Fig 1: CTCF and ß-catenin recruit AHCTF1 to the oncogenic super-enhancer to promote its ability to reach the nuclear pore.a Co-immunoprecipitation analyses of physical interactions between CTCF, ß-catenin, NUP133 and AHCTF1. IgG negative control. b Quantification of the CTCF-bound complexes shown in (a). c Co-immunoprecipitation analyses of the physical interactions between AHCTF1 and CTCF in WT HCT-116 cells in the absence or presence of BC21. d ChIP analyses of the binding of AHCTF1 to the oncogenic super-enhancer in DMSO control or BC21-treated WT HCT-116 cells. e ChIP analyses of CTCF and AHCTF1 binding to the CCAT1-specific CTCFBS in cells transfected with siGFP or siCTCF. The signals were normalized to the siGFP controls. The average siCTCF-mediated reduction in CTCF expression was 85% (Supplementary Fig 3d). f ChIP analyses of AHCTF1 binding to the CTCFBS and the CCAT1 promoter within the OSE in WT HCT-116 and mutant (D3/E4) cells. g The knock-down of AHCTF1 expression by siRNA using a siGFP as control. h 3D DNA FISH analyses of the proximity between the OSE and the nuclear periphery in HCT-116 cells in the presence or absence of AHCTF1 (reduced to 72% in comparison to controls7). The bars represent the sum of two independent experiments (219 and 201 alleles, respectively) for siGFP and siAHCTF1-treated cells. i Box-and-whisker plots show median values, interquartile ranges and Tukey whiskers of the distribution of the OSE within 0.7 µm from the nuclear periphery. j In situ proximity ligation assay (ISPLA) of the proximity between CTCF and AHCTF1 in the absence or presence of BC21 in WT HCT-116 cells. Overviews of the DMSO, BC21, and no primary antibody control motifs (upper row), with the the lower row shows magnifications of focal planes marked in the upper row. Bar = 5 µm. k The quantification of the ISPLA signals. The data is based on three independent experiments counting a total number of 710 alleles. C CTCF antibody, A AHCTF1 antibody, No ab no primary antibody. All the data (except for h) represent the average of three independent experiments with indicated standard deviations. The p values for (b–g, k) were calculated by the two-tailed Student’s t test whereas the p value for (i) was calculated using the two-sided KS test.
Fig 2: Three-dimensional super-resolution imaging over an adjustable axial range. a, b 3D super-resolution images of the nucleoporin Nup133 in HeLa cells reconstructed by ZOLA for a saddle point PSF with oil immersion objective and a tetrapod PSF with water immersion objective, respectively. Color indicates depth z. The axial range is 2 µm in a, showing the bottom portion of the nucleus, and 5 µm in b, allowing to visualize almost the entire nucleus. The (x', z) view shows a projection from the region of interest enclosed by the violet dashed rectangle. Magnified views of pink boxed regions show nuclear pores visible as ring-like structures. Scale bars are 5 µm for the main images, and 0.5 µm for insets and (x', z) projections. c, e 3D super-resolution images of microtubules in a U-373 MG cell. The same cell was imaged first with an astigmatic PSF (c), then with a saddle point PSF (e). The astigmatic PSF enables an axial range of 1 µm, allowing to visualize the bottom of the cell. The saddle point PSF enables an axial range of 2.5 µm, allowing to visualize the full cell. The (x', z) view shows a projection from the region of interest enclosed by the violet dashed rectangle. Scale bars are 5 µm for the main images, 0.5 µm for insets and (x', z) projections. d, f Histograms (2D and 1D) show the distribution of lateral and axial (z) coordinates of localizations across microtubule filaments at the three positions indicated by the pink rectangles in images c and e above. The number of localizations (N) and the mean z-coordinate (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar z_{}^{}$$\end{document}z¯) are indicated. Black curves show the probability densities of axial and lateral coordinates expected for optimal precision, based on the average theoretical precision limits (Cramér–Rao lower bounds \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar \sigma _{xy}^{\mathrm {CR}}$$\end{document}s¯xyCR and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar \sigma _z^{\mathrm {CR}}$$\end{document}s¯zCR of lateral and axial localization errors are indicated) and the diameter of immunolabeled tubulin filaments (see Supplementary Fig. 8). Full width at half maxima of the probability densities are indicated below double arrows. The good match between the theoretical probability densities and the experimental histograms indicates that ZOLA achieves optimal precision at all depths
Fig 3: MN size correlates with nuclear lamina protein levels and gene density inversely correlates with nuclear lamina gaps.(A) Images of lamin B1 (LmnB1) levels on intact (H3K27ac+) single chromosome MN containing indicated chromosome 20 h post Cdk4/6i release. LmnB1 images are a single section and merged images are maximum-intensity projections. Merge includes H3K27ac (blue) and FISH (magenta). Scale bar = 3 µm. (B) Quantification of MN LmnB1 intensity normalized to nucleus intensity. Lamin B1 intensity correlates with chromosome length, not gene density. Mean from each replicate shown (color squares) with individual measurements (gray circles). One-way ANOVA, P-value < 0.0001; pairwise comparison with Bonferroni adjustment, P-value < 0.01, n.s. P > 0.05; N = 3, n = (33, 29, 29, 37, and 43). (C) LmnB1 intensity correlates with MN area for single and multiple chromosome MN. Chromosome number determined by CREST signal. Dotted line indicates equal MN and nucleus LmnB1 intensity. Spearman’s correlation (solid line) r = 0.63, P-value < 0.0001. (B) N and n for single chromosomes same as panel (B). For multi-chromosome MN, N = 3, n = (41, 9, 2). (D) Maximum intensity projections of Nup133 foci on intact single chromosome MN. MN integrity determined by H3K27ac+ signal (not shown). Scale bar = 3 µm. (E) Quantification of Nup133 density (foci number/area) on MN normalized to nucleus density. Mean from each replicate shown (color squares) and individual measurements (gray circles). One-way ANOVA, P-value < 0.0001; Bonferroni adjusted pair-wise comparison, *** P-value < 0.001, n.s. P > 0.05. N = 3, n = (21, 23, 24, 24, and 21). (F) Nup133 density correlates with MN surface area for single and multiple chromosome MN. Spearman’s correlation (solid line), r = 0.59, P-value < 0.0001. (E) N and n for single chromosomes same as panel (E). For multi-chromosomes, N = 3, n = (16, 2). (G) Example images of nuclear lamina organization in intact single chromosome MN containing either HSA-18 or -19 labeled with antibodies to lamin A (LmnA). Left = maximum intensity projections of bottom half of z-stack (z-step size = 0.15 µm). Middle = 3D skeletonization of lamin A structure. Right = detected nuclear lamina gaps (green, pink). Detected gaps were filtered by size (not shown) and intensity. Only gaps where difference between mean fluorescent intensity inside the gap compared with outside was <0.5 (green) were retained. Scale bar = 2 µm. (H) Quantification of nuclear lamina organization in single chromosome intact MN. Chi-square; P-value > 0.05. N = 3, n = (25, 23, 21, 26, and 20).
Fig 4: Seh1 depletion affects the association of the GATOR2 complex with mitotic chromosomes. (A) Seh1 depletion across different experiments only mildly affects kinetochore levels of Nup107 complex in chromosomes of DT40 cells. Profile plot showing the behaviour of Nup107 complex members (Nup133, Nup107, Nup85, Nup96, Nup160, Elys, Nup37, Nup43, Sec13) (red line). (B) Seh1 depletion strongly affects association of the GATOR2 complex with chromosomes in DT40 cells. Mio is shown as a red line while WDR24 and WDR59 are shown as blue lines.
Fig 5: Nuclear envelope composition of micronuclei containing one or multiple chromosomes.(A) Lamin B1 (LmnB1, green) image is a single slice of intact MN (H3K27Ac+, blue) and corresponding nucleus. Merged image is a maximum intensity projection with H3K27Ac, FISH (red), and CREST (gray) labeling added. (B) Example images of Nup133 (gray) labeling on intact MN (H3K27ac+, not shown), and corresponding nucleus. Merged image is of DAPI (blue), FISH, or CREST (red). All images are maximum intensity projections. In zoomed imaged of the HSA 1–containing MN, arrowheads denote areas lacking Nup133 foci, consistent with lamina gaps. (C) Example of an intact MN (H3K27ac+) containing two centromeres (CREST foci) and lamin A (LmnA, gray) gaps. Images are maximum intensity projections of the top half of the z-stack. Merged image is a full maximum intensity projection of DAPI (blue), H3K27ac (green), CREST (red), and lamin A. (D) Area of individual nuclear lamina gaps in single chromosome intact MN of indicated identity. Line indicates the median. One-way ANOVA P-value < 0.01; Bonferroni adjusted pair-wise comparison, **P-value < 0.01, *P-value < 0.05; N = 3, n = (55, 74, 52, 66, and 24). (E) Quantification of nuclear lamina gap number per each intact single chromosome MN with at least one gap, normalized to MN area. N = 3, n = (25, 23, 21, 26, and 20). All scale bars = 10 and 3 µm (zoomed image).
Supplier Page from Abcam for Anti-NUP133 antibody [EPR10808(B)]