High-confidence TRF2-binding sites (TRF2_HC peaks) determined by ChIP-Seq in HT1080 cells were significantly enriched in PG4-motif sequences (B), and conversely, PG4-motif sequences were enriched within TRF2_HC peaks (C). Nonoverlapping PG4 motifs were considered for analysis; for control analysis, 100 regions of identical length for each TRF2 peak were taken. *, p < 0.05; **, p < 0.01 (Fisher's exact test). Error bars, S.D.

Figure 3.

Sequences within TRF2 peaks form G-quadruplex motifs. A, table showing the location, distance from the TSS, and G4 motifs (WT and respective G4 mutant sequences of promoter PG4 motifs that overlap in TRF2 high-confidence peaks). B, CD plots of PG4-motif sequences shown in the above table; base-substituted mutant sequences that would not adopt G4 motifs were used as controls.

Figure 4.

TRF2 occupancy on G4-motif sites on gene promoters within cells. A, telomeric enrichment was tested for TRF2 ChIP samples performed in HT1080 cells. Telomeric signal was normalized to signal from ALU probe. B, TRF2 occupancy at gene promoter sites in HT1080 cells (endogenous TRF2 and TRF2-overexpressed) quantified using ChIP-qRT PCR. TRF2 ChIP/IgG (mock) enrichment was normalized to 1% input; CTCF promoter (which does not harbor a TRF2 peak) was used as negative control; error bars correspond to S.D., and statistical significance was calculated by paired t test (*, p < 0.05; **, p < 0.01). For the TRF2-overexpressed condition (in red), all p values were <0.01.

Figure 5.

TRF2-dependent transcriptional outcomes. A, gene expression of target genes measured by qRT-PCR in untransfected and TRF2-overexpressed conditions in HT1080 cells. GAPDH expression was used for normalization; error bars correspond to S.D., and statistical significance was calculated by paired t test (*, p < 0.05; **, p < 0.01). B, effect of TRF2 domain mutations on expression of target genes. HT1080 cells were transfected with TRF2 WT or domain deletion mutants delB and delM, and expression was compared with control untransfected cells. Error bars, S.D. for three independent experiments. C, effect of TRF2 domain mutations on expression of target genes in TRF2-silenced background. HT1080 cells with stable TRF2 silencing were transfected with TRF2 WT or domain deletion mutants delB and delM, and expression was compared with SCR control and TRF2 shRNA–transfected cells. Error bars, S.D. for three independent experiments. D, H3K27me3 (suppression mark) and activation marks H3K4me3 and H3K4me1 enrichment (normalized to total H3) was tested within promoters in HT1080 cells following TRF2 overexpression relative to untransfected cells. Error bars, S.D. from two independent experiments.

Figure 6.

TRF2 occupancy at gene promoters is sensitive to intracellular G-quadruplex–binding ligand 360A. A, TRF2 occupancy at gene promoter sites was checked by ChIP qRT-PCR in HT1080 cells following overexpression of TRF2 in the presence or absence of 360A. Error bars, S.D. from three independent experiments; CTCF promoter was used as a negative control. B, TRF2 level was checked by Western blotting in the presence of the ligand 360A in both untransfected and TRF2 transient overexpression conditions. Overexpression was also confirmed by probing for DDK tag. GAPDH was used as loading control. C, expression of target genes was analyzed by qRT-PCR in HT1080 cells following overexpression of TRF2 in the presence or absence of 360A. GAPDH expression was used for normalization; error bars, S.D. from three independent experiments. Statistical significance was calculated by paired t test (*, p < 0.05; **, p < 0.01).