Fig 2: SETDB1 LOF leads to H3K9me3 redistribution between A/B compartments and affects both large-scale and local chromatin topology (A) Hi-C interaction matrices at 100-kb resolution showing examples of chromatin compartment switches in control and SETDB1LOF A549 cells for Chr. 4: 73.0–115.0 Mb (left panel) and Chr. 12: 102.0–133.0 Mb (right panel). Switching regions are highlighted with grey boxes and black arrows. Eigenvector (PC1 track) for compartments A (red) and B (blue) is shown. Lower panels: ChIP-seq profiles (RPKM- and input-normalized ratio) for H3K9me3 and H3K4me3 in control and SETDB1LOF A549 cells are shown according to the UCSC genome browser. (B) Scatter plot of PC1 values at 100-kb resolution for the control and SETDB1LOF A549 cells. S – total length of the regions that switched compartment state (absolute z-score of ?PC1 > 1.645) from B to A (red) or A to B (blue). (C) Pie chart showing the dynamics of compartment bins in SETDB1LOFvs. control A549 cells. N – number of 100-kb bins. (D) Distribution of RPKM-normalized H3K4me3 and H3K27ac ChIP-seq signals within stable A and B compartment bins and in switched from B to A, or A to B compartment bins in control (cyan) and SETDB1LOF (pink) A549 cell lines (ns, not significant; **P-value < 0.01; ****P-value < 0.0001, Mann–Whitney U-test). (E) Distribution of RPKM-normalized H3K27me3 and H3K9me3 ChIP-seq signals within stable A and B compartment bins and in switched from B to A, or A to B compartment bins in control (cyan) and SETDB1LOF (pink) A549 cell lines (**P-value < 0.01, ****P-value < 0.0001, Mann–Whitney U-test). (F) Left: saddle plots of contact enrichments in cis and in trans between 100-kb genomic bins belonging to A and B compartments in control and SETDB1LOF A549 cells. Numbers represent the compartment strength values calculated as described in (76). Right: subtraction of A549 SETDB1LOF from control saddle plots for cis and trans contacts. (G) Hi-C interaction matrices at 20- (top) and 5-kb (bottom) resolutions showing the PCDH gene cluster on Chr. 5: 135.5 – 145.0 Mb (left panels) and ZNF genes on Chr. 19: 50.0–58.0 Mb (right panels). Strengthened TAD boundaries in the SETDB1LOF as compared to control A549 cells are highlighted with black arrows. Eigenvector (PC1 track) for compartments A and B is shown in red and blue, respectively. Genes, RNA-seq profile (CPM-normalized coverage), ChIP-seq profiles (RPKM- and input-normalized ratio) for H3K9me3, H3K27me3, H3K4me3, H3K27ac, and CTCF in control and SETDB1LOF A549 cells are shown according to the UCSC genome browser.

Fig 3: Role of FOXD3 in SUV39H1-mediated regulation of MSR and MERVL STRING analysis to predict putative interaction between FOXD3, SUV39H1, SUV39H2, SETDB1 and SETDB2. FOXD3 was significantly predicted to interact with SUV39H1 and SETDB1 (P-value < 0.01). The criteria used for predicting interaction are depicted.GFP immunoprecipitation analysis using Foxd3 cKO mESCs expressing GFP-FOXD3. Immunoblot results using antibodies against SUV39H1, SETDB1 and GFP are depicted. The experiment was repeated three times using biological replicates.Volcano plot depicting significantly upregulated and downregulated repeats in Suv39h dn ES cells compared with wild-type ES cells determined by RNA-Seq analysis of nuclear RNA. MSR (GSAT_MM), MERVL-int, L1MdA and MT2_Mm are labelled.ChIP-qPCR depicting enrichment of H3K4me3 (top panel) and H3K27me3 (bottom panel) over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data are represented as average percentage input of 3 biological replicates. Error bars represent SEM (*P < 0.05 paired t-test).Schematic depicting the location of Foxd3 siRNAs (pink) along the Foxd3 locus (upper panel). Immunofluorescence detected by FOXD3 antibody in Foxd3i cells. Nuclei are stained with DAPI. Scale bar = 10 µm (lower left). RT–qPCR analysis of Foxd3 expression in control and Foxd3i cells. Data are represented as fold change compared with control (scrambled) after normalisation to Gapdh. N = 3 biological replicates (lower right). Error bars represent SEM. (*P < 0.05 paired t-test).Bar graph depicting Foxd3 expression at different stages of embryonic development. Y-axis depicts average FPKM from 4 independent single cell RNA-Seq data sets. Error bar represents standard deviation. (*P < 0.05, **P < 0.005, unpaired t-test).Bar graph depicting relative Foxd3 expression in MERVL-positive and MERVL-negative cells. The data are represented as the average read counts obtained from RNA-Seq data from three biological replicates. Error bars represent SEM. Statistical test: unpaired t-test. Source data are available online for this figure.

Fig 4: SETDB1 LOF induces H3K9me3 and H3K27me3 redistribution within the nuclear space and along the genome (A) Representative immunofluorescence images of control and SETDB1LOF A549 cells stained with anti-H3K9me3, anti-Lamin A/C, and anti-H3K27me3 antibodies. DNA was labeled with DAPI. Scale bar, 10 µm. (B) Nuclear distribution of H3K9me3, Lamin A/C, and H3K27me3 in control (cyan) and SETDB1LOF (pink) A549 cells. Intensities are plotted as a function of distance from the nuclear center (N = 3 independent experiments; mean intensity ± SEM; 92–104 of cells analyzed; ***P-value < 0.001, Kolmogorov–Smirnov test). The region with maximum Lamin A/C intensity is indicated with a grey dashed line. (C) Pile-ups of the H3K9me3 and H3K27me3 enrichments at the differentially expressed genes in control and SETDB1LOF A549 cells. Up- and down-regulated genes are shown in red and blue, respectively. (D) Pie chart of the genome-wide dynamics of H3K9me3-enriched regions in SETDB1LOF versus control A549 cells. The percentages indicate the fraction of changed H3K9me3 peaks from the total peaks number. S – the total length of changed H3K9me3 regions. (E) Correlation between H3K27me3 and H3K9me3 enrichment in 100-kb genomic bins in control and SETDB1LOF A549 cells. Spearman's correlation coefficients are indicated for bins with increased and decreased H3K9me3 levels separately. (F) Pile-ups of the H3K9me3 and H3K27me3 enrichment at increased H3K9me3 regions in control and SETDB1LOF A549 cells. (G) Pie charts of the overlap between increased, decreased, and stable H3K9me3 enriched regions with constitutive LADs from (62).

Fig 5: SETDB1 loss-of-function compromises oncogenic potential of lung adenocarcinoma cells decreasing migration and proliferation potential, and affecting 3D genome organization.