Fig 2: YcgC represents a new family of KDACs.(a) Five representative YcgC homologs with protein sequence homology ranging from low to high. (b) Amino acid sequence homology analysis between YcgC and five selected YcgC homologs from other bacteria. The consensus strength among the six homologous proteins at each amino acid position of YcgC is indicated with colored bars. Red, orange, green, light blue, dark blue, and blank bars represent 100, 80, 60, 40, 20, and 0% consensus strength, respectively. (c,d) Changes in global E. coli acetylation profiles upon induction of the five YcgC homologs. The five selected YcgC homologs were cloned, transformed into E. coli, and induced to overexpress. Global acetylation profiles of each induced strain were detected with a pan monoclonal antibody (Cell Signaling, #9441) and a pan polyclonal antibody (PTM-Biolabs, PTM-105), as shown in c and d, respectively. WT E. coli cells were also processed in parallel as a comparison. An antibody against myelin basic protein was used as a loading control. WT: Wild type.DOI: http://dx.doi.org/10.7554/eLife.05322.015

Fig 3: KAT6A acetylates SMAD3 at K20 and K117. A) Immunoprecipitation (IP) and mass spectrometry (MS) analyses of KAT6A-associated proteins in MDA-MB-231 cells. B) Co-immunoprecipitation (Co-IP) of exogenous KAT6A with SMAD3 in HEK293T cells. C) Co-IP of endogenous KAT6A with SMAD3 in MDA-MB-231 cells. D) In vitro GST pull-down analysis. Purified GST-KAT6A or GST proteins were incubated with cell extracts from MDA-MB-231 cells stimulated with or without TGF-ß1 (5 ng mL-1, 2 h). E) Schematics of SMAD3 wild type (WT), D1 (MH1 and Linker domain) mutant, and D2 (MH2 domain) mutant. F) IP and WB of KAT6A interaction with SMAD3 WT or mutants. EV, an empty vector control. G) IP and WB for analyzing the acetylated SMAD3 in MDA-MB-231 and BT-549 cells treated with or without TGF-ß1 (5 ng mL-1, 2 h). A pan acetylation antibody (9441S, CST) was used. H) KAT6A knockdown (KD) decreases the acetylation (Ac-Lys) of SMAD3 in MDA-MB-231 cells. I) Effect of acetyltransferase activity-deficient mutants G657E and C543G/G657E of KAT6A on SMAD3 acetylation. J) MS analysis of acetylation sites of SMAD3 by KAT6A. In vitro acetylated SMAD3 by KAT6A as in panel (D) was purified and then subjected to MS analysis. K) Effect of K20R, K117R, or K20/117R mutation of SMAD3 on KAT6A-mediated SMAD3 acetylation. L) In vitro KAT analysis using recombinant active KAT6A and (His)6-SMAD3 WT, K20R, or K117R mutant protein. Acetylation of K20 or K117 was determined by using anti-K20ac or anti-K117ac antibodies, respectively. M) IP and WB of KAT6A association with WT SMAD3, the acetylation-mimetic K20/117Q, or acetylation-deficient K20/117R mutant. Data are representative of three independent experiments with similar results.

Fig 4: Effect of AA on TET2 lysine mutations in myeloid neoplasia.a Lollipop plot of TET2 missense mutations of lysine residues from a cohort of 4930 patients1 with Myeloid Neoplasms. b Characterization of TET2 lysine mutants identified in patients. The indicated TET2MT were generated by site directed mutagenesis and ectopically expressed in HEK293T cells. Cells were harvested for protein extraction 72 h post-transfection and analyzed by western blot analysis using anti Myc tag antibody (Cell Signaling, Cat# 9402 s, 1:3000). c Dot blot for 5hmC/5mC with or without AA (100 µM) treatment for 24 h. d Frequently missense mutations in lysine residues of TET2 are present on the DNA binding interface. Cartoon representation of lysine residues K1299, K1310 and K1905 are depicted in red in sphere model. TET2CD (PDB ID 4NM6) was used to generate the figure in Pymol (Deleno Scientific). e Lysine acetylation inhibits TET2 activity. In vitro acetylation reactions were performed for TET2CD with or without P300/CBP and in the presence or absence of Acetyl-CoA. Anti-acetyl Lysine antibody (Cell Signaling, Cat# 9441, 1:1000) was used for the detection. SDS-PAGE was also stained to visualize the total proteins. Acetylated and nonacetylated TET2CD were used for in vitro activity assay in the presence of 5mC-DNA substrate, 2-OG and Fe2+. 5hmC and 5mC levels were assessed by 2D-UPLC-MS/MS. f In vitro acetylation reactions were performed for TET2CD with P300/CBP in the presence (+CoA) or absence of Acetyl-CoA (-CoA). Acetylated (+CoA) and nonacetylated (-CoA) TET2CD were used for in vitro activity assay. 100 µM of Fe3+ and increasing concentrations (0, 100, and 200 µM) of AA were added in the reaction buffer. 5hmC and 5mC levels were assessed by dot blot. g Mass spectrometric analysis was performed for the acetylated TET2CD protein. K1299, K1310, and K1905 were identified as acetylated lysine. h Schematics of the regulation of TET2 activity by acetyltransferases and deacetylases. Compounds used to regulate acetyltransferases and deacetylases activity are indicated. Data are shown as mean ± SEM of the triplicate and are representative of two independent set of experiments; statistical significance (p values) from two tailed t test are indicated (e–f).

Fig 5: G9a-mediated methylation inhibits interactions between RUNX3 and CBFß/p300.A Co-immunoprecipitation (IP) assay of CBFß and RUNX3 in HEK293 cells transfected with control vector and G9a plasmids (WT, ?SET). Immunoprecipitates were analyzed by immunoblotting with anti-CBFß, anti-RUNX3, anti-G9a, and anti-ß-actin antibodies. B Co-immunoprecipitation (IP) assay of p300 and RUNX3 in HEK293 cells transfected with control vector, p300 (HA-p300) and G9a plasmids (WT, ?SET). Immunoprecipitates were analyzed by immunoblotting with anti-acetyl Lysine (#9441, Cell Signaling Technology), anti-RUNX3, anti-p300 (ab10485, abcam), anti-G9a, and anti-ß-actin antibodies. C Co-immunoprecipitation (IP) assay of CBFß and RUNX3 in HEK293 cells transfected with G9a plasmid (pEGFP-G9a) and indicated RUNX3 mutants. Immunoprecipitates were analyzed by immunoblotting with anti-CBFß, anti-RUNX3, anti-G9a, and anti-ß-actin antibodies. D Co-immunoprecipitation (IP) assay of p300 and RUNX3 in HEK293 cells transfected with G9a plasmid (pEGFP-G9a) and indicated RUNX3 mutants. Immunoprecipitates were analyzed by immunoblotting with anti-RUNX3, anti-p300, anti-acetyl Lysine, anti-G9a, and anti-ß-actin antibodies. Protein levels were quantified from the western blot images (n = 3), and data are shown as the mean ± SD. *p < 0.05 (CBFß and RUNX3) and #p < 0.05 (Ace-RUNX3) by one-way ANOVA with Tukey’s posthoc correction. E Co-immunoprecipitation (IP) assay of RUNX3 and importins in HEK293 cells transfected with RUNX3 (Flag-RUNX3), importins (Myc-importin-a1, Myc-importin-a7), and/or CBFß (HA-CBFß) plasmids. Immunoprecipitates were analyzed by immunoblotting with anti-Myc (ab9106, abcam), anti-HA (SC-7392, Santa Cruz), anti-Flag (F1804, Sigma), anti-CBFß (ab124693, abcam), anti-RUNX3, and anti-Tubulin (SC-5286, Santa Cruz) antibodies. F Co-immunoprecipitation (IP) assay of RUNX3 and importin-a1 in HEK293 cells transfected with RUNX3 (Flag-RUNX3), importins (Myc-importin-a1), and/or G9a (pEGFP-G9a) plasmids. Immunoprecipitates were analyzed by immunoblotting with anti-Myc, anti-HA, anti-GFP, anti-RUNX3, anti-importin-a1, anti-G9a, and anti-ß-actin antibodies. G Co-immunoprecipitation (IP) assay of RUNX3 and importin-a7 in HEK293 cells transfected with RUNX3 (Flag-RUNX3), CBFß (HA-CBFß), importin (Myc-importin-a7) and/or G9a (pEGFP-G9a) plasmids. Immunoprecipitates were analyzed by immunoblotting with anti-Myc, anti-HA, anti-Flag, anti-GFP, anti-RUNX3, anti-CBFß, anti-importin-a7, anti-G9a, and anti-ß-actin antibodies.