Fig 1.
KAP1 is a binding partner of SIRT1 complex.
(A and B) HEK293T cells transfected with empty vector, FLAG-tagged KAP1, or FLAG-tagged SIRT1 were lysed and immunoprecipitated with anti-FLAG antibody. Whole cell extracts (WCE) and immunoprecipitates were blotted with the indicated antibodies. (C) HEK293T cell lysates were subjected to immunoprecipitation with control IgG or anti-KAP1 antibody. The SIRT1-KAP1 interaction was examined by western blot using anti-SIRT1 antibody. (D) FLAG-tagged SIRT1 was co-transfected with GAL4-tagged full-length (FL) KAP1 or the truncation mutants of KAP1 into HEK293T cells, followed by immunoprecipitation with anti-FLAG antibody. The interaction between KAP1 truncation mutants and full-length SIRT1 was determined by western blot analysis. (E) FLAG-tagged full-length (FL) SIRT1 or the SIRT1 deletion mutants were transfected into HEK293T cells and immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were analyzed by anti-KAP1 antibody.
Fig 2.
SIRT1 deacetylates KAP1 in vitro and in vivo.
(A) SIRT1 deacetylates KAP1 in vitro. Exogenous FLAG-tagged KAP1 and SIRT1 were purified by anti-FLAG immunoprecipitation. Combination of purified proteins was incubated in deacetylation buffer supplemented with or without NAD+ cofactor. Ex527 (40μM) was added to block the deacetylase activity of SIRT1. (B) SIRT1 deacetylates KAP1 in vivo. FLAG-tagged KAP1 was transfected into control or SIRT1 depleted HEK293T cells. Cells were treated with or without IR 1 hour before harvest. Cell lysates were subjected to immunoprecipitation using anti-FLAG antibody, followed by western blot analysis to assess the total KAP1 acetylation level. Relative density of the overall acetyl lysine was quantified using ImageJ software. Acetylation level was normalized to corresponding FLAG band. (C) HEK293T cells transfected with FLAG-tagged KAP1 were treated with DMSO or Ex527 (20μM) for 6 hours before harvest. Cell lysates were then immunoprecipitated with FLAG-conjugated agarose beads, and the immunoprecipitates were blotted with anti-acetyl lysine antibody to determine the total acetylation level. (D and E) HEK293T cells were transfected with FLAG-tagged KAP1 and treated with DMSO or Ex527 (20μM) for 6 hours before harvest. Recombinant KAP1 was purified and sent for mass spectrometric analysis. Acetyl residues with >2-fold enhancement after inhibitor treatment were considered to be SIRT1 targeted sites. (F) Site-directed mutagenesis was applied to generate 4KR mutant. FLAG-tagged WT-KAP1 or 4KR mutant were transfected into control or SIRT1 depleted HEK293T cells. Total acetylation levels of recombinant WT-KAP1 and 4KR mutant were assessed by immunoblotting.
Fig 3.
Acetylation does not crosstalk with IR-induced pSer824 and does not affect the co-repressor activity of KAP1.
(A) Control and SIRT1 depleted HEK293T cells were harvested at indicated time points following 10Gy-IR. IR-induced KAP1 pSer824 was examined by phosphorylation specific antibody. (B) Cells transfected with FLAG-tagged WT-KAP1 or 4KR mutant were harvested at indicated time points following 4Gy-IR. Exogenous KAP1 was immunoprecipitated and pSer824 was examined by phosphorylation specific antibody. (C) Cyclin D3 luciferase construct was co-transfected with combinations of HA-tagged E2F1, FLAG-tagged KAP1, and 4KR mutant. Luciferase activity was measured 48 hours post transfection. (D) Cells transfected with FLAG-tagged WT-KAP1 or KAP1 phospho mutants (S824A and S824D) were harvested at indicated time points following 10Gy-IR. Recombinant KAP1 was purified by immunoprecipitation and the total acetylation level was assessed by immunoblotting.
Fig 4.
Deacetylation promotes KAP1 function in NHEJ-mediated repair.
(A and B) HA-tagged KAP1 transfected U2OS cells were fixed at 1 hour and 8 hours following 2Gy-IR. BRCA1 and 53BP1 focus formation was assessed by immunostaining. The right panel shows the quantification of average foci per cell. (C) The DR-GFP reporter consists of a direct repeat of a mutated GFP gene, which is separated by one I-SceI cleavage site, and a wild-type donor sequence as the template for HR repair. Successful repair of I-SceI induces DNA cleavage by HR-mediated pathway results in GFP-positive cells, and therefore the HR efficiency can be quantified by flow cytometric analysis. (D) pEGFP-Pem1-Ad2 reporter contains a direct repeat of GFP gene, which is interrupted by an insertion of adenovirus exon. Pre-digestion of HindIII removes the inserted exon and create a DNA breakage on the reporter. NHEJ-mediated pathway re-ligates the DNA lesion and results in GFP-positive cells. NHEJ repair efficiency can then be quantified by flow cytometric analysis. (E) KAP1 was depleted by 3’UTR targeting shRNA in HEK293T cells. DR-GFP and I-SceI were co-transfected with HA-tagged WT-KAP1 or 4KR mutant, and the HR repair efficiency was quantified at 48 hours post transfection. (F) Linearized NHEJ reporter was co-transfected with WT-KAP1 or 4KR mutant into KAP1 depleted cells, and the NHEJ repair efficiency was quantified at 48 hours post transfection. ** P≤0.01, *** P≤0.001.
Fig 5.
4KR mutant stabilizes the formation of 53BP1 foci on DSB sites.
(A) FLAG-tagged WT-KAP1 or 4KR mutant were transfected into KAP1 depleted HEK293T cells. Cells were harvested 1 hour following 10Gy-IR, and cell lysates were used for anti-53BP1 immunoprecipitation. Protein-protein interaction between 53BP1 and KAP1 was determined by immunoblotting. (B and C) Quantifications of 53BP1 and BRCA1 focus formation in HA-tagged WT-KAP1 or 4KR reconstituted U2OS cells (assessed 1 hour and 8 hours after 2Gy-IR). (D) Model of SIRT1-KAP1 regulatory mechanism in DSB repair pathways. ** P≤0.01, *** P≤0.001.