(a) L1 retrotransposition activity is elevated in SIRT6 KO cells. WT and SIRT6 KO MEFs were transfected with a L1-EGFP reporter plasmid (Supplementary Fig. 1). GFP-positive cells, indicating de novo retrotransposition events, were scored by fluorescence-activated cell sorting. This experiment was repeated five times, error bars indicate s.d. (b) Overexpression of SIRT6 is sufficient to suppress L1 retrotransposition in HDF cells, as measured using the L1-EGFP reporter assay. Cells were transfected with 5 μg of the L1-EGFP reporter and either a control (HPRT) or SIRT6-encoding plasmid; n=3, error bars indicate s.d. (c) L1 mRNA is more abundant in SIRT6 KO cells than WT cells. Total cellular RNA was extracted from WT and SIRT6 KO MEFs. L1 mRNA was quantified by qRT–PCR; quantification was normalized to actin mRNA levels; n=4, error bars indicate s.d. (d) L1 mRNA is more abundant in the tissues of SIRT6 KO mice than the tissues of WT littermates. Total cellular RNA was extracted from the indicated tissues of WT and SIRT6 KO mice. L1 mRNA was quantified by qRT–PCR, L1 expression was normalized to actin expression; n=4, error bars indicate s.d. (e) MEFs in the SIRT6 KO background had higher L1 ORF2 DNA content than WT cells. L1 copy number was probed using qPCR with primers spanning ORF2, and normalized to 5S genomic content; n=3, error bars indicate s.d. (f) SIRT6 represses transcription of the L1 5′-UTR. A L1-5′-UTR reporter was either transiently transfected (black bars) or chromosomally integrated (open bars) into HDF cells. Overexpression of SIRT6 in these cells repressed the transcriptional activity of the L1 5′-UTR by ~55–70%, relative to control, HPRT overexpression, experiments. N=5, error bars indicate s.d. (g) SIRT6 localizes to the 5′-UTR of endogenous L1 elements in the genome. ChIP was performed using the indicated antibodies and primers spanning the indicated regions of the L1 locus. Representative ChIP experiment results are shown, the ChIP was repeated three times. Where appropriate, statistical significance was determined by use of the Student’s t-test.