RECQL4, the protein mutated in Rothmund-Thomson syndrome, functions in telomere maintenance

Abstract

Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.

FIGURE 1.

Fragile telomeric ends in RTS patient cells. A and B, microscopic images showing representative metaphase spreads from control cell GM00323 (panel 1) and RTS cell AG05013 (panel 2) (A) and control cell GM01864 (panel 1) and RTS cell AG18371 (panel 2) (B). DAPI (blue) was used for nuclear staining, and the red dots represent the telomeric ends. Some of the fragile telomeres are shown by white arrows. Scale bar represents 95 μm for A and 75 μm for B. C, representative images of chromosomes containing fragile telomeres. Fragile telomeric ends are shown by white arrows. D and E, percent fragile telomeres/chromosome in sex- and age-matched M00323 and AG05013 cells (D) and GM01864 and AG18371 cells (E). The differences in occurrence of fragile telomeres between control and RTS cells are statistically significant (p = 0.006 (D) and p = 0.004 (E); n = 50). The error bars represent mean ± S.D.

FIGURE 2.

Telomeric 53BP1 foci in RECQL4 knockdown U2OS cells. A, Western blot showing the level of RECQL4 in scrambled shRNA-treated (Scramble) and RECQL4-targeted shRNA-treated (RECQL4 KD) U2OS cells. Bands corresponding to RECQL4 and actin are shown by arrows. B, confocal microscopic images of representative cells showing TRF1 (red) and telomere (green) signals in scrambled shRNA-treated (panels 1 and 2, Scramble) and RECQL4 shRNA-treated (panels 4 and 5, RECQL4 KD) U2OS cells. Colocalized foci are visible as yellow dots in panels 3 and 6. Nuclear staining (with DAPI) is shown in the merged images. Scale bar = 10 μm. C, average numbers of TRF1 signals/cell in scrambled and RECQL4 KD U2OS cells. The error bars represent mean ± S.D., n = 50. D, confocal microscopic images showing colocalization of 53BP1 foci (green) and TRF1 (red) signals in scrambled (panels 1–3) and RECQL4 KD U2OS cells (panels 4–6) and in aphidicolin-treated scrambled U2OS cells (panels 7–9). Nuclear staining with DAPI is shown in the merged image. Some of the colocalized foci are marked with white arrows. Close-up images of some of the colocalized foci are shown next to panel 6. Scale bar = 5 μm. E, histograms showing the frequency distribution of telomeric 53BP1 foci (TIF) in scrambled, RECQL4 KD, and aphidicolin-treated scrambled U2OS cells (n = 70).

FIGURE 3.

Telomeric 53BP1 foci in RECQL4 knockdown HeLa and U2OS cells. A, Western blot showing the level of RECQL4 in scrambled shRNA-treated (Scramble) and RECQL4-targeted shRNA-treated (RECQL4 KD) HeLa cells. Bands corresponding to RECQL4 and actin are shown by arrows. B, confocal microscopic images showing colocalization of 53BP1 foci (green) and TRF1 (red) signals in scrambled (panels 1–3) and RECQL4 KD HeLa cells (panels 4–6). Nuclear staining with DAPI is shown in the merged image. Some of the colocalized foci are marked with white arrows. Close-up images of some of the colocalized foci are shown next to panel 6. Scale bar = 5 μm. C, histograms showing the frequency distribution of telomeric 53BP1 foci (TIF) in scrambled and RECQL4 KD HeLa cells (n = 70). D, Western blot showing the level of RECQL4 in control siRNA-treated (Control) and RECQL4-targeted siRNA-treated (RECQL4 KD) U2OS cells. Bands corresponding to RECQL4 and actin are shown by arrows. E, confocal microscopic images showing colocalization of 53BP1 foci (green) and TRF1 (red) signals in control (panels 1–3) and RECQL4 KD U2OS cells (panels 4–6). Nuclear staining with DAPI is shown in the merged image. Some of the colocalized foci are marked with white arrows. Close-up images of some of the colocalized foci are shown next to panel 6. Scale bar = 5 μm. F, histograms showing the frequency distribution of telomeric 53BP1 foci in control and RECQL4 KD U2OS cells (n = 70).

FIGURE 4.

Telomeric abnormalities in RECQL4-depleted U2OS and HeLa cells. A, percent fragile telomeres/chromosome in scrambled, aphidicolin-treated scrambled, RECQL4 KD, and aphidicolin-treated RECQL4 KD U2OS cells. The differences in occurrence of the event between scrambled and RECQL4 KD U2OS cells (**, p = 0.002, n = 50), between aphidicolin-treated scrambled and RECQL4 KD U2OS cells (*, p = 0.01, n = 30), and between RECQL4 KD and aphidicolin-treated RECQL4 KD U2OS cells (*, p = 0.03, n = 50) are statistically significant. Error bars show the standard deviation from the average of three independent experiments. B, percent fragile telomeres/chromosome in scrambled and RECQL4-targeted shRNA HeLa cells and control and RECQL4-targeted siRNA U2OS cells. The differences in occurrence of the event between scrambled and RECQL4 KD HeLa cells (*, p = 0.02, n = 30) and control and RECQL4 KD U2OS cells (**, p = 0.001, n = 30) are statistically significant. Error bars represent the standard deviation from an average of three independent experiments. C, percent T-SCE/chromosome in scrambled and RECQL4-targeted shRNA (RECQL4 KD) U2OS cells. The differences in occurrence of the event between scrambled and RECQL4 KD cells are statistically significant (**, p = 0.002, n = 30). The error bars represent mean ± S.D. of three independent experiments.

FIGURE 5.

RECQL4 localizes at telomere. A, FACS analysis showing cell cycle progression states of U2OS cells immediately after double thymidine block (panel 1, G₁), 4 h after double thymidine block (panel 2, S), and immediately after nocodazole treatment (panel 3, M). B, histograms showing the percentage of telomeric foci colocalized with RECQL4 at G₁, S, and M phases. The error bars represent mean ± S.D., n = 50. C, three-dimensional images of four representative colocalized RECQL4 (green) and TRF1 (red) foci. Scale bar = 0.74 μm. D, histogram showing Pearson's correlation coefficients of 50 randomly selected S phase cells.

FIGURE 6.

Telomeric D-loop substrates. A, complete list of oligos used to prepare the D-loop structures. B, structures of the D-loop substrates used in this study. The full structure of DL1, composed of three oligos, SS1, BT, and BB, is shown, and the telomeric region is marked. The telomeric regions of DL4 and DLmx are also shown. The rest of the structure remains the same in these three substrates.

FIGURE 7.

Interaction of RECQL4 with telomeric and non-telomeric D-loops. A, autoradiogram showing binding of 0, 7.5, 15, and 30 nm RECQL4 with 0.5 nm DL1 (lanes 1–4) and 0.5 nm DLmx (lanes 5–8). Bound products are marked as a, b, and c. B, autoradiogram showing the unwinding activity of 0, 5, 10, and 25 nm RECQL4 on telomeric (lanes 1–4, 0.5 nm DL1) and non-telomeric D-loops (lanes 5–8, 0.5 nm DLmx) and oxidatively damaged telomeric D-loops (lanes 10–13, 0.5 nm DL4) in the presence of excess (25×) single-stranded DNA. Δ (lane 9), represents heat-denatured DLmx. C, autoradiogram showing the helicase activity of 5, 10, and 25 nm helicase-dead mutant RECQL4 (RECQL4M) on 0.5 nm DL1 and 0.5 nm DL4 in the presence of excess (25×) single-stranded DNA. D, histogram showing the unwinding activity of 0, 5, 10, and 25 nm RECQL4 on DL1, DLmx, and DL4 in the presence of excess (25×) single-stranded DNA. The error bars represent mean ± S.D., n = 3.

FIGURE 8.

RECQL4 physically and functionally interacts with TRF2. A, autoradiogram showing the unwinding activity of RECQL4 (10 nm) on 0.5 nm DL1 in the presence of 0, 5, 10, and 20 nm TRF1 (lanes 1–5) and TRF2 (lanes 6–9) in the presence of 25× excess single-stranded DNA. Δ (lane 12), represents heat-denatured DL1. 20 nm TRF1 (lane 10) or TRF2 (lane 11) alone does not have any unwinding activity on DL1. B, histogram showing the unwinding activity of RECQL4 (10 nm) on 0.5 nm DL1 in the presence of 0, 5, 10, and 20 nm TRF1 and TRF2 in the presence of 25× excess single-stranded DNA. The error bars represent mean ± S.D., n = 3. C, autoradiogram showing the effect of 5, 10, and 20 nm TRF1 (lanes 1–5) or TRF2 (lanes 6–10) on annealing activity of RECQL4. 0.5 nm radiolabeled oligo SS1 and 2.5× excess oligo BB were used as annealing substrates. D, in vitro pulldown of TRF2 by RECQL4. Lane 1, represents input, showing the bands corresponding to RECQL4 and TRF2. Lane 2, represents co-IP with IgG controls. Lane 3, represents co-IP with antibodies specific to RECQL4. Bands corresponding to anti-RECQL4 and anti-TRF2 antibodies are marked with arrows.

FIGURE 9.

RECQL4 and WRN synergistically unwind telomeric D-loops. A, gel showing the effect of 10 and 5 nm RECQL4 on the unwinding activities of 10 nm WRN and 5 nm BLM, respectively. 0.5 nm DL1 (lanes 1–5) and DL4 (lanes 7–11) were used as substrates. Lanes 6 and 12, represent the unwinding activity of RECQL4 alone on DL1 and DL4, respectively. Δ (lane 13), indicates heat-denatured DL4. B, quantitative analysis of the gel showing the effect of RECQL4 on WRN and BLM unwinding activities on DL1 and DL4 at a 1:1 molar ratio of the corresponding proteins. The error bars represent mean ± S.D., n = 3. C, in vivo co-IP of WRN by FLAG-tagged RECQL4 in U2OS cells. The bands corresponding to anti-WRN and anti-FLAG antibodies are shown. Lanes 1 and 2, the bands in RECQL4-FLAG and empty vector-transduced cells, respectively. The corresponding IPs with FLAG, in the presence of EtBr, are shown in lanes 3 and 4, respectively. D, schematic summarizing the proposed synergistic role of RECQL4 and WRN in telomere maintenance. The DNA replication and repair machinery fails to proceed through the telomeric D-loop, and WRN and RECQL4 are recruited by the shelterin complex (most probably by TRF2, as both of these RecQ helicases interact with it directly) to resolve this structure and thus maintain telomere integrity.