Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes (original) (raw)
Figure 5
Centromere function of induced dicentrics.
(A) Scheme of experimental strategy to produce de novo dicentrics for which centromere function and mitotic stability were monitored every 2 weeks (wks) for a total of 20 weeks. Centromere function was assayed by immunostaining for various centromere proteins (CENPs). Centromeric DNA regions were identified using CENP-B immunostaining or FISH with α-satellite–specific DNA probes. (B) Assessment of centromere function using immunostaining for CENP-A (green) and CENP-B (red). CENP-A identifies functional centromeres. CENP-B is an α-satellite DNA binding protein that binds to both active and inactive centromeres. After 40 hours (40h) of dnTRF2 expression, dicentrics were formed, including iROBs (B) and dicentrics involving non-acrocentric chromosomes (B′). Each type of dicentric, denoted by arrows, had two active centromeres. Scale bars = 7.5 µm. (C) At 4 days (4d) after dicentric formation, functionally dicentric chromosomes were still observed, including on chromosomes with two distantly located centromeres (C, arrow). Some cells also contained structurally tricentric chromosomes, as shown in (C′, arrow). In this case, one centromere was inactivated, since it exhibited immunostaining for CENP-B only. Scale bar in C = 5 µm; in C′ = 7.5 µm. (D) Several types of functionally dicentric chromosomes persisted even at 20 days (20d) after formation. These included iROBs (D) and non-acrocentric dicentrics with large inter-centromeric distances (D′). However, some functionally monocentric dicentrics were observed (D″), primarily among the non-acrocentric class of dicentrics. Arrows denote the chromosome fusion in each panel. Scale bars = 7.5 µm. (E) Assessment of centromere function on an irob(13;14) after 14 weeks of continuous culture (∼100 cell divisions). Centromeres were detected using FISH with α-satellite probes. CENP-A staining (green) appeared at the CEN14 (E, red). (E′) CENP-A (red) was also present at CEN13 (green), indicating that this iROB was functionally dicentric. (E″) This image shows the same iROB detected only with FISH probes to illustrate that CEN13 (green) and CEN14 (red) were spatially distinct. (F) Functionally monocentric iROBs were also detected during the timecourse experiment. Another irob(13;14), different than the one in (E), showed evidence of centromere inactivation. In (F), CENP-A (green) did not overlap with CEN14 (red). (F′) CENP-A (red) and CEN13 (green) co-localized, indicating the CEN13 was the functional centromere and CEN14 had been inactivated. (F″) This image shows the same iROB detected only with CEN13 (green) and CEN14 (red) FISH probes to illustrate that the centromeric arrays were spatially distinct.