Fig 2: 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 3: SETDB1 LOF changes the shape and mechanical properties of the A549 cell nuclei (A) Representative DAPI-stained nuclei of control (cyan) and SETDB1LOF A549 (pink), and NHBE (grey) cells showing different nuclear morphology. Numbers denote the nuclear irregularity index of the corresponding nucleus. Bar, 10 µm. (B) Quantification of irregularity index (left panel), circularity index (middle panel), and nucleus area (right panel) for control (cyan), SETDB1LOF A549 (pink), and NHBE cells (grey). Each point represents the measurement for individual nuclei, horizontal lines correspond to median, error bars represent the interquartile range (ns, not significant; ****P-value < 0.0001, Kruskall-Wallis test with Dunn's correction for multiple comparisons). (C) Quantification of NE fluctuations for control (cyan) and SETDB1LOF (pink) A549, and NHBE cells (grey). Each point represents the average for each individual cell on 2 to 4 kymographs of the square root of the NE mean square displacement relative to its mean position; horizontal lines correspond to the median, error bars represent the interquartile range (ns, not significant; ****P-value < 0.0001, one-way ANOVA–Kruskal–Wallis test). (D) Quantification of DNA displacement for control (cyan) and SETDB1LOF (pink) A549, and NHBE nuclei (grey). Each point represents the average for each individual cell on 10 timeframes of the displacement between two consecutive frames; horizontal lines correspond to the median, error bars represent the interquartile range (ns, not significant; ****P-value < 0.0001, one-way ANOVA- Kruskal–Wallis test). (E) Representative particle image velocimetry (PIV) of DNA within nuclei in control and SETDB1LOF A549, and NHBE cells showing the overall flow of DNA displacement. Scale bar, 5 µm. (F) Graphical representation of the SETDB1 LOF-induced effects in lung adenocarcinoma cells.

Fig 4: 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 5: SETDB1 is required for MPP8 recruitment in mESCs, while H3K9me3 is dispensable for maintaining LINE1 repression.a, b MPP8 (a) and H3K9me3 (b) ChIP-qPCR at two MPP8 binding sites that overlap LINE1 elements (L1_mus3, Kcnq1ot1 locus), L1Md_F2, chr. 2), two non-LINE1 MPP8 binding sites (Srrm2, Zfp617) and two negative control (NegCtrl) loci not bound by MPP8 (Utp6, Pmp22) in indicated cell lines grown in 2i/LIF. E14 and TT2 cells served as parental control for Suv39h1/h2 dKOs and G9a/Glp dKOs, respectively. Conditional Setdb1 KO (Setdb1 cKO) cells were treated with 500 nM 4-hydroxytamoxifen (OHT) for 3 days (n = 2 independent experiments). c, d Representative genome browser tracks of H3K9me3 ChIP-seq in indicated Mpp8mAID; OsTIR1 cell lines grown in 2i/LIF ± 500 µM IAA for 6 h (c) or 48 h (d); MPP8 ChIP-seq in Mpp8mAID; OsTIR1 cells ± 500 µM IAA for 16 h; DNA methylation profiles in ESCs grown under serum/LIF or 2i/LIF culture conditions (0 = unmethylated, 1 = fully methylated CpG sites; data taken from Habibi et al.35); Repeatmasker track showing the location of relevant LINE1 elements. e Model for MPP8-mediated maintenance of stem cell self-renewal in ground-state pluripotent stem cells. In wild-type MPP8-expressing mESCs MPP8 is recruited to chromatin by SETDB1. MPP8 binds to TASOR through its C-terminal part, enabling the integrity of the core complex, and to chromatin and SETDB1 through the N-terminal part containing the chromodomain. This configuration allows maintenance of LINE1 repression and hence stem cell self-renewal of mESCs. Removal of the C-terminal part evokes destabilization of the complex as binding of MPP8 to TASOR is lost, leading to de-repression of evolutionary young LINE1 elements and loss of stem cell identity. In cells that lack MPP8’s N-terminal part stable binding of MPP8 and TASOR is lost from chromatin. MPP8 can no longer bind to SETDB1 and maintenance of H3K9me3 is lost. However, the intact HUSH core complex continuously represses the LINE1 elements and support self-renewal of mESCs, demonstrating that stable association with chromatin and maintenance of H3K9me3 are not required. Source data are provided as a Source Data file.