Extended Data Fig. 12: Long-term effects on chromatin and expression after chromosome bridge formation.
From: Heritable transcriptional defects from aberrations of nuclear architecture
(a) MN-body-like structures in the daughter cells after chromosome bridge formation. Top, schematic presentation of the experiment. Bottom left, representative images of immunofluorescence analysis for MDC1 and RNAP2-Ser5ph, showing MDC1-positive nuclear bodies (dashed magenta line) after chromosome bridge formation and cell division of RPE-1 cells expressing TRF2-DN (see Methods). We observed a high frequency of cells with MDC1-positive nuclear structures of varying size (~10%) that likely represent reincorporated chromosome bridges. This number is expected, since the frequency of chromosome bridge formation is ~30% per cell division under the conditions described in Methods11. Scale bar, 5 µm. Right, quantification of the RNAP2-Ser5ph levels in the MDC1-positive nuclear structures after bridge chromosome reincorporation compared to the PN control. Performed and analyzed as in Fig. 3c (n = 309, from two experiments). (b) Genome-wide ATAC signal variation in control (i-x, left) and bridge (I-XII, right) clones in 1 Mb (top), 5 Mb (middle), and 10 Mb (bottom) intervals. The ATAC change in each interval (1 Mb increment) is assessed by normalizing the observed total ATAC signal (only from peaks) in each interval by the mean ATAC density of the null distribution generated by random permutations of individual peaks (see Methods, n = 2637 of 1 Mb genomic intervals). Bins with less than 10 ATAC peaks/Mb are excluded. Box plots indicate the first (bottom edge) and third (top edge) quartiles and the median (horizontal line), with whiskers indicating 1.5x the interquartile range. In each plot, red dots represent bins overlapping with the region of 27-38 Mb of Chr.4 that displays the most significant ATAC reduction across all bridge clones (see below). (c) Average ATAC signal variation in 10 Mb intervals across all 12 bridge clones. We only consider 10 Mb regions with 100 or more peaks. As the calculation is performed on all 10 Mb intervals with 1 Mb increment, a single region with a significant reduction in ATAC signal may result in multiple 10 Mb intervals with significant ATAC reduction; these consecutive 10 Mb bins are merged. Bins with the most significant ATAC reduction (fold change < 0.8) mostly come from two regions: Red dots are from the 4p region (26-38 Mb) shown in Fig. 5; purple dots are from a region from Chr.13q (54-76 Mb). Among 10 Mb regions with ATAC signal < 0.85, two are from Chr.4 and Chr.13: Chr.4:129-139 Mb (red circles) and Chr.13:78-94 Mb (purple circles). The other regions with ATAC signal < 0.85 are likely to have a non-epigenetic origin: Two regions (Chr.3:88-99 Mb, green dots; Chr.6:58-70 Mb, blue dots) span centromeres and have low confidence; another region on Chr.12p (12-30 Mb, light green dots) shows a similar reduction in the control clones and the variation is likely related to 12p gain or uniparental disomy that are frequent subclonal alterations in RPE-1 cells. The significant reduction in ATAC signal in Chr.4 and Chr.13 is unlikely to reflect random technical variation as they are specific to the bridge clones. For the Chr.13 region, we do not exclude a biological source for this variation, for example, an unidentified trans signaling effect that is related to bridge formation, breakage, or downstream evolution. It is known that certain genomic regions display more intrinsic variability of ATAC-Seq signals63 and such regions may be more prone to effects from chromosome bridge formation or breakage. Box plots indicate the first (bottom edge) and third (top edge) quartiles and the median (horizontal line), with whiskers indicating 1.5x the interquartile range. (d) Scatter plot of the fold change of ATAC signal (log2 transformed) and the P-value of ATAC signal variation estimated from permutations in bridge Clone I (two-sided permutation test, up to 5 million permutations without additional adjustment; see Methods). The two red dots are both from Chr.4:27-38 Mb. The cap of p-value at 2 x 10−7 reflects 5 million permutations performed for each interval.
