Fig 1: KAT5-induced FTL acetylation suppresses ferroptosis. (A,B) STRING analysis revealed TFCP2 interacted acyltransferase (A) and methyltransferases (B) (confidence = 0.4). (C) Co-immunoprecipitation experiments performed in PC9 cells using IgG, anti-KAT5 and anti-TFCP2 antibodies, and further analysis of KAT5, TFCP2 and GAPDH expression by IB. (D–G) The enrichments of H4K12Ac and H4K16Ac at –2k, TFCP2 (FOXA1) motif or 2k regions of FTL (D,F) or FTH (E,G) promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in WT (D,E) or KAT5–/– (F, G) PC9 cells with erastin (10 μM) treated for indicated times. IgG was used as the parallel control. (H) FTH expression were measured using IB in WT, YAP–/–, TFCP2–/– and KAT5–/– PC9 cells with erastin (10 μM) treatment for indicated hours. (I–K) Labile iron, cell death and lipid ROS were measured in PC9 cells with or without KAT5 overexpression combined with FTL knockdown before erastin (10 μM, 24 h or 16 h) treatment. (L,M) Labile iron, cell death and lipid ROS generation were measured in PC9 and H1299 cells with or without FTL knocked down. Pentamidine (30 μM) and TH1834 (100 μM) were used to pretreat cells for 8 h before further treated with erastin (10 μM, 24 h). (N) The enrichments of YAP, TFCP2 and FOXA1 at –2k, TFCP2 (FOXA1) motif or 2k regions of FTL promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in PC9 cells with or without KAT5 overexpressed. (O) Enrichment of YAP at TFCP2 (FOXA1)-binding motif within the FTL promoter in WT or KAT5–/– PC9 cells with or without YAP, TFCP2 and FOXA1 overexpressed was measured by ChIP-qPCR. (P) Co-IP experiments were performed using anti-YAP in PC9 cells with KAT5 overexpression or knockdown with or without erastin treatment (10 μM, 24 h). The YAP level in each co-IP samples was adjusted to the same protein content. The indicated proteins in co-IP samples or WCL were measured by IB. The data are shown as the mean ± SD from three biological replicates (including IB). *P < 0.05, **P < 0.01 indicates statistical significance. Data in (I–M,O) were analyzed using a one-way ANOVA test. Data in (N) were analyzed using student’s t-test.
Fig 2: Overexpression of KAT5 crippled the impacts of VPS72 silencing on the PI3K/AKT signaling in HuH7 cells. Analysis of p-PI3K, PI3K, p-AKT and AKT expression using western blot analysis. Results were generated from three independent experiments and data were expressed as mean ± standard deviation (SD). ***P < 0.001.
Fig 3: KDM2B is acetylated by Tip60. (A) KDM2B is acetylated in osteosarcoma cancer cell lines (MG‐63 and HOS), and inhibitors of HDAC, that is, Trichostatin A (TSA) and Nicotinamide (NAM) promote the acetylation. MG‐63 and HOS cells were transduced with lentiviral Flag‐KDM2B and thus treated by TSA and/or NAM for 24 h. Cell lysates were immunoprecipitated with anti‐Flag antibody and immunoblotted with antibodies specific for acetyl‐lysine (Ac‐K) and Flag. (B) KDM2B is acetylated by Tip60. Flag‐KDM2B was transfected into HEK293 cells alone or in combination with Tip60 expression vector. Immunoprecipitation was performed with anti‐Flag antibody, and immunoblotting was carried out by using anti‐Ac‐K and anti‐Flag antibodies. (C) Tip60 induces the acetylation of endogenous KDM2B. MG‐63 and HOS cells were transfected with shRNAs specific against Tip60 (sh‐Tip60). A lentiviral vector inserting with non‐targeting sequences was transfected as a negative control (sh‐cont.). Endogenous KDM2B proteins were immunoprecipitated with anti‐KDM2B antibody, followed by immunoblotting with anti‐Ac‐K and anti‐KDM2B antibodies. (D) HOS cell lysates were immunoprecipitated with anti‐Tip60 antibody followed by immunoprecipitated with antibodies against KDM2B and Tip60. (E) MG‐63 cell lysates were immunoprecipitated with anti‐HA antibody followed by immunoprecipitated with antibodies against Myc and Flag to detect Tip60. (F) Glutathione S‐transferase (GST) pull‐down was performed to test the association between GST‐KDM2B and human HA‐tagged Tip60. *P < 0.05, **P < 0.01 vs the non‐treated control group
Fig 4: The interactome of MRG proteins highlights the association with several distinct complexes.A, schematic representation of MRG15 spliced variants, MRGX, and the yeast Eaf3 functional domains. B, short chromodomain MRG15 binds preferentially H3K36me3 in vitro. Recombinant GST-tagged MRG15 isoforms or GST or beads were incubated with LON followed by washes and Western blotting. Anti-H4 and anti-H3K79me3 are used as control. C, 3xFLAG-2xStrep elutions of indicated purifications from K562 cells were migrated on 4 to 12% SDS-PAGE and analyzed by silver staining. MRGX and MRG15 spliced variants bind the acetyltransferase NuA4/TIP60 complex. Subunits were identified by mass spectrometry (supplemental Table S1). Mock is a purification from cells expressing an empty tag. D and E, purified complexes from (C) were analyzed by Western blot with the indicated antibodies to confirm the equivalent presence of known subunits of the NuA4/TIP60 complex (D) and the other known interactors of MRG proteins (E) (∗nonspecific band). GST, glutathione-S-transferase; H3K36me, methylation of lysine 36 on histone H3; MRG15, MORF-related gene on chromosome 15; NuA4, nucleosome acetyltransferase of H4; TIP60, Tat interactive protein 60 kDa.
Fig 5: Identification of the TINTIN complex in human cells, composed of BRD8–MRGBP–MRG15/X and new subunit EP400NL.A, purification of native NuA4/TIP60 complex from K562 cells. Strep elutions of indicated purifications were analyzed on 4 to 12% SDS-PAGE followed by silver staining. MRGBP and spliced variants of BRD8 (1BRD, one bromodomain, 2BRD, two bromodomains, and CRISPR for N-terminal-tagged BRD8) are linked to NuA4/TIP60 complex. B, Western blots of purified complexes in (A) confirmed the association with the NuA4/TIP60 complex and the new subunit EP400NL. C, Western blots of gel filtration fractions 17 to 33 (calibrated Superose 6, FPLC) with FLAG elutions from EPC1, BRD8, and MRGBP AAVS1 K562 cells (EPC1 purified fraction is used as a control reflecting the native NuA4–TIP60 complex (64)). Arrows point to specific annotated complexes with different elution profiles based on size. D, 4 to 12% SDS-PAGE followed by silver staining of annotated fractions from (C) showing the NuA4/TIP60 complex (17), the TINTIN complex (23), and a dimer MRGBP–MRG15 or MRGX (33). See also supplemental Table S5 for mass spectrometry results. EP400NL, EP400 N-terminal like; MRGBP, MRG-binding protein; NuA4, nucleosome acetyltransferase of H4; TINTIN, Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes; TIP60, Tat interactive protein 60 kDa.
Supplier Page from Abcam for Anti-KAT5 / Tip60 antibody