Fig 2: CPT inhibits KMT1A enzymatic activity in an in vitro reconstituted system(A) HMTase assay with GST-KMT1A, [3H]-SAM cofactor, and either GST or GST-H3(N) substrate as indicated. Reactions contained either increasing concentrations of CPT (0.25 μM, 0.5 μM, 1 μM, 4 μM, and 5 μM) or maximum volume of DMSO, as indicated. H3(N) was visualized by coomassie staining, and 3H-[Me]-H3(N) by autoradiography. (B) Same as (A) except reactions were carried out using 2.5 μM or 5.0 μM of CPT, CPT-11 and SN38 as indicated. (C) HMTase assay with reactions carried out similarly as in (A), except samples were precipitated on filter paper, washed, dried, and subjected to scintillation counting. Counts per minute (CPM) were normalized by subtraction of background signal as measured by a control reaction lacking enzyme. Error bars represent ±SEM from reactions performed in duplicate.

Fig 3: The 1–167 domain of Suv39h1 is responsible for promoting HP1α sumoylation.(a) Far-western blot using His-Suv39h1 full-length (WT) or truncated versions as baits and purified recombinant GST-Ubc9 as prey. We revealed bound GST-Ubc9 using anti-GST antibodies. Along each construct, interaction (+) or absence of interaction (−) with Ubc9 is indicated. (b) In vitro HP1α sumoylation assay. Top: western blot analysis of the sumoylation reaction mixture with anti-HP1α antibodies revealed sumoylated GST-HP1α (SUMO-1-GST-HP1α, box) and unmodified GST-HP1α. We performed quantitative analysis of this assay. The histogram shows the comparison of the ratio between SUMO-1-GST-HP1α and unmodified HP1α signal from western blot in the presence of Suv39h1-WT or truncated versions (Supplementary Fig. 5c). Error bars represent s.d. from three independent experiments. Bottom: results summary.

Fig 4: SUV39H1 increased DNMT3A expression in cervical cancer cells. a, b Detection the expression of SUV39H1, H3K9me3 and DNMT3A in SiHa and HeLa cells which overexpressed SUV39H1 by western blotting. c, d Western blotting analysis of SUV39H1, H3K9me3 and DNMT3A in SiHa and HeLa cells 48 h after transfection with siRNAs against SUV39H1. e Detection of the expression of H3K9me3 and DNMT3A in SiHa and HeLa cells by western blotting. f Schematic representation of the four regions of the DNMT3A promoter amplified in the ChIP-qPCR experiment. g, h Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-H3K9me3 ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for indicated four regions in the DNMT3A promoter in qPCR. The enrichment of H3K9me3 on DNMT3A promoter relative to IgG in SiHa and HeLa cells, and H3 against RPL30 was used as positive control. **P < 0.01

Fig 5: Suv39h1 induces constitutive heterochromatin a. Diagram showing the experimental design. b. A representative full z-series projection of 11 zygotes injected with HA-Suv39h1 mRNA, fixed and immunostained with anti-Suv39h1 and anti-HA 8 hours later from 3 independent experiments. Scale bar 10 μm. c. Summary of the dynamics of global levels of heterochromatic histone modifications in the preimplantation mouse embryo and the transient ectopic SUV39H1 expression. E, indicates embryonic day. d. Representative full z-series confocal projections for embryos manipualted as indiciated in a. The right-hand panel depicts a representative single confocal section of an individual nucleus indicated by the boxed region on the H3K9me3 panel. The white solid line highlights the segregation of the maternal and paternal genomes in the controls but not Suv39h1wt-injected embryos (see Supplementary Videos for 3D reconstructions). White dashed lines indicate the nuclear membrane. N = total number of embryos analysed across 6 independent experiments for Suv39h1wt and 2 for Suv39h1mut. Scale bar 20 μm and 10 μm for nuclear zooms. Where visible, the polar body is indicated by an arrow in the merge panel. e. Zygotes microinjected with dCas9-mClover plus sgRNAs targeting Major Satellites and HA-Suv39h1wt or HA-Suv39h1mut mRNA (18 hphCG) were fixed at the 2-cell stage (42h phCG) and immunostained with an anti-GFP antibody. A representative full z-series projection of the indicated number of embryos from 5 independent experiments is shown. Red arrows indicate DAPI-intense, major satellite-positive clusters. Scale bar 10 μm. f. Mean number (± S.E.M.) of CenpA foci per nuclei was performed by blind counting for the number of embryos in e. Statistical analysis was performed using the two-sided Mann-Whitney U test. g. As d. for H4K20me3. N = total number of embryos analysed across 3 independent experiments. The Suv39h1wt-injected group showed two patterns; either a strong increase around the nucleolar-like bodies (arrowheads) (group 1) or no H4K20me3 accumulation (group 2). The proportion of nuclei showing each pattern is indicated, determined through blind analysis. h. As d. for H3K64me3. N = total number of embryos analysed across 5 independent experiments for Suv39h1wt and 4 for Suv39h1mut. Statistical source data are shown in Source data fig. 4.