Extended Data Fig. 4: Analysis of models displaying altered H3K27me3 confinement in stem cells and cancers.
A. Western blot (left) and mass spectrometry (right) measurements of H3K36me2 and H3K27me3 abundance in WT and Nsd1-KO mESCs, revealing quantitative gain of H3K27me3 upon H3K36me2 loss. A representative western blot of n = 3 replicates is shown, and mass spectrometry data is from n = 2 biological replicates. B. Confinement scores reveal H3K27me3 focal distribution of WT mESCs is lost in Nsd1-KO lines. C. Density plot showing differential CGI enrichment of H3K27me3 (x-axis), RING1B (y-axis), and CBX2 (color code) between WT and Nsd1-KO mESCs. Each dot represents a CGI and the differential enrichment is plotted as log2 ratio of Nsd1 KO/WT. Loss of CBX2 binding correlates with decreases in H3K27me3 and Ring1b at CGIs upon Nsd1-KO. D. Average signals of transcription and chromatin features for CGIs & promoters in each of the four clusters in mESCs (see Fig. 4d), demonstrating the characteristic chromatin state of each cluster. Symbols indicate data sources: * = Chen 2022, \ = Kundu 2017, ^ = Healy 2019, ‘ = Mas 2018, ° = ENCODE, no symbol = this study. E. Confinement scores of Vector or EZH2-Y641N expressing mESCs for H3K27me3 CUT&RUN profiles. F. Genomic distribution of H3K27me3 (ChIP–seq coverage tracks in units of counts-per-million-alignments) at representative loci in germinal center B cells (Histone H1 double knockout vs WT) and acute lymphoblastic leukemia cells (NSD2-E1099K vs WT-resembling NSD2 loss by shRNA knockdown), demonstrating distinctive profiles of confined versus diffuse H3K27me3. CpG islands are denoted with green dashed bars. G. Measure of H3K27me3 ChIP–seq signal confinement (fragment cluster score at 1 kb separation, computed using the tool ‘ssp’, see methods), comparing confined (H1-KO, NSD2 mutant) versus diffuse profiles. Individual data points correspond to a replicate, with connected points indicating replicates from the same batch; connections not linking points indicate that multiple replicates were sequenced in a batch, and so the links are drawn between the average value per condition. H. Pile-up of Hi-C interactions among H3K27me3-enriched CpG islands, as defined above, portraying average pairwise contact strength between such regions (in units of enrichment, that is, observed / expected). Punctate enrichment signal in the center indicates elevated long-range interaction anchored at H3K27me3-enriched CGIs in cells with confined H3K27me3.
