Fig 1: Chromatin tracing reveals the fine folding architecture of inactive and active X chromosomes. (Target region: ChrX: 76,800,000–77,640,000, hg18). A Schematic illustration of the chromatin tracing approach (i), raw images of the chromatin tracing (ii), co-immunofluorescence of mH2A1 and co-RNA FISH of XIST (iii), and a reconstructed chromatin trace showing two domains separated by a white dashed line (iv). In ii, the white dashed line in the top panel outlines a cell nucleus. The yellow boxed region containing a copy of the targeted chromatin region is shown in the lower panels of ii. The four rows of panels correspond to Hybs-0, 1, 2, and 14 as indicated on the left. B Mean spatial distance matrix of the traced genomic region calculated from pooled X chromosome copies including both active and inactive X’s in IMR-90 cells. C Hi-C contact frequency matrix of the same genomic region as in B. CTCF and RAD21-binding peaks are illustrated in blue and pink respectively. D Comparison of mean spatial distance from chromatin tracing with contact frequency measured by Hi-C. E Separate mean spatial distance matrices from inactive (left) and active (right) X chromosomes. The yellow lines in B and E represent the ensemble TAD boundary
Fig 2: Effects of H2AFY knockdown on cell cycle and migration in HCC cells. (A) Cell cycle detected by flow cytometry in HepG2 and Hep3B cells after H2AFY knockdown. (B–D) Representative images of transwell (200×) and wound healing assays (40×) in HepG2 and Hep3B cells, and the quantitative result following H2AFY knockdown. (E) Western blot analysis of cell cycle, apoptosis, EMT related molecular markers in HepG2 and Hep3B cells transfected with H2AFY-shRNA or the negative control. (F) STAT3 signaling pathway was significantly enriched in high-H2AFY expression patients. (G) Evaluation of p-STAT3 and STAT3 expression in HepG2 and Hep3B cells after transfecting H2AFY-shRNA ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: Correlation of H2AFY expression and 22 immune cell types in HCC based on CIBERSORT. (A) The relative fraction of 22 immune cell types in TCGA-LIHC cohort. (B) The heat map showing relative immune cell fraction of HCC patients (C) Violin plots showing the difference of 22 immune cell types between high- and low-H2AFY expression patients *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 4: Down regulation of LMNB1 results in redistribution of macroH2A1 in mouse hepatocytes.(a) Knockdown efficiency as tested by qRT-PCR. Relative expression level of the scramble control was set as 1. (b) Total protein levels as shown by western blots using whole cell extract. B-Actin was used as loading control. (c) MacroH2A1, histone modifications (H3K27me3 and H3K9me3) and total histone H3 detected by western blots using histone extracts, Both H3 western blot and Comassie Blue Brilliant staining (CBB) are as loading controls. (d) Immunofluorescence of LMNB1 (green) and macroH2A1 (red) upon LMNB1 knockdown. Scale = 10 µm.
Fig 5: LMNB1 associates with histone variants macroH2A1.(a) Validation of proteomic data with western blotting. Whole cell extract (Input) and captured biotinylated proteins (BioID) were blotted with the indicated antibodies, in presence of biotin. Starting materials were equal for the samples with (+) and without (-) dox. GAPDH was used as a negative control. (b) Test of macroH2A1-LMNB1 association by BioID using macroH2A1 as the bait. Whole cell extract (Input) and macroH2A1 BioID products in Hela cells were blotted with the indicated antibodies. GAPDH was used as a negative control. (c) Sub-cellular localization of macroH2A1 or H3K4me3 (red) and LMNB1 (green) were shown by immunofluorescence and super-resolution microscope (SIM) in AML12. DNA was labeled with DAPI (blue). Scale = 10 µm.
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