Volume 68, Issue 2, Pages 323-335.e6 (October 2017) mTORC1 Phosphorylates Acetyltransferase p300 to Regulate Autophagy and Lipogenesis  Wei Wan, Zhiyuan You, Yinfeng Xu, Li Zhou, Zhunlv Guan, Chao Peng, Catherine C.L. Wong, Hua Su, Tianhua Zhou, Hongguang Xia, Wei Liu  Molecular Cell  Volume 68, Issue 2, Pages 323-335.e6 (October 2017) DOI: 10.1016/j.molcel.2017.09.020 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 mTORC1 Regulates p300 Activity (A) Acetylation of p300 and histone H3 in HeLa cells treated with amino-acid-free medium, the mTORC1 inhibitor Torin1, the mTORC1 inhibitor rapamycin (Rapa), the p300 inhibitor C646, the p300 activator CTB, or amino acids after amino acid deprivation. Immunoprecipitated p300 and lysate histone H3 were analyzed by western blot using anti-acetyl-lysine and anti-acetyl-histone H3, respectively. (B) Quantification of p300 activity in HeLa cells treated as indicated and measured using p53 peptide as a substrate with acetyl-CoA. p300 was immunoprecipitated from the cells with anti-p300. Data are presented as mean ± SEM of triplicates. ∗∗p < 0.01, ∗∗∗p < 0.001. (C) In vitro acetylation assay using purified GST-histone H3 and p300-Flag immunoprecipitated from HeLa cells in the presence of acetyl-CoA. (D–G) Acetylation of p300 and histone H3 in HeLa cells overexpressing the mTORC1 activator Rheb (D), in MEFs with or without deletion of the mTORC1 inhibitor TSC1/2 (E), in HeLa cells treated with rapamycin of 25 nM for 2 hr or with 250 nM for 6 hr (F), or in HeLa cells incubated with small interfering RNAs (siRNAs) against the mTORC1 subunit Raptor or the mTORC2 subunit Rictor (G). In (F), the phosphorylation of S6K1 and Akt in the cells was also shown. See also Figure S1. Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 mTORC1 Interacts with p300 (A) Co-immunoprecipitation of endogenous p300 with Flag-Raptor in treated HeLa cells expressing Flag-Raptor with or without Rheb-myc. Flag-Raptor was immunoprecipitated using anti-Flag, and the precipitates were analyzed using anti-p300. (B) Subcellular localization of LAMP2-GFP, mTOR, and p300 in HEK293 cells stably expressing LAMP2-GFP. The cells were amino acid starved for 50 min or re-stimulated with amino acids after the starvation for 10 min and then subjected to immunostaining using anti-mTOR and anti-p300. Scale bars, 10 μm. (C) HeLa cells transiently expressing LAMP1-GFP and RFP-Raptor were homogenized in extraction buffer without detergent. The post-nuclear supernatants were then incubated with a specific GFP antibody and subjected to organelle precipitation with protein A agarose. Fluorescent images of the agarose beads are shown; the green signal indicates that LAMP1-GFP-labeled lysosomes were bound to the agarose beads. Scale bars, 10 μm. The precipitated lysosomes were analyzed by western blot using the indicated antibodies. (D) Co-immunoprecipitation of p300 with Raptor in the nuclear or cytoplasmic fractions from HeLa cells. (E) Recombinant Flag-p300 was incubated with recombinant mTORC1 comprising Flag-mTOR, His-Raptor, and His-MLST8. Flag-p300 was then pulled down using anti-p300, and the bound mTOR and Raptor were detected by western blot using anti-Flag and anti-His. See also Figures S2 and S3. Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 p300 Is a Direct Phosphorylation Substrate of mTORC1 (A) Phosphorylation of p300 in HeLa cells treated with amino-acid-free medium, Torin1, or rapamycin. p300 was immunoprecipitated from cells with anti-p300 and analyzed by western blot using anti-phospho-serine/threonine and anti-phospho-tyrosine. Cell lysates were analyzed using anti-S6K1 and anti-phospho-S6K1. (B–D) Phosphorylation of p300 in HeLa cells incubated with Raptor siRNAs (B), in MEFs with or without TSC1/2 deletion (C), and in HeLa cells with insulin, amino acid deprivation, or addition of amino acids after deprivation (D). (E) In vitro kinase assay of mTORC1 using recombinant mTORC1 and recombinant p300, with or without ATP. (F) In vitro kinase assay using recombinant mTORC1, recombinant GST-p300C (amino acids 2,175–2,414), or p300C mutants, with or without ATP. (G) Phosphorylation of Flag-tagged p300 or p300 mutants expressed in HeLa cells. 4SA, all 4 serines were replaced by alanine; 4SD, all 4 serines were replaced by aspartic acid. See also Figures S4 and S5. Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 mTORC1-Dependent Phosphorylation Disrupts the Intra-molecular Inhibition of p300 (A) Activity quantification of the Flag-tagged p300 or p300 mutants in HeLa cells. (B) Acetylation of Flag-tagged p300 or p300 mutants and histone H3 in HeLa cells treated with p300 siRNA. (C) In vitro acetylation assay using purified GST-H3 and Flag-tagged p300 or p300 mutants immunoprecipitated from treated HeLa cells. (D) Recombinant p300 incubated with recombinant mTORC1 was immunoprecipitated and incubated with acetyl-CoA, and the autoacetylation of p300 was analyzed using anti-acetyl-lysine. (E) Domain architecture of p300. AIL, autoinhibitory loop; Bd, bromodomain; BRP, Bd, RING, and PHD module; CTD, C-terminal domain; HAT, histone acetyltransferase; NTD, N-terminal domain; PHD, plant homeodomain; RING, really interesting new gene (RING). (F and G) Flag-tagged p300 or p300 mutants were transfected into p300-knockdown HeLa cells. p300 (F) or p300 mutants (G) were immunoprecipitated by anti-Flag and the acetylation levels were analyzed by western blot using anti-acetyl-lysine. The acetylation of endogenous histone H3 in the cells is also shown. (H) Quantification of the acetyltransferase activity of Flag-tagged p300, p300-4SA, or p300-4SD with or without RING deletion in HeLa cells and measured using p53 peptide as a substrate with acetyl-CoA. (I and J) Purified p300 (HAT and CTD) or p300HC mutants were incubated with purified GST-tagged p300BRP with or without RING deletion (GST-BRP or GST-BRPΔRING). GST-BRP (I) or GST-BRPΔRING (J) was then immunoprecipitated by anti-GST, and the bound p300HC or p300HC mutants were detected by western blot using anti-p300, which recognizes the C terminus of p300. The statistical data are presented as mean ± SEM of triplicates. ∗p < 0.05, ∗∗p < 0.01. Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 mTORC1-Dependent Phosphorylation of p300 Inhibits Autophagy (A) Formation of GFP-LC3 puncta in HEK293 cells stably expressing GFP-LC3. The cells were transfected with Flag-tagged p300 or p300 mutants 48 hr after p300 RNAi. Amino acid starvation was carried out 16 hr after transfection. Scale bars, 10 μm. (B) Statistical analysis of the number of GFP-LC3 puncta per cell in (A). Data are shown as mean ± SEM; n = 30. ∗∗∗p < 0.001. (C) LC3I/LC3II and p62 levels in HEK293 cells treated as in (A). (D) Acetylation of GFP-LC3 in HEK293 cells stably expressing GFP-LC3 and treated as indicated. (E and F) Acetylation of GFP-LC3 (E) or co-immunoprecipitation of Atg7 with GFP-LC3 (F) in HEK293 cells stably expressing GFP-LC3. Cells were transfected with p300-Flag, p300-4SA-Flag, or p300-4SD-Flag 48 hr after p300 RNAi. See also Figure S6. Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 mTORC1-Dependent Phosphorylation of p300 Promotes Lipogenesis (A–C) Acetylation of Myc-SREBP-1c (A), binding of Myc-SREBP-1c and RNA polymerase II (Pol II) to the FASN gene promoter (B), or co-immunoprecipitation of Pol II with Myc-SREBP-1c (C) in HepG2 cells with or without transfection of Flag-tagged p300 or p300 mutants 48 hr after p300 RNAi. (D) Relative mRNA levels of FASN, SCD, and ELOVL6 in HepG2 cells treated as in (A). (E–G) Oil red O staining (E) and quantification of intracellular oil red O content (F) or fatty acids (G) in HepG2 cells with or without transfection of Flag-tagged p300 or p300 mutants 48 hr after p300 or TSC2 RNAi. Scale bars, 20 μm. The statistical results are presented as mean ± SEM of triplicates. ∗∗p < 0.01. Molecular Cell 2017 68, 323-335.e6DOI: (10.1016/j.molcel.2017.09.020) Copyright © 2017 Elsevier Inc. Terms and Conditions