SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation

Abstract

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

Figure 1. SIRT7 is a chromatin-associated H3K18Ac-specific deacetylase

a, Western analysis showing chromatin association of SIRT7 in 293T and HT1080 cells. Biochemical fractions S2, S3, and P3 are enriched for cytoplasm, nucleoplasm, or chromatin, respectively. b, Mass spectra showing deacetylation of H3K18Ac peptide by SIRT7 compared to negative control reaction lacking enzyme. Molecular weights of acetylated and deacetylated (arrows) peptides are 2650 and 2608 Daltons, respectively. c, Results of SIRT7 deacetylation reactions using acetylated histone peptides, determined by mass spectrometry as in (b). d, e, Western analysis of H3K18Ac deacetylation activity of wild-type (SIRT7) or mutant (SIRT7-HY) proteins on poly-nucleosomes in vitro, and inhibition by nicotinamide (NAM). f, Western analysis showing H3K18Ac levels in 293T cells transfected with Flag-tagged SIRT7, SIRT7-HY, or control empty vector. g, Changes in global histone acetylation levels in SIRT7 overexpressing versus control 293T cells, determined by quantitative mass spectrometry. Error bars represent standard error of the mean (S.E.M.) of three independent experiments.

Figure 2. SIRT7 binds to gene promoters and couples H3K18 deacetylation to transcriptional repression

a, Enrichment of SIRT7 in promoter proximal regions, determined by ChIP-sequencing. b, Representative SIRT7 ChIP-sequencing peak at the RPS20 gene TSS (arrow). c, ChIP-qPCR (mean +/− S.E.M.) showing SIRT7 occupancy in control or SIRT7 knockdown (S7KD1, S7KD2) HT1080 cells, compared to IgG negative control samples. d, ChIP-qPCR (mean +/− S.E.M.) showing H3K18Ac hyperacetylation in S7KD HT1080 cells. e, Increased expression of SIRT7 target genes in S7KD HT1080 cells determined by qPCR (mean +/− S.E.M.). Signals were normalized to GAPDH expression. f, Western blots of cell extracts corresponding to samples in e.

Figure 3. SIRT7 is stabilized at target promoters by interaction with the ETS family transcription factor ELK4

a, Comparison of the SIRT7 consensus motif to the ELK4 consensus motif (e-value: 9.66e-9). b, Western analysis showing co-immunoprecipitation (co-IP) of FLAG-tagged SIRT7 and HA-tagged ELK4 expressed in 293T cells. c, Western blots showing co-IP of endogenous SIRT7 and ELK4 proteins. d, Westerns blots showing knockdown of ELK4 from HT1080 cells with two independent siRNAs. e, Partial reduction of SIRT7 occupancy at target promoters in ELK4 KD HT1080 cells determined by ChIP (mean +/− S.E.M.). f, ELK4 depletion attenuates SIRT7-mediated transcriptional repression in HT1080 cells, as determined by qPCR. Error bars represent S.E.M. of three independent experiments.

Figure 4. SIRT7 depletion reverses cancer cell phenotypes and inhibits tumour growth in vivo

a Western blots showing SIRT7 levels from stable cell lines used in (b). b, Reduced anchorage-independent growth of SIRT7 knockdown cells when plated in soft agar, and reconstitution with wild-type but not mutant SIRT7. Data represent mean +/− S.E.M. of three independent experiments. c, Western analysis showing impaired H3K18 deacetylation induced by E1A in S7KD HT1080 cells. Rel. H3K18Ac: relative levels of H3K18Ac in mock-treated versus E1A expressing cells, normalized to total H3 levels. d, SIRT7 depletion impairs E1A-mediated loss of contact inhibition in primary IMR90 fibroblasts determined by flow cytometry. DNA content (2N or 4N, as determined by propidium iodide (PI) staining) is indicated. e, Representative imaging of tumours derived from SIRT7-knockdown or control cells, following subcutaneous xenograft transplants in immunodeficient mice, 16 days post injection. f, Tumour volume (mean +/− S.E.M.; n=5) as in (e), measured over 35 days.