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. 2018 Aug 1;37(15):e98997.
doi: 10.15252/embj.201898997. Epub 2018 Jul 11.

Shelterin Promotes Tethering of Late Replication Origins to Telomeres for Replication-Timing Control

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Free PMC article

Shelterin Promotes Tethering of Late Replication Origins to Telomeres for Replication-Timing Control

Shiho Ogawa et al. EMBO J. .
Free PMC article

Abstract

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere-binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere-like sequence near the origins. Here, we showed using a lacO/LacI-GFP system that Taz1-dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication-timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication-timing control and telomeric association of Taz1-dependent late origins, and this requirement was bypassed by a minishelterin Tpz1-Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin-mediated telomeric association of the origins at the onset of S phase.

Keywords: Rap1; Taz1; chromatin organization; replication timing; telomere.

Figures

Figure 1
Figure 1. Taz1‐dependent late replication origins are localized at the nuclear periphery

The locations of early and late replication origins relevant to this study are schematically shown on Schizosaccharomyces pombe chromosome II. The early origins ars2004, ori2024, and ori2123, the Taz1‐dependent late origins AT2088 and ori2100, and the Taz1‐independent late origin ars727 are indicated. The location of the centromere (cen2) is depicted by a yellow square, and the telomeres are indicated by black arrowheads.

Schematic images for the analysis of the subnuclear locations of LacI‐GFP‐tagged replication origins in comparison with the mCherry‐Ish1 at the nuclear envelope under a fluorescence microscope. Among 21 focal planes with 0.1‐μm focus intervals, a single plane that contained the strongest lacO/LacI‐GFP signal was chosen for quantitative analyses.

Representative images of lacO/LacI‐GFP (green) at the AT2088 late origin (upper 4 panels) and at the ars2004 early origin (lower 4 panels) merged with Ish1‐mCherry (red) are presented. The optical section images were processed as described in “Materials and Methods”. The scale bar indicates 5 μm.

Images were classified into two categories: Class 1 (peripheral), GFP signals that overlapped with Ish1‐mCherry; and Class 2 (non‐peripheral), GFP signals that did not overlap with or had separated from Ish1‐mCherry. The proportions of Class 1 (blue) and Class 2 (pale blue) of the AT2088 late origin and the ars2004 early origin are presented in pie charts.

The Class 1 and Class 2 proportions of ori2100 (Taz1‐dependent late origin), ars727 (Taz1‐independent late origin), ori2024 (early origin), and ori2123 (early origin) are shown in pie charts.

The effect of a base substitution (AT2088‐S2632) in the telomere‐like sequence near the AT2088 on the peripheral localization of AT2088 and the effects of deletion of taz1 + on the localizations of late origins AT2088 and ori2100, and the early origin ars2004 were examined. Representative images of lacO/LacI‐GFP (green) and Ish1‐mCherry (red) are presented with the proportions of Class 1 (peripheral, blue) and Class 2 (non‐peripheral, pale blue). The scale bar indicates 5 μm.

Figure EV1
Figure EV1. Intra‐nuclear locations of LacI‐GFP‐tagged early and late origins in comparison with Ish1‐mCherry during G1/S and G2 phases
Class 1 (peripheral, blue) and Class 2 (non‐peripheral, pale blue) proportions of the Taz1‐dependent late origins AT2088 and ori2100 and the early origins ars2004, ori2024, and ori2123 were determined separately during G1/S phase (bi‐nuclear) and G2 phase (mono‐nuclear) cells, and are shown in pie charts. P‐values were calculated using Fisher's exact test.
Figure EV2
Figure EV2. Time‐lapse images of AT2088‐LacI‐GFP merged with Ish1‐mCherry during M phase
Representative images of AT2088‐LacI‐GFP (green) and Ish1‐mCherry (red) obtained in 3‐min intervals before and after the onset of anaphase. Optical section images were projected by the maximum intensity method using the SoftWoRx 5.5 software on the DeltaVision Elite system. The scale bar indicates 1 μm.
Figure 2
Figure 2. G1/S phase‐specific telomeric association of late origin AT2088

Schematic images are shown for the analysis of the location of AT2088‐LacI‐GFP in comparison with Taz1‐mCherry at the telomeres. Among 21 vertical planes with 0.1‐μm intervals, a single plane that contained both a strong lacO/LacI‐GFP (green) signal and the closest Taz1‐mCherry (red) signal is chosen for analysis. For quantitative analysis of the locations of AT2088 locus in comparison with telomeres, the distance between the center of AT2088‐LacI‐GFP signal and that of the closest Taz1‐mCherry signal (telomere) in a single focal plane was measured.

The LacI‐GFP signal at AT2088 (left), the Taz1‐mCherry foci at the telomeres (middle), and the merged image (right) in a single focal plane are presented. The scale bar indicates 1 μm.

Merged images of LacI‐GFP and Taz1‐mCherry are presented as in (B). The scale bar indicates 1 μm.

The distances between the center of AT2088‐LacI‐GFP signal and that of the closest Taz1‐mCherry signal (telomere) were measured in a single focal plane in G1/S and G2 phase nuclei of wild‐type and AT2088‐S2632 cells (as in A). The results (μm) are presented in the scatter plot. Lines indicate means ± SD. The numbers of nuclei analyzed were n = 60 and n = 119 for G1/S and G2 phases in wild type, respectively, and n = 70 and n = 103 for G1/S and G2 phases in AT2088‐S2632 cells, respectively. P‐values were calculated using the Kruskal–Wallis test in GraphPad Prism 6. ****< 0.0001.

Schematic drawings of the lacO/LacI‐GFP (AT2088) and Taz1‐mCherry (telomere) in G1/S and G2 phases based on the results of Figs 1 and 2 are presented.

Figure EV3
Figure EV3. Time‐lapse images of AT2088‐LacI‐GFP merged with that of telomeric Taz1‐mCherry during late M phase
The images of AT2088‐LacI‐GFP (green) and Taz1‐mCherry (telomeres, red) obtained in 3‐min intervals are presented together with bright‐field images (left panels). The right and left cells correspond to late M and G2 phase cells, respectively. Images in a single focal plane are presented. The scale bar indicates 5 μm. At 0, 9, and 12 min, AT2088‐LacI‐GFP signal (green) overlapped, at least partly, with the Taz1‐mCherry signal (red), generating yellow spot or boundary. They were found as adjacent but non‐overlapping signals at the other time points.
Figure 3
Figure 3. Telomere anchoring to the nuclear membrane is not required for telomeric association of the Taz1‐dependent origin and late replication control

Localization of AT2088‐LacI‐GFP in comparison with Taz1‐mCherry was analyzed in bqt4Δ cells. Representative images of a single focal section (top) with enlarged images for the regions “a” (middle) and “b” (bottom) in G1/S phase cells are shown. One of the two nuclei in the G1/S phase cell containing “a” is out of focus. The scale bar indicates 5 μm (top panels) or 1 μm (middle and bottom panels).

The distances between the AT2088‐LacI‐GFP focus and the closest Taz1‐mCherry in G1/S (n = 85) and G2 (n = 101) phases of wild‐type cells and G1/S (n = 68) and G2 (n = 112) phases of bqt4Δ cells are shown in the scatter plots. Lines indicate means ± SD. P‐values were calculated using the Kruskal–Wallis test. ****< 0.0001 and **< 0.01.

Representative images of lacO/LacI‐GFP (green) at the AT2088 merged with Ish1‐mCherry (red) in bqt4Δ cells are presented. The proportions of Class 1 (peripheral, blue) and Class 2 (non‐peripheral, pale blue) during G1/S and G2 phases of bqt4Δ cells together with the results of wild type (as shown in Fig EV1) are shown in the pie charts. P‐values were obtained using the Fisher's exact test. ****< 0.0001.

Effect of bqt4 + deletion on replication timing of early and late origins. Wild‐type and bqt4Δ cells synchronously released from the G2/M block were labeled with BrdU for 90 min at 25°C in the presence of HU. Replication (%) was quantified as described in “Materials and Methods” by qPCR using primers for nonARS, early origin ars2004, late origins ori2100 and AT2088, and subtelomeric origin TAS59. The mean values obtained from three independent experiments are presented ±SEM.

Figure 4
Figure 4. Shelterin components Rap1 and Poz1 are required for replication‐timing control and telomeric association of Taz1‐dependent late origins

Effects of shelterin component deletion on replication timing of late origins. Wild‐type, taz1Δ, rap1Δ, and poz1Δ cells were synchronously released from the G2/M block and labeled with BrdU for 90 min in the presence of HU. Replication (%) was analyzed as described in Fig 3. The mean values obtained from three independent experiments are presented ±SEM.

Localization of AT2088‐LacI‐GFP in comparison with Taz1‐mCherry was analyzed in rap1Δ cells. Representative images of a single focal section (top) with enlarged images for the regions “G1/S” (middle) and “G2” (bottom) are shown. The scale bar indicates 5 μm (top panels) or 1 μm (middle and bottom panels).

Distances between the AT2088‐LacI‐GFP focus and the closest Taz1‐mCherry (telomere) during G1/S (n = 60) and G2 (n = 108) phases of rap1Δ cells and G1/S (n = 64) and G2 (n = 86) phases of poz1Δ cells together with the results of wild‐type cells (as in Fig 2) are shown in the scatter plots. Lines indicate means ± SD. P‐values were obtained by the Kruskal–Wallis test. ****< 0.0001, ***< 0.001, and **< 0.01.

Representative images of lacO/LacI‐GFP (green) at the AT2088 merged with Ish1‐mCherry (red) in rap1Δ cells. Class 1 (peripheral) and Class 2 (non‐peripheral) proportions in G1/S and G2 phases rap1Δ cells together with the results of wild type (as shown in Fig EV1) are shown in pie charts. P‐values were calculated using Fisher's exact test. ****< 0.0001 and *< 0.05.

Figure EV4
Figure EV4. Localization of Flag‐Taz1 at Taz1‐dependent late origins in rap1Δ cells
Chromatin immunoprecipitation with anti‐Flag antibody was conducted in logarithmically growing wild‐type and rap1Δ cells expressing Flag‐Taz1 as described previously (Tazumi et al, 2012). Localization of Flag‐Taz1 was analyzed by qPCR using sets of primers for nonARS (gray), AT2088 (cyan), tel‐3 (violet), and tel‐0.3 (purple). The mean values obtained from three independent experiments are presented ±SEM. P‐values in the magnified results were obtained by Sidak's multiple comparisons test in GraphPad Prism 6. **< 0.01 and *< 0.05.
Figure EV5
Figure EV5. Localization of Rap1‐Flag at the proximity of the telomere but not at the Taz1‐dependent late origin
Chromatin immunoprecipitation with anti‐Flag antibody was conducted in G2/M‐arrested rap1‐Flag cells (0 min) and those in G1/S phase (80 min after release). Localization of Rap1‐Flag was analyzed by qPCR using sets of primers for nonARS, AT2088, tel‐3, and tel‐0.3. The mean values obtained from three independent experiments are presented ±SEM. P‐values were calculated using Sidak's multiple comparisons test in GraphPad Prism 6. ****< 0.0001.
Figure 5
Figure 5. Minishelterin restores replication‐timing control and telomeric association of Taz1‐dependent late origins in the absence of Rap1 and Poz1

Effect of minishelterin expression on the replication timing of late origins. A minishelterin containing the Tpz1‐Taz1 fusion protein is schematically presented. Wild‐type and rap1Δ poz1Δ double‐mutant cells with or without expression of the Tpz1‐Taz1 fusion protein were synchronously released from the G2/M block and labeled with BrdU for 90 min in the presence of HU. Replication (%) was analyzed as described in Fig 3D. The mean values obtained from three independent experiments are presented ±SEM.

Representative images are shown for AT2088‐LacI‐GFP in comparison with Taz1‐mCherry in rap1Δ poz1Δ double‐mutant cells with (bottom) or without (top) Tpz1‐Taz1 expression. The scale bar indicates 5 μm.

The distances between the AT2088‐LacI‐GFP focus and the closest Taz1‐mCherry (telomere) in G1/S (n = 76) and G2 (n = 103) phases of rap1Δ poz1Δ cells and in G1/S (n = 82) and G2 (n = 99) phases of cells expressing the Tpz1‐Taz1 fusion protein together with the results of wild‐type cells (as in Fig 2) are shown in scatter plots. Lines indicate means ± SD. P‐values were calculated using the Kruskal–Wallis test. ****< 0.0001 and **< 0.01.

Localization of AT2088‐LacI‐GFP in comparison with Ish1‐mCherry was analyzed in rap1Δ poz1Δ cells with or without expression of a Tpz1‐Taz1 minishelterin. Class 1 (peripheral) and Class 2 (non‐peripheral) proportions are shown in pie charts. P‐values were calculated using Fisher's exact test. ****< 0.0001.

Figure 6
Figure 6. Model for replication‐timing control by shelterin‐mediated telomeric association
A model for control of Taz1‐dependent late origins by shelterin‐mediated localization near the telomeres is shown. Taz1 binds to telomeric repeats (red arrowheads) at the telomeres, recruiting Rap1 and Rif1 to the telomeres. Telomeres are anchored to the nuclear membrane through the interaction of Rap1 with nuclear membrane protein Bqt3/4 during the interphase. Rif1 recruits PP1 that acts as a counter‐phosphatase for DDK‐phosphorylation of Mcm2‐7 subunits. Subtelomeric late replication origins are strongly suppressed by PP1. Because Rif1 has a cloud‐like localization around the telomeres (Appendix Fig S5), PP1 is likely to be enriched around telomeres, forming a “PP1‐zone”. Taz1 also binds to the telomere‐like repeat (two arrowheads) near the replication origins in chromosomal arm regions throughout the cell cycle. During M phase, Taz1‐bound origins as well as telomeres dissociate from the nuclear membrane. At the end of M phase, Taz1‐bound origins associate with telomeres, possibly through interaction of Taz1 with the shelterin component Rap1. During G1/S phase, telomeres are anchored to the nuclear membrane, and Taz1‐dependent origins tethered around the telomeres are suppressed by PP1.

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