Fig. 4: Damaged MN bodies are more likely to have persistent transcription defects.
From: Heritable transcriptional defects from aberrations of nuclear architecture
a, Transgenerational tracking of MN chromosome fate. Top, cartoon of the gene expressing megaDam. Bottom, scheme of manipulations that restrict DamMN expression to the first interphase when MN form. b, Representative images of re-incorporated MN with high (top) and low (bottom) DNA damage in MN bodies. Top, MN body (m6A-Tracer, dashed magenta outline) with high γH2AX signal (top quartile, see c on the right) but low RNAP2-Ser5ph labelling. Bottom, MN body with low γH2AX signal (bottom quartile) and normal RNAP2-Ser5ph labelling. c, Aggregate data of relative RNAP2-Ser5ph intensities in MN bodies with DNA damage levels (Extended Data Fig. 10e; left to right, n = 220, 111, 220 and 112 from 4 experiments). Boxes are median with 95% CI; Kruskal–Wallis with Dunn’s multiple comparisons. d, Correspondence between high damage level and low transcriptional activity for re-incorporated MN chromosome 1 using the U2OS 2-6-3 nascent transcription reporter. Top, scheme of the experiment. During live imaging, cells with the reporter in a micronucleus were identified by LacI–SNAP and followed through cell division to identify MN body formation. Transcription activity of the reporter was tracked in live cells (MCP–Halo foci) and, after re-incorporation, was scored for γH2AX-marked DNA damage. Bottom left, MS2 signal used to detect reporter transcription activity as in Fig. 2e (grey line indicates the mean and s.e.m. of FI in controls as shown in Extended Data Fig. 8a). Bottom right, after live imaging, cells were fixed to detect γH2AX and RNAP2-Ser5ph (which correlated with the MS2 signal). Shown are representative images. Yellow arrowheads indicate MN bodies. e, Summary of the transcriptional output and presence of damage for 49 re-incorporated chromosome 1 with the reporter. The correlation between transcription defect (Fig. 2d) and DNA damage is significant (11 experiments, P < 0.0001, two-sided Fisher’s exact test). Scale bars, 5 µm (b,d).
