Figure 5: H3.3cs1-induced senescence is mediated by decreased transcriptional output of cell cycle genes and reduced H3K4me3.

(a) Heat map showing gene expression changes of 914 genes as determined by RNA-seq in H3.3cs1-induced senescence (early and late time points) relative to those that are altered in OIS (versus growing). Note the overlap of downregulated genes between H3.3cs1-induced senescence and OIS. (b) (Top) venn diagram of genes downregulated in OIS, H3.3cs1 (early) and H3.3cs1 (late) as compared with growing IMR90s. Fisher exact test; P=2.418E−117 OIS versus H3.3cs1 (early); P=3.477E−162 OIS versus H3.3cs1 (late); P=2.404E−117 H3.3cs1 (early) versus H3.3cs1 (late). (Bottom) venn diagram of genes upregulated in OIS, H3.3cs1 (early) and H3.3cs1 (late) as compared with growing IMR90s. Fisher exact test; P=3.414E−4 OIS versus H3.3cs1 (early); P=2.883E−7 OIS versus H3.3cs1 (late); P=1.783E−7 H3.3cs1 (early) versus H3.3cs1 (late). (c) ChIP Enrichment Analysis (ChEA) of H3.3cs1-induced senescence (top) and OIS (bottom)-downregulated genes. Note that E2F family members and KDM5B (JARID1B) gene targets are enriched in both. The −log of the P value associated with each term is plotted. (d) Venn diagrams illustrating the overlap between H3.3cs1-downregulated genes and genes that lose H3K4me3 in senescence and JARID1A/1B-regulated genes in senescence¹⁵. Fisher exact test of P values indicated in the diagram. (e) ChIP-quantitative PCR for H3K4me3 at cell cycle genes and SASP-related genes in IMR90s expressing H3.1FL (blue) or H3.3cs1 (red). Two regions near the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter used as positive controls; intergenic regions used as negative controls. Fold enrichment of H3K4me3 ChIP/input is plotted. Mean±s.e.m.; two-tailed paired sample t-test; P values as indicated.