Extended Data Fig. 8: Analysis of nascent transcription from reincorporated micronuclei. | Nature

Extended Data Fig. 8: Analysis of nascent transcription from reincorporated micronuclei.

From: Heritable transcriptional defects from aberrations of nuclear architecture

Extended Data Fig. 8

(a) Control U2OS 2-6-3 reporters to assess nascent transcription of normally expressing reporters in the main nucleus. Normalized FI of MS2 signal (MCP-Halo) were measured from reporters that were in the main nucleus during both generation 1 and 2 (n = 23 LacI reporters). Grey bar: mitosis. Error bars: mean +/− SEM). Red line: minimum detectable normalized MS2 value of the controls (see Methods). (b) Example of a MN with late G2 rupture in generation 1 that recovered transcription after reincorporation into a daughter nucleus in generation 2. Performed and analyzed as in (a), above. Grey line: mean intensity of the control reporters in main nucleus. (c) Aggregated data of nascent transcription from reincorporated MN assessed by the U2OS 2-6-3 reporter, similar to (a) and Fig. 2e and f. Normalized FI of the MS2 signal (MCP-Halo) were measured from reporters that were in a MN in generation 1 and then incorporated into a daughter nucleus in generation 2. Top, a subset of cells with MN that ruptured during generation 1 interphase and then recovered transcription after reincorporation into a daughter nucleus in generation 2 (n = 7 analyzed out of 19 similar cases). Note that prior to mitosis there is variable MS2 signal because of variable MCP-Halo accumulation in intact micronuclei and because of variability in the timing of MN NE rupture. Bottom, aggregate data for a similar subset of samples where the MN ruptured during generation 1 interphase and then displayed a generation 2 transcription defect after reincorporation (black line, n = 9 analyzed out of 20 similar cases). Note: (1) for ease of visualization, error bars (mean +/− SEM) are shown only for the experimental samples, but not the controls; (2) when there was no detectable MS2 signal in an experimental sample, we assigned the minimal detectable normalized value in control cells (1.7, see Methods) to this sample (This explains the complete overlap between the black and red lines after the 10-hour timepoint). (d) Images from a timelapse series for the experiment in (b), above. Green arrowhead: MN rupture. Red arrowheads: MS2 expression from the reporter after reincorporation into a daughter nucleus in generation 2. Time: hours post release from the G2 block. Scale bars 5 µm. (e) Validation that MN-bodies originate from MN chromosomes, using same-cell live/fixed imaging. Left, images from a time-lapse series (U2OS 2-6-3 system, see Figs. 2e–g, 4d). A cell with a MN harboring Chr.1 (with the reporter integrated in Chr.1p, yellow arrowheads) was identified. The MN ruptured in the interphase that it was formed. After mitosis, the MN chromosome was reincorporated into a daughter cell PN (blue arrowheads, LacI-SNAP) but was not expressed (magenta arrowheads, MCP-Halo), even though it was in a normal nuclear environment. Right, at the end of the time-lapse imaging (t = 42.5 h) cells were fixed and the same cells were analyzed by immunofluorescence microscopy, revealing a large γH2AX-positive MN-body is at the ___location of the reincorporated MN chromosome identified by LacI-SNAP (open magenta arrowheads). Scale bars 5 µm. (f) Validation of the method to assess MN rupture with the U2OS 2-6-3 reporter system. For all experiments with this reporter, loss of the general nuclear MCP-Halo signal (MCP-Halo contains an NLS) was used to determine the time of MN NE rupture. We verified that MCP-Halo signal loss from MN corresponds to RFP-NLS by two-color live-cell imaging in U2OS 2-6-3 cells expressing both MCP-Halo and RFP-NLS (n = 40 from four experiments; two-tailed Spearman’s correlation).

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