Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells

Abstract

Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. Using live-cell imaging, we show that expression of TRF1 or yellow fluorescent protein (YFP)-TRF1 fusion protein above endogenous levels prevents proper telomere resolution during mitosis. TRF1 overexpression results in telomere anaphase bridges and aggregates containing TRF1 protein and telomeric DNA. Site-specific protein cleavage of YFP-TRF1 by tobacco etch virus protease resolves telomere aggregates, indicating that telomere associations are mediated by TRF1. This study provides novel insight into the formation and resolution of telomere associations.

FIGURE 1:

Generating cell populations expressing defined TRF1 protein levels. (A) Schematic of constructs encoding YFP-TRF1 (top) and TRF1 translated separately from YFP (bottom), driven by a CAG promoter. (B) FACS plots showing wild-type control, vector control (IRES-YFP), TRF1_IRES-YFP, and YFP-TRF1 transfected cells at 24 h posttransfection and gates used to sort negative, low, high, and high++ YFP populations. Numbers in gates indicate fold difference in median YFP levels compared with the low population. (C) Western blot analysis of wild-type control, vector control (IRES-YFP), and TRF1_IRES-YFP low and high sorted cell populations (lanes 1–4, respectively) and (D) YFP-TRF1 low (lane 1) and high (lane 2) sorted cell populations using anti-TRF1 antibody or anti-YFP antibody (D, lane 3). Bottom, GAPDH loading control. The molecular weights of TRF1 (solid arrowhead) and YFP-TRF1 (open arrowheads) are indicated. (E) Localization of low YFP-TRF1 (green) over the cell cycle with DAPI DNA stain (blue). Foci doublets in metaphase (insets, arrows) and singlets in anaphase (insets, arrowheads). Images are maximum-intensity projections.

FIGURE 2:

TRF1 overexpression induces anaphase bridges containing TRF1, mitotic bypass, and TRF1 aggregates. (A) Time-lapse images of cells expressing low or high YFP-TRF1 (green) levels and H2B-RFP (chromosomes; red). Note the formation of YFP-TRF1 bridges (arrows) between segregating chromosomes, which persist into interphase (bottom). Images are maximum-intensity projections (Supplemental Movies S1 and S2). (B) Quantification of YFP-TRF1 bridges and mitotic bypass from two independent experiments. Percentages are given in brackets. (C) Time-lapse images showing transient YFP-TRF1 bridge (arrowheads). Images are of a single z-section (Supplemental Movie S3). (D) Examples of persistent TRF1 bridges between daughter cells in interphase. (E) YFP-TRF1 (green) bridges overlap with telomere FISH probe labeling telomeric DNA (red). Note that images of YFP-TRF1 in fixed cells were acquired before telomere FISH, and images were juxtaposed from previously recorded slide coordinates. (F) Time-lapse images showing mitotic bypass. Note that this cell appears to progress from metaphase directly to interphase without undergoing cell division. (G) Example of an interphase cell expressing high++ YFP-TRF1 exhibiting several large, intense aggregates of YFP-TRF1 foci (arrowheads) connected by thin stretches of YFP-TRF1. Scale bar, 5 μm.

FIGURE 3:

TRF1 overexpression induces telomere associations. Examples of metaphase chromosome spreads in mouse ES cells transiently expressing high or greater YFP levels for (A) vector control (IRES-YFP) or (B) TRF1_IRES-YFP at ∼72 h posttransfection. Telomere DNA was labeled by a telomere FISH probe conjugated to Cy5 (green), and DNA was stained with DAPI (blue). (C) Column scatter plot showing percentage of single or joined telomeres at long arm. The p value was calculated using an unpaired two-tailed t test. Mean ± SEM is indicated. (D) Examples of telomere associations between metaphase chromosomes (arrowheads) in TRF1_IRES-YFP overexpressing cells and (E) YFP-TRF1 overexpressing cells at ∼72 h posttransfection. Inverted DAPI images (E, left). Insets show telomere aggregates for which each telomere aggregate is surrounded by multiple (up to seven) radially distributed chromosomes. Gamma was set to 0.5 for images in E to visualize lower-intensity signals.

FIGURE 4:

Resolution of telomere aggregates by TRF1 cleavage. (A) Schematic of constructs for TRF1 cleavage assay: cleavable FKBP-YFP-TRF1_TEV showing TRF1 domains with cleavage site (purple) inserted in flexible linker, noncleavable RFP-TRF1 to visualize telomeres, and CFP-TEV protease driven by a CAG promoter (or a PGK promoter in D). (B) Projection images of cells expressing high levels of cleavable FKBP-YFP-TRF1_TEV (green) and low levels of noncleavable RFP-TRF1 (red) for three time points before and after detection of CFP-TEV protease (blue). RFP-TRF1 foci selected using automatic thresholding of fluorescence intensity (bottom), with each color representing one selected focus. (C) Column scatter plot showing average intensity of foci (sum) on a log scale for three time points before and after detection of CFP-TEV protease or CFP control, where each dot represents an individual selected focus, and each color represents an individual cell. The number of selected foci (n) for each time point is indicated in brackets. Time-lapse movies of nine and seven cells are represented for CFP-TEV protease and a CFP control, respectively (legend). The p values for T = 1 compared with T = 4–6 was calculated using an unpaired two-tailed t test. Error bars, SEM. (D) Projection images from a time-lapse movie of cells expressing high levels of cleavable FKBP-YFP-TRF1_TEV (green) and low levels of noncleavable RFP-TRF1 (red) upon transfection with PGK-CFP-TEV protease, showing time after transfection. Note that FKBP-YFP-TRF1_TEV fluorescence becomes diffuse (indicating that cleavage has occurred), and noncleavable RFP-TRF1 aggregates become smaller and more uniform (insets; see Supplemental Movie S4). Scale bar, 5 μm.

FIGURE 5:

Model of TRF1-mediated telomere associations. (A) Cis-TRF1 conformation showing YFP on N-terminus and TEV cleavage site inserted in flexible linker domain. (B) Trans-TRF1 conformation may promote associations between telomeres under conditions in which telomere-binding sites are saturated with high levels of TRF1. (C) Model showing TRF1-mediated telomere associations in a cellular context and potential consequences. Telomere associations may occur by increase in TRF1 levels, decrease in telomere length (assuming TRF1 levels remain relatively constant), or under conditions that promote the trans-TRF1 conformation. Two main types of telomere associations may form: between sister telomeres and between telomeres from different chromosomes. Persistent telomere bridges may recruit DNA repair proteins, such as the helicase RTEL1, for resolution and repair. Telomere bridges may lead to DNA breakage within chromosomes or telomeres, and broken ends may continue to fuse and break in subsequent cell cycles, giving rise to genomic instability.