The SIRT2 deacetylase regulates autoacetylation of p300

Abstract

Autoacetylation of the p300 histone acetyltransferase controls the transition between VP16-mediated chromatin acetylation and preinitiation complex (PIC) assembly. Currently, it is unknown if and how autoacetylated p300 is deacetylated. We found that the NAD(+)-dependent histone deacetylase SIRT2 deacetylates p300 in vitro and in cells. SIRT2 deacetylates lysine residues in the catalytic domain of p300 and restores binding of p300 to the PIC. RNAi-mediated depletion or chemical inhibition of SIRT2 in cells results in accumulation of acetylated p300. The altered ac-p300/p300 ratio in SIRT2-depleted cells results in decreased p300 recruitment to an integrated VP16-responsive gene and inhibition of transcription. We conclude that p300 undergoes a dynamic cycle of autoacetylation and deacetylation.

Figure 1

SIRT2 Deacetylates p300. A.) Cellular extracts contain an NAD⁺ dependent p300 deacetylase activity. Dialyzed nuclear (NE) or cytoplasmic extract (CE) from equal number of cells were incubated with ³H ac-p300 in the presence of NAD⁺ as indicated. Error bars represent the standard deviation. B.) Coomassie-blue stained gel of purified Sirtuins (300ng). C.) SIRT2 deacetylates p300 in vitro. Filter binding assays of purified Sirtuins incubated with ³H ac-p300. Error bars represent the standard deviation. D.) SIRT1, 2 and 3 deacetylate histones. Sirtuins were incubated with NAD⁺, ac-p300 and HeLa core histones. Deacetylation was monitored by immunoblot.

Figure 2

SIRT2 deacetylation of p300 restores p300 binding to GAL4-VP16-Mediator complexes. A.) SIRT2 deacetylates p300. Ac-p300 or ac-p300 HAT domain treated with SIRT2 in the presence of NAD⁺ or Nicotinamide (NIC) as indicated. Deacetylation was assayed by immunoblot with anti-acetyl-lysine antibody. B.) Amount of lysine acetylation detected in p300 MudPIT analyses. The percent lysine acetylation represents the number of spectral counts for acetylated lysine residues divided by the total spectral counts for lysine residues. The data is expressed as an average +/− standard deviation. A two-tailed student's t-test was performed and gave a p-value = 0.009691. C.) Representative lysines from MudPIT analysis. Data are shown as percent of peptides acetylated divided by the total number of peptides identified for each residue. D.) Immobilized template assay. Immobilized template was prebound by GAL4-VP16 followed by incubation with Mediator and p300, ac-p300, or SIRT2 treated ac-p300 as indicated. Binding was analyzed by immunoblot. Titrations represent 2-fold steps. E.) SIRT2 restores p300 binding to Mediator. Immobilized DNA was prebound by GAL4-VP16 and Mediator followed by incubation with p300, ac-p300, or SIRT1-7 treated ac-p300 as indicated. Input indicates levels of p300 and ac-p300 included in each immobilized template reaction.

Figure 3

SIRT2 deacetylates p300 in vivo. A.) Depletion of SIRT2 by shRNA increases ac-p300. U2OS cells treated with shGFP, OBS #1, OBS #2 and shSIRT2. The p300 was immunoprecipitated and analyzed by acetyl-lysine immunoblot. The immunoblot signals correspond to mRNA levels measured by qPCR (data not shown) B.) Knockdown of SIRT2. Whole cell extracts from panel a immunoblotted for SIRT2 levels. Titrations are 3-fold steps. C.) Expression of shRNA resistant FLAG-SIRT2 in U2OS cells restores p300 deacetylation. Immunoprecipitated p300 analyzed for acetylation by immunoblot. WT= wildtype SIRT2, SR= shRNA resistant SIRT2. D.) Expression of FLAG-SIRT2 and SR FLAG-SIRT2. Whole cell extract immunoblots. Titrations are 3-fold steps. E.) Depletion or inhibition of SIRT2 increases cellular ac-p300. U2OS Cells treated with Nicotinamide (NIC), shGFP, or shSIRT2 were lysed and p300 immunoprecipitated. Immunoprecipitates were immunoblotted with anti acetyl-lysine antibody.

Figure 4

Ablation of SIRT2 regulates VP16-dependent recruitment of p300 and gene expression. A.) Increased acetylated p300 levels result in decreased p300 recruitment. qPCR of ChIP analysis of p300 binding to Luciferase promoter in Tet-ON U2OS cells treated with shSIRT2 shRNAs. Error bars represent standard deviation of three biological replicates. B.) Loss of p300 recruitment inhibits Luciferase expression. qPCR analysis of Luciferase RNA was normalized to β-Actin (p=.031 using two-tailed student’s T test). Error bars represent standard deviation of three biological replicates.