Fig 2: Model for the involvement of RECQL4 in OGG1-mediated removal of 8-oxoG and its regulation by SIRT1 deacetylase. In response to oxidative stress, RECQL4 becomes hyperacetylated, which enhances its interaction with OGG1 to promote 8-oxoG repair. After repair, SIRT1 outcompetes OGG1 from interaction with RECQL4 to return it to a hypoacetylated state. However, 8-oxoG repair is impaired in RECQL4- deficient cells, because of the loss of stimulatory effect of RECQL4 on OGG1, which leads to increased genomic 8-oxoG lesions.

Fig 3: SIRT1 interacts with and deacetylates RECQL4. (A) Immunoprecipitation (IP) in cells expressing GFP or GFP-tagged WT-RECQL4 with GFP-TRAP beads analyzed by immunoblotting (IB) with anti-GFP and anti-SIRT1 antibodies. Representative blot is shown from two independent experiments. (B) Immunoprecipitation (IP) performed in cells expressing vector alone or FLAG-tagged FL-RECQL4, NT-RECQL4, and CT-RECQL4 with FLAG-M2 magnetic beads and analyzed by immunoblotting (IB) with the indicated antibodies shows that the N-terminal domain of RECQL4 interacts with SIRT1 in cells. Representative blot is shown from two independent experiments. (C) Deacetylation of RECQL4 by SIRT1 in an in vitro deacetylation assay. Recombinant RECQL4 (1 µg) was first acetylated by CBP (0.1 µg) for the first 1 h, then acetylated RECQL4 was incubated with NAD+ (50 µM), and different amounts of recombinant SIRT1 (0.5, 1, 2 U) at 30°C for an additional 1 h. Acetylated RECQL4 was probed with anti-acetylated lysine antibody. Total amounts of RECQL4 were assessed with an anti-RECQL4 antibody. NAM served as a SIRT1 inhibitors. A representative gel is shown on the left. The graph on the right shows quantification of relative acetylated RECQL4 levels normalized to total RECQL4. Data are shown as mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001, using unpaired two-tailed Student's t test. (D) Deacetylation of wild type RECQL4 (WT) and RECQL4 (KQ) mutant by SIRT1 in an in vitro deacetylation assay. 3 × FLAG-tagged RECQL4 proteins were purified from normal U2OS cells. FLAG-tagged RECQL4 proteins (1 µg), were first acetylated by CBP (0.1 µg) for the first 1 h, then acetylated proteins were incubated with NAD+ (50 µM), and recombinant SIRT1 (2 U) at 30°C for an additional 1 h. Acetylated FLAG-tagged RECQL4 proteins were probed with anti-acetylated lysine antibody. Total amounts of FLAG-tagged RECQL4 proteins were assessed with an anti-FLAG antibody. A representative gel is shown on the left. The graph on the right shows quantification of relative acetylated RECQL4 protein levels normalized to total. Data are shown as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, using unpaired two-tailed Student's t test.

Fig 4: GR acetylation is regulated by p300/CBP and SIRT1 in a dexamethasone-dependent mannerWestern blot analyses with pan acetyl-lysine (ac-K) and FLAG antibodies, showing acetylation levels of immunoprecipitated FLAG-GR under the following experimental conditions.(A) The indicated cell lines were treated with 1 μM Dex or vehicle for 6 h.(B) HeLa cells transiently overexpressed the individual lysine acetyltransferases (KATs) and were treated with 1 μM Dex for 16 h. Immunoblot of whole cell lysates indicates expression levels of the overexpressed KATs.(C) HeLa cells treated with vehicle or 1 μM Dex in combination with the pan sirtuin inhibitor NAM (10 mM) or the selective SIRT1 inhibitor Ex527 (20 μM) or the class I and II HDACi TSA (1 µM) for 6 h.(D) HeLa cells were transfected with siRNAs against the indicated histone deacetylases followed by treatment with vehicle or 1 μM Dex for 16 h. Western blotting of whole cell lysates show knockdown efficiency of the individual HDACs.(E) In vitro deacetylation assay. Immunopurified, hyperacetylated FLAG-GR from BHK-21 cells, was incubated with 1 or 3 units (U) of recombinant human (rh)SIRT1 in the presence or absence of NAD+ and Ex527 as indicated.See also Figure S1.

Fig 5: Acetylation-deficient GR sustains transcription longer than the wild-type(A) Western blot showing doxycycline-induced expression of wild-type and mutant GR in stably transduced HepG2 GR−/− cells treated with vehicle or 1 μM Dex for 6 h. Quantification below shows relative GR/ACTB levels normalized to WT veh sample from n = 4 independent experiments. n.s = non-significant; Welch’s ANOVA with Dunnett’s T3 post hoc test.(B) Luciferase assay detecting MMTV-driven luciferase expression in cells treated as in (A) expressed as fold induction to respective vehicle controls.(C) ChEA3 analysis of the 100 commonly regulated DEGs showing top 10 enriched transcription factors (TFs). Y axis shows names of TFs and X axis indicates negative logarithm of Fischer’s exact test (FET) p-values.31(D) Gene ontology analysis indicating biological processes enriched among the common DEGs.(E) Heatmap showing expression of all Dex-regulated genes common between WT-, K154R- and K154Q-GR mutants as identified by RNA-seq analysis.(F) Western blot showing GR-K154 acetylation, GR and SIRT1 levels in HepG2 cells over a time course of 1 μM Dex treatment (0–24 h). ACTB serves as loading control.(G) Ratios of acK154 to GR (orange) or GR to ACTB (blue) western blot signals from (F) were quantified and normalized to 24 h Dex sample or vehicle (0 h Dex) sample respectively and represented as relative signal.(H) RT-qPCR showing expression of GILZ as log2 fold change in cells treated as in (F).(I) RT-qPCR showing expression of GILZ as log2 fold change to vehicle after 6 h, 12 h and 24 h Dex treatments in HepG2 GR−/− + WT-GR or HepG2 GR−/− + K154R cell lines.Data represent mean from n = 4, except for (G, H) where n = 3, independent experiments. Error bars = S.E.M ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, n.s = non-significant; One way ANOVA- Fischer’s LSD test (G, H, I).See also Figure S4, Tables S1 and S2.