SETD2 ablation promotes KRASG12C-driven lung tumorigenesis in vivo.

A, Schematic of the Setd2LoxP/LoxP conditional allele. In the presence of Cre recombinase, exon 3 is deleted to disrupt Setd2 expression. B, Confirmation of Setd2LoxP/LoxP conditional allele by PCR on DNA isolated from mouse tail biopsies from indicated mouse genotypes, expected product sizes are marked. C, Schematic of generation of LUAD model driven by Cre-recombinase inducible conditional oncogenic KrasG12C mutation and deletion of p53 (KCP) and Setd2 (KCP;Setd2). D, Experimental design to assess effects of SETD2 ablation on LUAD pathogenesis in KCP model. E, Representative HE and IHC staining with indicated antibodies of lung tumors from KCP and KCP;Setd2 mutant mice at 10 weeks after Ad-Cre induction (n=6/group). H3K36me3 serves as a proxy of SETD2 ablation in tumor cells in KCP;Setd2 mutant mice. P values determined by two-tailed unpaired t-test; boxes: 25th to 75th percentile, whiskers: min. to max., center line: median; scale bars: 100 µm. F-I, Quantification of tumor number, tumor burden, proliferation (Ki67+) and cell death (cleaved Caspase3+) in KCP and KCP;Setd2 samples as in e. J, Kaplan-Meier survival curves of KCP control (n=8, median survival 151 days) and KCP;Setd2 mutant mice (n=8, median survival 92 days) mutant mice. P values determined by the log-rank test.

SETD2 methylates unmodified, H3K36me1-, and H3K36me2-modified nucleosomes, but not those bearing H3K36me3

A, Schematic of SETD2 domain structure, with the catalytic region used in the biochemical assays (SETD2SET) indicated. B, SETD2SET in vitro methylation reactions with radiolabeled 3H-SAM on unmodified (H3), H3K36me1, H3K36me2, or H3K36me3 recombinant nucleosomes (rNuc) substrates as indicated. K36me: methylated H3K36. Top, autoradiography; bottom, Coomassie blue staining. C, Methylation (MTase-Glo) assays (see methods) with enzyme and substrates as in (B). SAH concentration serves as a measurement of methylation. Activity is normalized to control conditions. Data are means ± SEM from 3 independent replicates. P values determined by one-way ANOVA. D, Methylation reactions as in (B) using NSD2SET as the enzyme and the indicated rNucs as substrates. Top, autoradiography; bottom, Coomassie blue staining. E, Western blot analysis with the indicated antibodies on the indicated rNucs as in (B). F, SETD2SET methylation assays as in (B) using non-radiolabeled SAM and methylation detected by Western analyses using the antibodies characterized in (E). H3 is shown as a loading control.

Histone poly-acetylation enhances SETD2SET activity

A, Schematic showing the tetra-acetylated rNuc tested in the study. B, In vitro methylation reactions with SETD2SET as in Fig. 2B using the indicated tetra-acetylated rNucs. Top, autoradiography; bottom, Coomassie blue staining. C, MTase-Glo assays as in Fig. 2C using the indicated tetra-acetylated rNucs. Data are means ± SEM from 12 independent replicates. P values determined by one-way ANOVA. D, Methylation reactions as in (B) using NSD2SET as enzyme. Top, autoradiography; bottom, Coomassie blue staining. E, Methylation reactions as in (B) using DOT1L as enzyme. Top, autoradiography; bottom, Coomassie blue staining.

H3K27 acetylation enhances SETD2SET binding and activity

A, Schematic showing the different histone H3 acetylated rNuc tested in the study. B, In vitro methylation reactions with SETD2SET as in Fig. 2B using the indicated acetylated rNucs. Top, autoradiography; bottom, Coomassie blue staining. C, MTase-Glo assays as in Fig. 2C using the indicated acetylated rNucs. Data are means ± SEM from 6 independent replicates. P values determined by one-way ANOVA. D, Nucleosome pulldown assay using biotinylated unmodified or H3K27ac nucleosomes as bait to assess binding to GST-SETD2SET. Bound protein was detected by Western blotting with indicated antibodies. E, Electrophoretic mobility shift assay (EMSA) with increasing concentrations of SETD2SET (0–5 μM) incubated with the indicated rNuc (250 nM) and SETD2SET binding to rNuc detected by staining DNA with Sybr Gold.

Histone ubiquitination does not affect SETD2SET activity

A, Schematic showing the different histone ubiquitinated rNuc tested in the study. B, In vitro methylation reactions with SETD2SET as in Figure 2B using the indicated ubiquitinated rNucs. Top, autoradiography; bottom, Coomassie blue staining. C, SETD2SET methylation assays as in Fig. 2F using non-radiolabeled SAM and methylation detected by Western analysis using the indicated antibodies. H3 is shown as a loading control. Note that H3K14ub and H3K18ub interfere with the αH3K36me2 antibody from recognizing H3K36me2. D, MTase-Glo assays as in Fig. 2C using the indicated rNucs. Data are means ± SEM from 6 independent replicates. P values determined by one-way ANOVA. E, Methylation reactions as in (B) using NSD2SET as enzyme. Top, autoradiography; bottom, Coomassie blue staining.