HMGB2 preferentially binds to SASP gene loci in senescent cells. (A) HMGB2 ChIP-Seq data (GEO accession no. GSE85057) were cross-referenced with publically available microarray datasets. 89 genes were bound by HMGB2 and had increased expression in senescent cells. The significance of gene overlap was estimated and showed a significant enrichment in HMGB2-bound up-regulated genes compared with a random set of genes (P = 0.0027). The identified genes were subjected to pathway enrichment analysis by DAVID software. (B) Heatmap of SASP genes preferentially bound by HMGB2 during senescence. The ratio of HMGB2 ChIP signal and expression of the indicated genes in senescent (S) and control (C) is listed in dataset #1 (GEO accession no. GSE40349) and dataset #2 (GEO accession no. GSE60652). Green represents the signal intensity in the HMGB2 ChIP-Seq in senescent cells versus control. Red represents up-regulated genes, whereas blue represents down-regulated genes. Yellow represents the false discovery rate (FDR). (C) Representative tracks from HMGB2 ChIP-Seq for IL1β, IL8, and IL6. (D) IMR90 cells were infected with retrovirus encoding oncogenic RAS to induce senescence or controls. Cells were selected for 3 d with 1 µg/ml puromycin. 4 d later, IL1β, IL8, and IL6 mRNA expression was determined. B2M was used as an internal control. (E) Same as D, but HMGB2 ChIP was performed, and HMGB2 binding to IL1β, IL8, and IL6 was determined by quantitative PCR and normalized to a nonpeak region control. (F) Expression of IL1β and IL6 mRNA was determined by qRT-PCR in senescent G4 mTerc^−/− and wild-type (WT) control mouse ear fibroblasts. B2M expression was used as an internal control. (G) Same as F, but HMGB2 ChIP was performed. HMGB2 binding to IL1β and IL6 was determined by quantitative PCR and normalized to a nonpeak region (NPR) control. For all panels, graphs shown are the mean and SEM of triplicates from a representative experiment that was independently repeated at least three times. *, P < 0.05 versus control.