Extended Data Fig. 6: NSD3(T1242A)-mediated H3K36me2 in LUSC oncogenic reprogramming.
From: Elevated NSD3 histone methylation activity drives squamous cell lung cancer
a, Independent CUT&RUN replicate for H3K36me2 as in Fig. 3i. b, Normalized gene expression levels from the indicated groups from PSCN cell line RNA-seq datasets. dDEGs, downregulated DEGs; uDEGs, upregulated DEGs, from Extended Data Fig. 5j. P values determined by two-tailed robust t-test (Methods). dDEGs n = 234, uDEGs n = 229, all genes n = 16,091. Boxes, 25th to 75th percentiles; whiskers, minimum to maximum; centre line, median. c, CUT&RUN profile for H3K36me2 and H3K27me3 in PSCN cells with or without sgNSD3 over average of intergenic regions on a genome-wide scale. d, CUT&RUN profile for a monoclonal IgG (against GFP) as in Fig. 3i for all genes and dDEGs as indicated in PSCN cells with or without sgNSD3 is shown as a negative control. e, Genome browser view of CUT&RUN signals for H3K36me2 and H3K27me3 on the indicated genes and conditions. Arrow indicates the direction of gene transcription. f, Loss of occupancy of NSD3(T1242A) and H3K36me2 at the indicated NSD3 target genes (Irgm1 and Prkaa2) upon NSD3 depletion. Btg2 and Gadd45g are not NSD3 target genes and are shown as control regions that do not change in response to NSD3 depletion. ChIP–qPCR analysis of V5–NSD3(T1242A) (left) and H3K36me2 (right) in the bodies of the indicated genes. The data were plotted as per cent enrichment relative to input. Mean ± s.d. from three independent experiments. P values determined by two-tailed unpaired t-test. g, NSD3(T1242A) and H3K36me2 occupy the promoter and gene body regions of target genes Irgm1 and Prkaa2. Top, schematic of general gene structure and site of primers for Irgm1 (left) and Prkaa2 (right) gene loci. ChIP–qPCR analysis of V5–NSD3(T1242A) (left) and H3K36me2 (right) in promoter (p1), gene body (p2) and transcription end site (TES; p3) regions of target genes. TSS, transcription start site. The data were plotted as per cent enrichment relative to input. Mean ± s.d. from three independent experiments. P values determined by two-tailed unpaired t-test. h, ChIP–qPCR analysis of NSD3(T1242A) (left) and H3K36me2 (right) occupancy at the gene body regions of Irgm1 and Prkaa2 gene loci in reconstituted cells as described in Extended Data Fig. 5l. The enrichment was normalized to the sgControl sample and presented as fold change relative to the sgControl sample. Mean ± s.d. from three independent experiments. P values determined by one-way ANOVA with Tukey’s post hoc test.
