Figure 3: SIRT6 mono-ADP ribosylates KAP1 to induce L1 silencing. | Nature Communications

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

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

Figure 3

(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. Retrotransposition events were scored by fluorescence-activated cell sorting, asterisk indicates P>0.05 when compared with control, n=5; NS, not significant. (b) SIRT6 mono-ADP ribosylates KAP1, but not MeCP2 or HP1α. WT 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 three times and a representative result is shown. (c) Depletion of KAP1 by short hairpin RNA in HDF cells phenocopies SIRT6 depletion. Cells were stably transfected with either control or KAP1 targeting short hairpin RNAs (Supplementary Fig. 6B); 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 three times and 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 three times and a representative result is shown. Where appropriate, statistical significance was determined by use of the Student’s t-test.

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