Figure 5

SIRT6 deacetylates H3K56 and is required for recruiting RNAP II to the HO-1 gene promoter. (A) Co-IP assay using protein extracts from HEK293T cells expressing Flag-SIRT6 indicated that SIRT6 formed a protein complex with RNAP II and TAF II-p135. (B) ChIP-qPCR performed in SIRT6^−/− hMSCs transduced with Flag-SIRT6 or Flag-luciferase (control) indicated association of SIRT6 with HO-1 promoter and enhancers. Data were presented as mean ± SEM, n = 3, *P < 0.05, **P < 0.01. (C) ChIP-qPCR assay showing SIRT6-dependent recruitment of RNAP II at HO-1 promoter. Data were presented as mean ± SEM, n = 3, *P <0.05. (D) Western blotting analyses of H3K56Ac, H3K9Ac, and H3K4me3 in WT and SIRT6-deficient hMSCs. Histone 3 (H3) was used as the loading control. (E) Immunofluorescence (left) and statistical (right) analyses of H3K56Ac levels in WT and SIRT6-deficient hMSCs. Scale bar, 100 μm. (F) ChIP-qPCR analysis of the enrichment of H3K56Ac and H3K9Ac at HO-1 promoter in WT and SIRT6-deficient hMSCs. Data were presented as mean ± SEM, n = 3, NS, not significant, **P < 0.01. (G) ChIP-qPCR analysis of H3K56Ac at HO-1 promoter in WT or SIRT6-deficient hMSCs transduced with lentiviral vector encoding SIRT6 (WT), SIRT6 (HY), or luciferase (control). Data were presented as mean ± SEM, n = 3, **P < 0.01.