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ARID1A spatially partitions interphase chromosomes

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Science Advances  22 May 2019:
Vol. 5, no. 5, eaaw5294
DOI: 10.1126/sciadv.aaw5294
  • Fig. 1 The SWI/SNF complex interacts with the condensin II complex.

    (A) Validation of the interaction between the SWI/SNF and condensin II complexes by co-IP in RMG1 cells with endogenously FLAG-tagged ARID1A. Nuclear fractions were subjected to IP using an anti-FLAG antibody and immunoblotting using the indicated antibodies. (B) Expression of the indicated SWI/SNF and condensin II subunits in ARID1A wild-type OVCAR429 and RMG1 cells with or without ARID1A knockout determined by immunoblotting. (C) Co-IP analysis between NCAPH2 and BAF155 using an anti-NCAPH2 antibody from nuclear extracts prepared from RMG1 cells treated with or without DNase I. (D) Venn diagram showing the overlap of ChIP-seq peaks among NCAPH2, ARID1A, H3K27ac, and H3K4me3 in RMG1 cells. (E) Heatmap clustering of ChIP-seq profiles of NCAPH2, ARID1A, SNF5, H3K27ac, and H3K4me3 in RMG1 cells. The number of binding sites in each of the four clusters is indicated. (F) Average profiles of the ChIP-seq signal for the indicated antibodies within four distinct clusters found by k-means clustering. Immunoglobulin G, IgG.

  • Fig. 2 ARID1A loss does not affect the interaction between the SWI/SNF complex and condensin II.

    (A) Parental control and ARID1A knockout (KO) RMG1 cells were subjected to co-IP analysis using an antibody against a core SWI/SNF subunit BAF155 and examined for interactions with the indicated subunits of the SWI/SNF and condensin I and II complexes by immunoblotting. Asterisks indicate the nonspecific bands in the immunoblotting. (B) Glycerol sedimentation (10 to 30%) assay of the SWI/SNF and condensin II complexes from parental control and ARID1A knockout RMG1 cells. The red box indicates the cosegregation of the SWI/SNF and condensin II complexes. (C) Sucrose sedimentation (10 to 50%) assay of the SWI/SNF and condensin II complexes in ARID1A-mutated TOV21G OCCC cells. The red box indicates the cofractionation of SWI/SNF and condensin II complexes.

  • Fig. 3 ARID1A loss redistributes the condensin II complex preferentially at enhancers.

    (A and B) Heatmap clustering of ChIP-seq profiles of ARID1A, NCAPH2, H3K27ac, and H3K4me3 in the indicated control (Ctrl) and ARID1A knockout RMG1 cells based on changes in NCAPH2 binding. The number of binding sites in each of the five clusters was indicated (A). Average profiles of the ChIP-seq signal for the indicated antibodies were generated on the basis of the five clusters (B). (C) Changes in NCAPH2 binding signal in parental control and ARID1A knockout RMG1 cells in regions with H3K4me3 or H3K27ac marks. Percentages represent proportion of binding sites that have at least a 1.2-fold change in binding signal cutoff indicated by blue and red lines. (D) Validation of NCAPH2 knockdown. RMG1 cells with inducible short hairpin RNA against the human NCAPH2 gene (shNCAPH2). Expression of NCAPH2, SMC4, ARID1A, and β-actin was examined by immunoblotting. DOX, doxycycline. (E) Same as (B) but for the H3K27ac mark in the indicated control and shNCAPH2 RMG1 cells. Note that the input is the same as those used in (B).

  • Fig. 4 ARID1A-driven condensin II redistribution correlates with changes in gene expression.

    (A) Venn diagram showing the overlap of changes in gene expression induced by ARID1A knockout or NCAPH2 knockdown in RMG1 cells determined by RNA-seq analysis. (B) Average gene expression changes show positive correlation with changes in NCAPH2 binding and gene expression induced by ARID1A knockout. (C and D) Schematic of 3C primers covering the enhancer and proximal promoter anchoring of the down-regulated CDH6 gene locus aligned with ChIP-seq tracks for H3K27ac and NCAPH2 in control and ARID1A knockout RMG1 cells (C). 3C reveals that ARID1A knockout in RMG1 cells elicits a significant reduction of the interaction frequency between the enhancer and the promoter of the CDH6 gene locus (D). (E and F) Same as (C) and (D) but for the up-regulated LAMA4 gene. Error bars represent means ± SD. ***P < 0.0001.

  • Fig. 5 ARID1A loss enhances TAD border strength and can drive compartment switching.

    (A) Changes in the strength of TAD borders induced by NCAPH2 knockdown or ARID1A knockout in RMG1 cells. (B) Average ChIP-seq peak intensity of ARID1A, NCAPH2, H3K27ac, and H3K4me3 across the TADs. (C) ARID1A ChIP-seq signal intensity at the TBs divided on the basis of the strength of TBs, which become stronger, unchanged, or weaker after ARID1A knockout. (D) Overall distribution of the indicated euchromatic A-compartments and heterochromatic B-compartment and compartments switch induced by NCAPH2 knockdown or ARID1A knockout. (E) Distribution of ARID1A and NCAPH2 ChIP-seq signal in regions with compartments switch compared to those without compartments switch induced by ARID1A knockout in RMG1 cells. (F) An example of a region on chromosome 3 (chr3) showing a B-to-A compartment switching that is associated with the HGD gene locus, as indicated by the box. (G) Validation of an increase in NCAPH2, a decrease in H3K9me2, and an increase in H3K27ac on the HGD locus that showed a B-to-A compartment switch by ChIP analysis using the indicated antibodies or an IgG control. ***P < 0.0001 and **P < 0.001.

  • Fig. 6 ARID1A loss promotes intermixing of small chromosomes.

    (A) Mean interchromosomal interaction changes across chromosomes. Number of chromosome pairs that showed significant [false discovery rate (FDR) < 5%] increase of interaction for each chromosome are indicated on the top of bars. Red bars highlight chromosomes with >50% significantly increased interchromosomal interactions. (B) Correlation between ARID1A ChIP-seq signal reads within significant peaks per megabase of chromosome length and changes in interchromosomal interaction. (C) NCAPH2 redistribution (relative change in number of NCAPH2 peaks in ARID1A knockout versus control RMG1 cells) across chromosomes. (D) Correlation between NCAPH2 redistribution and interchromosomal interactions. (E) Representative images of 3D chromosomal painting showing chr18 (green), chr19 (purple), and chr22 (white) in parental control and ARID1A knockout RMG1 cells. (F) Volumes of chr18, chr19, and chr22 (calculated on the basis of 3D chromosome painting from at least 790 nuclei) in parental control and two independent ARID1A knockout RMG1 clones. Error bars = mean with SD. (G and H) Distribution of 3D chromosome overlap area (G) or contact frequency (H) (from at least 747 nuclei) between chr19 and chr22 in parental control and ARID1A knockout RMG1 cells. Error bars = mean with SEM. P value was calculated by Mann-Whitney U test. Note that chr18 was excluded from the analysis in (B) to (D). Correlation was calculated by Pearson analysis. ***P < 0.001.

  • Table 1 Coordinates for the indicated paints for chr18, chr19, and chr22 used in the current study.
    ChromosomeStartEndTotal oligos
    1814,921,48678,016,66188,197
    19238,39259,093,25049,788
    2216,054,66351,220,47238,360

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaaw5294/DC1

    Fig. S1. The SWI/SNF complex colocalizes with the condensin II complex at enhancers.

    Fig. S2. ARID1A loss does not affect the interaction between the SWI/SNF and condensin II complexes.

    Fig. S3. ARID1A loss redistributes condensin II complex binding at enhancers to regulate gene expression.

    Fig. S4. ARID1A suppresses insulation of TADs.

    Fig. S5. ARID1A partitions chromosomal territories.

    Fig. S6. ARID1A loss increases chromosome and nuclei volume and promotes small chromosome intermixing in trans.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The SWI/SNF complex colocalizes with the condensin II complex at enhancers.
    • Fig. S2. ARID1A loss does not affect the interaction between the SWI/SNF and condensin II complexes.
    • Fig. S3. ARID1A loss redistributes condensin II complex binding at enhancers to regulate gene expression.
    • Fig. S4. ARID1A suppresses insulation of TADs.
    • Fig. S5. ARID1A partitions chromosomal territories.
    • Fig. S6. ARID1A loss increases chromosome and nuclei volume and promotes small chromosome intermixing in trans.

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