CDK9 acetylation in vitro and in vivo. (A) In vivo acetylation. (Left) Western blot analysis of Flag immunoprecipitates (IP) from 293T cells transfected with the indicated plasmids using an anti-acetylated lysine antibody. (Right) The same immunoprecipitates analyzed with an anti-Flag antibody as the loading control. WB, Western blot; Ac, acetylated; α, anti. (B) Same as in panel A except for cell treatment with TSA. The exposure time is shorter than that for panel A. (C) Acetylation of CDK9 upon expression of various HATs. (Top two sections) Same conditions as those for panel A. The basal level of CDK9 acetylation is not appreciable in these blots since the exposure time is significantly shorter than that for panel A. (Bottom two sections) Western blot analysis to show GCN5 and p300 transfected protein levels. WCL, whole-cell lysate. (D) Acetylation of CDK9 depends on the integrity of the GCN5 and p300 catalytic domains. The experiment was performed as in panel C upon transfection of the indicated plasmids. The top two sections show different exposures of the same blot. mut, mutant. (E) In vitro CDK9 acetylation. Autoradiography (top) and Coomassie blue staining (bottom) of products of in vitro HAT assays with GST-CDK9 and GST-GCN5 or GST-p300 HATs.

GCN5-mediated acetylation of CDK9 at lysines 44 and 48 in vitro and vivo. (A) GST-CDK9 fragments used for the acetylation assay shown in panel B. The position of lysines found positive for acetylation is indicated by an asterisk. (B) In vitro mapping of CDK9 acetylated lysines. Autoradiography (top) and Coomassie blue staining (bottom) of products of in vitro HAT assays with recombinant GCN5 and CDK9 full-length or mutant proteins are shown. (C) Integrity of K44 and K48 is essential for GCN5-mediated CDK9 acetylation in vivo. A Western blot (WB) analysis of Flag immunoprecipitates from 293T transfected with the indicated plasmids is shown. IP, immunoprecipitation; Ac-CDK9, acetylated CDK9; α, anti. (D) Integrity of K44 and K48 is essential for p300- and P/CAF-mediated CDK9 acetylation in vivo. (E) Anti-Ac-CDK9 antibody detects endogenous Ac-CDK9. HeLa whole-cell lysates (WCL) were immunoprecipitated with the anti-Ac-CDK9 antibody and immunoblotted with an anti-CDK9 antibody. Control IP was performed with the preimmune serum. (F) Levels of acetylation of CDK9 variants with individual substitutions at K44 and K48. The experiment was performed as in panel C. (G) Conservation of K48 in other CDKs.

Acetylation of CDK9 inhibits kinase activity. (A) Mutation K48R inhibits CDK9 kinase activity. An immunoprecipitation kinase assay from 293T cells transfected with Flag-tagged wild-type CDK9, K44R, K48R, and K44,48R is shown. The graph reports the quantification of labeled GST-CTD (means ± standard deviations [SD] of three independent experiments), and the picture at the bottom shows a representative experiment. The Western blot (WB) in the lower panel shows the levels of protein expression. WCL, whole-cell lysates; α, anti. (B) CDK9 K44,48R (KR) reduced ATP binding activity. Transfected wild-type (wt) or K44,48R Flag-CDK9 was immunoprecipitated (IP) and labeled with the ATP analog FSBA in the presence or absence of excess ATP. The graph shows the ratio between FSBA-labeled protein and total immunoprecipitated Flag protein, determined after densitometric quantification (means ± SD from three independent experiments). The gels show the results of a representative experiment. (C) GCN5-mediated acetylation inhibits CDK9 kinase activity. Immunoprecipitation kinase assay of 293T cells transfected with the indicated plasmids. The experiment was performed as in panel A. (D) Same procedure as in panel C, also using the catalytically inactive GCN5 mutant (mut). (E) Inhibition of CDK9 enzymatic activity by GCN5 does not impair binding to cyclin T1 and HEXIM1. Shown is a Western blot analysis of Flag immunoprecipitated from the same extracts as in panel C. (F) Cyclin T1 and HEXIM1 show indistinguishable binding to either endogenous total CDK9 or acetylated CDK9 (AcCDK9). Lysates from cells transfected with Flag-HEXIM1 (top) or Flag-cyclin T1 (bottom) were immunoprecipitated for the respective proteins and probed by Western blotting using either anti-total CDK9 or anti-acetylated CDK9 antibodies. (G) Cyclin T1 shows indistinguishable binding to either total or acetylated CDK9. Cells transfected with CDK9 and HA-GCN5 were immunoprecipitated with anti-total CDK9 or anti-acetylated CDK9 antibodies or preimmune serum as a negative control (see panel C) and then analyzed by Western blotting with anti-cyclin T1 or anti-CDK9 antibodies.

Subnuclear localization of acetylated CDK9. (A) Total endogenous CDK9 (detected with an anti-CDK9 antibody) and transfected GFP-CDK9 show a diffuse nuclear localization in HeLa cells. IF, immunofluorescence; α, anti. (B) Both endogenous and transfected acetylated CDK9 (Ac-CDK9; detected with the novel anti-Ac-CDK9 antibody) have a speckled nuclear localization; the amount of acetylated protein detectable in these speckles increases upon cell treatment with TSA, which also determines a relocalization of GFP-CDK9 into speckles. (C) Transfection of enzymatically active GCN5 increases the amount of Ac-CDK9. Cells were transfected with either wild-type HA-GCN5 (row a) or its catalytically inactive mutant (row b), followed by immunofluorescence with the anti-Ac-CDK9 antibody and with an anti-HA antibody. Cells not transfected with GCN5 (not visible with the anti-HA antibody) are indicated by arrows. In these cells, as well as in cells transfected with mutant GCN5, the levels of acetylated CDK9 are remarkably reduced compared to those in cells transfected with enzymatically active GCN5. (D) Ac-CDK9 fractionates in the insoluble nuclear fraction together with PML. Shown is a Western blot (WB) analysis after biochemical fractionation of 293T cells transfected with Flag-CDK9 and HA-GCN5. PML localization in the insoluble fraction was verified. (E) Endogenous Ac-CDK9 resides in the nuclear matrix fraction. Monocytic U1 whole-cell lysates (WCL) were fractionated to obtain the soluble (Sol) and insoluble (Ins) nuclear fractions, and the levels of endogenous Ac-CDK9 were analyzed directly by Western blotting (top) or after immunoprecipitation (IP) with the anti-Ac-CDK9 antibody followed by Western blotting with an anti-total CDK9 antibody (bottom). Partitioning of Ac-CDK9 in the insoluble compartment did not change after micrococcal nuclease (Mnase) addition, a treatment known to solubilize loosely attached factors, such as MCM-3, used as a control. PARP-1, poly(ADP-ribose) polymerase 1. (F) Most endogenous CDK9 does not colocalize with endogenous PML (row a); however, overexpression of PML-IV forces its redistribution into nuclear bodies (row b). In contrast, Ac-CDK9 spontaneously colocalizes with both endogenous (row c) and transfected (row d) PML in nuclear bodies.

Acetylation impairs CDK9 transcriptional activity. (A) Schematic representation of the pG6(5′Pro) reporter vector used for the transcription assays. (B) Expression of wild-type GCN5, but not of its inactive mutant (mut), inhibits CDK9-dependent reporter gene transcription. The graph reports CAT analysis of extracts of U2OS cells transfected with pG6(5′Pro) and the indicated plasmids (means ± standard deviations); the bottom section shows a Western blot (WB) analysis of protein levels. All experiments were performed at least in triplicate. DBD, DNA binding domain; α, anti. (C) GCN5-mediated acetylation increases VP16-driven transcription. (D) Expression of GCN5 represses the positive effect that wild-type CDK9 exerts on cyclin T1-driven transcription. (E) Silencing of endogenous GCN5 by RNAi increases CDK9-driven transcription. si, small interfering; Luc, luciferase.

Acetylated CDK9 associates with the transcriptionally silent HIV-1 genome. (A) Induction of HIV-1 transcription in U1 cells after TPA stimulation. The levels of HIV-1 mRNA were measured by real-time PCR using TaqMan primers and a probe corresponding to the Nuc1A region; the results are expressed as a ratio between HIV-1 mRNA and the cellular 18S rRNA. (B) Positions of primers selected for the amplification of HIV-1 chromatin. The position of the LTR, including the transcription start site, is indicated, along with the known nucleosomal arrangement at the 5′ genome region. Numbers below investigated segments indicate the location of the 5′ primer used for amplification. (C to H) For each analyzed region, the amount of immunoprecipitated chromatin using the indicated antibodies was first normalized according to the input amount of chromatin and then expressed as enrichment over the amount in the B13 control genomic region. Each graph shows control (white bars) and TPA-induced (black bars) chromatin immunoprecipitations.