(A) Western analysis of SIRT6 protein in HeLa cells stably expressing SIRT6 shRNA (sh2)
(B) SIRT6 is required for H3K9 deacetylation at promoters of RELA target genes. ChIP with α-H3K9Ac and α-H3 antibodies was performed. H3K9 acetylation at RELA target gene promoters (mean ± s.e.) is shown relative to untreated control samples and normalized to total H3 levels.
(C) SIRT6 is required to limit RELA occupancy at the promoter of RELA target gene promoters. ChIP with α-RELA antibodies was performed following a 30 minute TNF-α pulse; RELA occupancy (mean ± s.e.) at promoters relative to untreated control samples is shown.
(D)-(E) SIRT6-mediated deacetylation of nucleosomes inhibits nucleosome binding to RELA. Western analysis of H3K9Ac levels on nucleosomes following incubation with SIRT6 in NAD-dependent deacetylation or mock reactions (D). The acetylated or SIRT6-deacetylated nucleosomes were used in nucleosome binding assays with increasing amounts of GST-RELA protein (E). Extent of nucleosome binding can be estimated by comparing levels of bound (**) and unbound (*) nucleosomes. Reduced binding is observed in the SIRT6-deacetylated samples (compare 10 ug RELA samples)