Journal: Nature Structural & Molecular Biology

Article Title: Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis

doi: 10.1038/s41594-023-00943-7

Figure Lengend Snippet: a , Volcano plots as in Fig. , highlighting pluripotency-associated factors that are enriched (red) or not significantly changed (black) on WT (left plot) or Suv39h dn (right plot) ESC mitotic chromosomes. b , Esrrb immunolabeling (red) of WT and Suv39h dn flow-sorted chromosomes 19 (left panel) and X (right panel), where DAPI counterstain is shown in light gray. Scale bar, 5 μm. Esrrb mean intensities were measured across individual chromosomes; the mean ± s.d. is shown ( n = minimum 50 chromosomes analyzed over 3 independent experiments). P values of statistically significant changes, measured by unpaired, two-tailed Student’s t -tests, are indicated. c , Esrrb ChIP–qPCR analysis in WT versus Suv39h dn mitotic and asynchronous ESCs. Enrichment (immunoprecipitated as a percentage of input (% IP)) was measured at Esrrb bookmarked sites ( Capn2 , Esrrb , Jam2-s1 , Jam2-s2 , Tbx3 and Tet2 ), Esrrb lost sites (bound only in interphase ; Mgat3 and Twistnb ) or control sites that do not bind Esrrb ( Esrrb-3 ′ and Actb ). The mean + s.d. results are shown. For interphase cells n = 3 biological replicates, for mitotic cells n = 4 biological replicates (except Capn2 , Esrrb , Rex1 and Jam2-s1 , where n = 5). d , Live-cell imaging of Esrrb–tdTomato mouse ESCs pretreated with DMSO (upper panel) or 100 nM of chaetocin (lower panel) cultured with SiR-DNA (red). Arrows show Esrrb localization to mitotic chromatin. Scale bar, 5 μm. Esrrb–tdTomato mean intensities on mitotic DNA (gated based on SiR-DNA signal) and in interphase nuclei were quantified for each condition; the mean ± s.d. is shown. For mitotic chromosomes: n = 25 (DMSO) or n = 35 (chaetocin) cells analyzed; for interphase nuclei: n = 46 cells analyzed for both DMSO and chaetocin treatments, representing 3 independent experiments. b – d , P values of statistically significant changes, measured by unpaired, two-tailed Student’s t -tests, are indicated. and precise n numbers are provided.

Article Snippet: Real-time qPCR (BioRad, CFX96 system with CFX manager software) was performed in technical triplicate for each biological replicate, using SYBR Green PCR Master Mix (QIAGEN) and the primers listed in Supplementary Table .

Techniques: Immunolabeling, Two Tailed Test, ChIP-qPCR, Immunoprecipitation, Control, Live Cell Imaging, Cell Culture