TRF1 Depletion Reveals Mutual Regulation Between Telomeres, Kinetochores, and Inner Centromeres in Mouse Oocytes

Abstract

In eukaryotic chromosomes, the centromere and telomere are two specialized structures that are essential for chromosome stability and segregation. Although centromeres and telomeres often are located in close proximity to form telocentric chromosomes in mice, it remained unclear whether these two structures influence each other. Here we show that TRF1 is required for inner centromere and kinetochore assembly in addition to its role in telomere protection in mouse oocytes. TRF1 depletion caused premature chromosome segregation by abrogating the spindle assembly checkpoint (SAC) and impairing kinetochore-microtubule (kMT) attachment, which increased the incidence of aneuploidy. Notably, TRF1 depletion disturbed the localization of Survivin and Ndc80/Hec1 at inner centromeres and kinetochores, respectively. Moreover, SMC3 and SMC4 levels significantly decreased after TRF1 depletion, suggesting that TRF1 is involved in chromosome cohesion and condensation. Importantly, inhibition of inner centromere or kinetochore function led to a significant decrease in TRF1 level and telomere shortening. Therefore, our results suggest that telomere integrity is required to preserve inner centromere and kinetochore architectures, and vice versa, suggesting mutual regulation between telomeres and centromeres.

Keywords: TRF1; centromere; kinetochore; oocyte; telomere.

FIGURE 1

TRF1 depletion accelerates polar body extrusion by abrogating the SAC. After microinjection of TRF1 dsRNA (dsTRF1) or control EGFP dsRNA (dsEGFP), the oocytes were cultured in M2 medium containing IBMX for 24 h and then transferred to IBMX-free M2 medium for up to 16 h. (A,B) Oocytes at MI stage (8 h after IBMX release) were fixed and stained with anti-TRF1 antibody. DNA was counterstained with DAPI (scale bar, 10 μm). TRF1 intensity was quantified and shown as mean ± SEM from at least four independent experiments with representative images. (C,D) Rates of GVBD and polar body extrusion (PBE) were scored. (E) Oocytes at the MI stage (8 h after IBMX release) were cultured in medium containing 20 μg/ml nocodazole and were scored 6 h later for PBE. (F–H) Chromosome spreads were prepared from oocytes at the MI stage and were stained with anti-Zw10 and anti-BubR1 antibodies. DNA was counterstained with DAPI (scale bar, 20 μm). The intensity of Zw10 and BubR1 was quantified from three independent experiments and shown with representative images. **p < 0.01, ***p < 0.001.

FIGURE 2

TRF1 depletion impairs kMT attachment and increases the rate of aneuploidy. Oocytes injected with dsEGFP or dsTRF1 were cultured in medium containing IBMX for 24 h and then transferred to IBMX-free medium for 8 h. (A–D) Oocytes at the MI stage were fixed and stained with anti-α-tubulin antibody and DAPI (scale bar, 10 μm). Spindle abnormality, chromosome misalignment, and metaphase plate width were quantified and are shown in representative images. Data are presented as mean ± SEM from three independent experiments. (E–G) After cold treatment, oocytes were fixed and stained with anti-centromere antibody (ACA), anti-α-tubulin antibody, and DAPI to visualize kinetochore, spindle, and DNA, respectively. The percentages of unattached kinetochores per oocyte and oocytes with abnormal kMT attachment were quantified, also shown in representative images (scale bar, 10 μm). Data are presented as mean ± SEM from three independent experiments. (H,I) Chromosome spreading of TRF1-depleted MII oocytes. Kinetochores and DNA were stained with ACA and DAPI, respectively (scale bar, 20 μm). The incidence of aneuploidy was quantified. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

TRF1 is required to maintain inner centromere and kinetochore integrity. Chromosome spreads were prepared from oocytes at the MI stage and were stained with anti-CENP-A, anti-Survivin, anti-Hec1, and anti-H3T3ph antibodies. Kinetochores and DNA were labeled with anti-centromere antibody (ACA) and DAPI, respectively (scale bar, 20 μm). (A–H) The intensities of CENP-A (normalized to DAPI or ACA), Survivin, and Hec1 were quantified, also shown in representative images. ns; not significant, ***p < 0.001.

FIGURE 4

TRF1 is associated with chromosome condensation and cohesion. (A–D) Chromosome spreads were prepared from oocytes at the MI stage and were stained with anti-SMC3 and anti-SMC4 antibodies. DNA was counterstained with DAPI. The normalized intensity of SMC3 and SMC4 was quantified and shown with the representative images (scale bar, 20 μm). ***p < 0.001.

FIGURE 5

Inhibition of inner centromere or kinetochore function decreases TRF1 level at telomeres. (A–C) Ndc80/Hec1 chromosome spreads were prepared from MI oocytes depleted Hec1 using the Trim-away method, and chromosome spreads were stained with anti-TRF1 antibody. Kinetochores and DNA were labeled with anti-centromere antibody (ACA) and DAPI, respectively (scale bar, 20 μm). (A) Representative images from three independent experiments are shown. (B,C) The normalized intensity of TRF1 at p-arms and q-arms was quantified. (D–F) Chromosome spreads were prepared from MI oocytes after treatment with DMSO (Control), AZ3146, or ZM447439 and stained with anti-TRF1 antibody. Kinetochores and DNA were labeled with anti-centromere antibody (ACA) and DAPI, respectively (scale bar, 20 μm). (D) Representative images from three independent experiments are shown. (E,F) The normalized intensity of TRF1 at p-arms and q-arms was quantified. (G,H) After treating with DMSO (Control), AZ3146, or ZM447439, oocytes were cultured for 12 h and subjected to telomere Q-FISH. Relative telomere FISH intensity was quantified and shown with representative images (scale bar, 20 μm). ns; not significant, ***p < 0.001.