Loss of HMGB2 allows for spreading of heterochromatin and promotes the inclusion of SASP gene loci in SAHF. (A) Cross-referencing of HMGB2 ChIP-Seq data and H3K9me3 ChIP-Seq data at the IL8 locus. Green indicates enrichment of HMGB2 or H3K9me3 binding in senescent cells, whereas orange indicates depletion of HMGB2 or H3K9me3 binding in senescent cells. The black bar indicates the IL8 genomic locus. (B) IMR90 cells were infected with retrovirus encoding oncogenic RAS alone or in combination with a lentivirus expressing an shRNA to HMGB2. Cells were selected for 3 d with 3 µg/ml puromycin. 4 d later, cells were stained for SAHF using DAPI. Bars, 10 µm. (C) Quantification of B. (D) Same as B, but 3D DNA-FISH was performed with a BAC containing the IL8 gene locus. White dashed lines indicate the nucleus. Bars: (top) 5 µm; (inset) 0.5 µm. (E) Quantification of 3D DNA-FISH. The distance between IL8 loci and the nearest SAHF was determined using ImageJ software (au, arbitrary units). At least 50 nuclei were quantified. Horizontal bars denote the comparison between RAS alone and RAS/shHMGB2. (F) Same as B, but H3K9me2 ChIP was performed, and H3K9me2 binding to IL8 was determined by quantitative PCR and normalized to a nonpeak region (NPR) control. (G) Scheme of how HMGB2 binds to SASP genes to promote their transcription (left). Loss of HMGB2 allows for spreading of repressive senescence-associated heterochromatin to inhibit SASP gene expression (right). 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; #, P < 0.05 versus RAS.