SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age

Abstract

L1 retrotransposons are an abundant class of transposable elements that pose a threat to genome stability and may have a role in age-related pathologies such as cancer. Recent evidence indicates that L1s become more active in somatic tissues during the course of ageing; however the mechanisms underlying this phenomenon remain unknown. Here we report that the longevity regulating protein, SIRT6, is a powerful repressor of L1 activity. Specifically, SIRT6 binds to the 5'-UTR of L1 loci, where it mono-ADP ribosylates the nuclear corepressor protein, KAP1, and facilitates KAP1 interaction with the heterochromatin factor, HP1α, thereby contributing to the packaging of L1 elements into transcriptionally repressive heterochromatin. During the course of ageing, and also in response to DNA damage, however, we find that SIRT6 is depleted from L1 loci, allowing the activation of these previously silenced retroelements.

Figure 1. SIRT6 mediates the transcriptional silencing of L1 loci

(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 FACS. This experiment was repeated 5 times, error bars indicate standard deviation (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 approximately 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 3 times. Where appropriate, statistical significance was determined by use of the Student's t-test.

Figure 2. In the absence of SIRT6 heterochromatin is perturbed at L1 sites

(a) Higher-order heterochromatin is disrupted in the genome of SIRT6 KO mice. Whole genomic DNA was extracted from WT and SIRT6 KO cells and then digested with the indicated units of micrococcal nuclease (MNase). Digested DNA was separated by gel electrophoresis. This experiment was repeated 3 times, a representative result is shown. (b) The L1 5′UTR is hypomethylated in SIRT6 KO cells. Whole genomic DNA was extracted from SIRT6 KO and WT fibroblasts; methylation levels were quantified using bisulfite primers following bisulfite conversion; n=3, error bars indicate s.d. (c) Multiple heterochromatin formation and maintenance proteins, namely H3K9me3, MeCP2 and KAP1 are specifically depleted from the L1 5′UTR in SIRT6 KO cells. ChIP was performed using the indicated antibodies in WT and SIRT6 KO cells. Primers spanning the mouse L1 5′UTR were used for quantification. These experiments were performed three times, representative results are shown. Quantification of the data by qPCR is shown in fig. S5. (d) SIRT6 interacts with multiple heterochromatin associated proteins, including KAP1, MeCP2 and HP1α. SIRT6 was immunoprecipitated from HDF cells, and the precipitate was probed with the indicated antibodies. These experiments were repeated 3 times, representative blots are shown; Input loaded at 5%. Where appropriate, statistical significance was determined by use of the Student's t-test.

Figure 3. SIRT6 mono-ADP ribosylates KAP1 to induce L1 silencing

(a) The mono-ADP ribosylation activity of SIRT6 is required to mediate L1 transcriptional silencing. HDF cells were transfected with the L1-EGFP reporter plasmid and the indicated expression vector. Retrotranspostion events were scored by FACS, asterisk indicates P>0.05 when compared to control, n=5; NS, not significant. (b) SIRT6 mono-ADP ribosylates KAP1, but not MeCP2 or HP1α. Wild type or catalytically inactive (S56Y) SIRT6 was incubated with the indicated substrates in the presence of radiolabelled NAD⁺. Mono-ADP ribosylation was detected by transfer of the radiolabel to the substrate. This experiment was repeated 3 times, a representative result is shown. (c) Depletion of KAP1 by shRNA in HDF cells phenocopies SIRT6 depletion. Cells were stably transfected with either control or KAP1 targeting shRNAs (Supplementary Fig. S6B); L1 mRNA levels were quantified by qRT-PCR; n=3, error bars indicate s.d. (d) SIRT6 fails to silence L1 transcription in the absence of KAP1. SIRT6 overexpression was not sufficient to reduce L1 transcription in KAP1-depleted cells; n=3, error bars indicate s.d.; NS, not significant. (e) In the absence of SIRT6, KAP1 does not stably interact with HP1α. KAP1 was immunoprecipitated from WT, SIRT6 KO, and SIRT6 KO expressing WT SIRT6 or SIRT6 activity mutants (R65A, ribosylation only; G60A, deacetylation only; S56Y, catalytically dead) cells and then probed with HP1α antibodies. This experiment was repeated 3 times; a representative result is shown. (f) SIRT6 promotes interaction between KAP1 and HP1α. KAP1 was incubated in the presence or absence of SIRT6 or SIRT6 activity mutants before addition of HP1α and PCR amplified L1 5′UTR; KAP1 was immunoprecipitated from each reaction and its affinity for HP1α was quantified by immunoblot. This experiment was repeated 3 times, a representative result is shown. Where appropriate, statistical significance was determined by use of the Student's t-test.

Figure 4. SIRT6 vacates L1 promoters in aged cells leading to de-repression of L1

(a) Senescent cells exhibit elevated L1 expression. Total cellular RNA was isolated from young and replicatively senescent HDF cells. L1 levels were quantified by qRT-PCR and normalized to actin expression; n=5, error bars indicate s.d. (b) Brain and liver tissue from old mice exhibits increased L1 expression. Total RNA was isolated from the indicated tissues from young (4 months old) and old (24 months old) mice. L1 levels were quantified by qRT-PCR and normalized to actin expression; n=3, error bars indicate s.d.; NS, not significant. (c) SIRT6 is depleted from the L1 5′UTR in senescent cells. ChIP with SIRT6 antibodies revealed that the protein is depleted from the L1 5′UTR in senescent cells, relative to young cells. Relative enrichment, after normalization to input and H3 levels is shown; n=3, error bars indicate s.d. (d) SIRT6 is depleted from the L1 5′UTR in old brain tissue. ChIP with SIRT6 antibodies revealed that the protein is depleted from the L1 5′UTR in old brain tissue, relative to young brain tissue. Relative enrichment, after normalization to input and H3 levels is shown; n=3, error bars indicate s.d. (e) Overexpression of SIRT6 partially restores L1 expression to youthful levels in senescent cells. Young and senescent cells were transfected with either a HRPT or SIRT6 encoding expression plasmid. Total RNA was isolated from these cells and L1 expression was quantified by qRT-PCR after normalization to actin expression; n=3, error bars indicate s.d. (f) DNA damage is sufficient to rapidly induce SIRT6 relocaliation. ChIP-qPCR with SIRT6 antibodies revealed that the protein is rapidly depleted from the L1 5′UTR in response to gamma-irradiation. (g) Overexpression of SIRT6 attenuates gamma-irradiation induced activation of L1 transcription. Cells were transfected with the indicated vector and then irradiated with gamma irradiation. L1 levels were quantified by qRT-PCR; n=3, error bars indicate s.d.; NS, not significant. Where appropriate, statistical significance was determined by the Student's t-test.

Figure 5. Model for age-related derepression of L1

In young cells, L1s are maintained in a silent state. SIRT6 plays a critical role in regulating the transcriptional repression of L1s in young cells by coordinating the packaging of the L1 5′UTR into transcriptionally repressive heterochromatin. In old and senescent cells, however, L1s become more active. This derepression of L1s in is part due to a failure to maintain the L1 5′UTR in a constitutively heterochromatic state. This failure to properly package the L1 5′UTR in heterochromatin stems from depletion of SIRT6 from the L1 5′UTR. Old and senescent cells accumulate DNA damage, causing SIRT6 to relocalize from the L1 5′UTR to DNA damage sites where it coordinates DNA repair. As a result of this relocalization, however, the repressive machinery at the L1 5′UTR becomes less efficient, allowing for activation of L1 elements. Additional factors that may contribute to reduced SIRT6-mediated L1 silencing are declining levels of nuclear NAD+ and lower levels of SIRT6 in senescent cells.