Interaction between acetylated MyoD and the bromodomain of CBP and/or p300

Abstract

Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

FIG. 1

Preferential association between acetylated MyoD and CBP and p300. Myogenic cell extracts were immunoprecipitated (ip) as shown in panel A and analyzed by Western blotting (wb) (B) using anti-acetylated lysine or anti-MyoD as indicated. irr, immunoprecipitation using an irrelevant antibody as a negative control.

FIG. 2

Nonacetylatable MyoD mutants do not associate with CBP in cells. Mutants used were m2, in which lysines 99, 102, and 104 were replaced by arginines, and m5, in which lysines 99, 102, 104, 112, 124, and 133 were replaced by arginines. (A) C3H 10T1/2 cells were transfected with expression vectors for wild-type (wt) or point-mutated (m2 or m5) MyoD together with an expression vector for wt CBP 1-2441. A MyoD empty vector vehicle was used in negative controls (co). Extracts from transfected cells were immunoprecipitated (ip) using an anti-CBP antibody or an anti-MyoD antibody as indicated. MyoD and CBP were detected by Western blotting (wb), as indicated. (B) Nonacetylated wt or mutant MyoD (m2) was incubated with GST-CBP-coated beads for 1 h and washed with TE buffer containing the indicated concentrations of KCl. Beads were analyzed by Western blotting using anti-MyoD antibodies. (C) Indicated doses of nonacetylated MyoD (wt or mutated) were incubated in TE buffer with GST-CBP-coated beads. Beads were analyzed by Western blotting using anti-MyoD antibodies. Input, the amount of each MyoD protein, estimated as 2 ng (1/25 of maximal amount used in this experiment).

FIG. 3

Acetylation increases MyoD affinity for CBP in vitro. (A) Equivalent amounts of wild-type (wt) and point-mutated (m2) MyoD were incubated with GST-CBP 1-2441-coated beads for 1 h, with or without acetyl-CoA (1 mM), and washed with TE buffer containing the indicated concentrations of KCl. GST-coated beads were used as a negative control. (B) Autoradiograms of the blots shown in panel A were analyzed by densitometry. Open circle, nonacetylated MyoD; solid square, acetylated MyoD. Increasing doses (2, 10, and 50 ng) of recombinant MyoD were incubated with GST-CBP absorbed onto glutathione-coated beads in the presence or absence of acetyl-CoA. The presence of MyoD in association with CBP was detected by Western blotting as described for Fig. 2.

FIG. 4

CBP bromodomain is involved in the interaction with acetylated MyoD. (A) Diagram of the CBP molecule, showing the C/H3 domain, bromodomain, and HAT domain. (B) Increasing doses (2, 10, and 50 ng) of recombinant MyoD were incubated with GST-CBP absorbed onto glutathione-coated beads in the presence or absence of acetyl-CoA; the presence of MyoD in association with CBP was detected by Western blotting. wt, wild type. (C to F) MyoD was incubated with wt CBP in the presence of acetyl-CoA (Ac-CoA) (or in the absence of Ac-CoA for the controls, as indicated), isolated from the enzyme, and used in a GST pull-down assay with beads coated with the indicated CBP mutants; the presence of MyoD in association with CBP was detected by Western blotting.

FIG. 5

CBP bromodomain is involved in physical and functional interaction with MyoD. (A) Nonmuscle cells (C3H 10T1/2) were transfected with expression vectors for MyoD and p300, either wild type (wt) or with the bromodomain deleted. Extracts were immunoprecipitated (ip) with an anti-p300 antibody and analyzed by Western blotting (wb) using an anti-MyoD antibody. (B) Expression of transfected proteins. Extracts of transfected cells were immunoprecipitated and analyzed by Western blotting using the indicated antibodies. (C) C3H 10T1/2 cells were microinjected with expression vectors for MyoD and for p300, either wt or with the bromodomain deleted, together with a rhodamine-coupled injection marker. After 40 h in differentiation medium, cells were fixed and immunostained with an antimyogenin antibody. Shown is the fraction of injected cells (rhodamine positive) which express myogenin; ▪, wt CBP; □, wt p300; ○, p300 delta Br.

FIG. 6

Impaired cooperation between nonacetylatable MyoD and CBP mutants. Nonmuscle cells (C3H 10T1/2) were transfected with an MCK-luciferase reporter construct, together with a pEMSV MyoD expression vector (100 ng) and indicated doses of CMV-CBP. Results of a representative experiment are shown. (A) Luciferase activity was measured in duplicate. r.l.u., relative light units; wt wild type. (B) Means of three independent experiments ± standard deviations are shown as the fold inductions by CBP (ratio between r.l.u. obtained in the presence of CBP and r.l.u. obtained in its absence). ●, wt MyoD; ▪, m2. (C) Western blot analysis of MyoD expression.

FIG. 7

MCK-associated histone H4 is acetylated on terminal differentiation. Chromatin was extracted from C2C12 muscle cells, either continuously growing or else differentiating, as indicated. Equivalent amounts of chromatin (as assessed by gel analysis of dilutions; data not shown) were immunoprecipitated using an anti-acetylated (ac) histone H4 (A and C) or an irrelevant antibody (ab) as a control (B). The MCK promoter (A and B) and the GAPDH promoter as an internal control (C) were detected by quantitative PCR analysis using increasing doses of a competitor DNA (no DNA [−]; 100 ag; 1, 10, and 100 fg; and 1 pg) amplified by the same primers. Arrows, bands corresponding to the MCK or GAPDH amplification products, as well as the bands corresponding to the competitor (comp) amplification products. (D) Nondenaturing gel analysis of serial dilutions of chromatin used for immunoprecipitation stained by ethidium bromide.

FIG. 8

A model for the association between acetylated MyoD and CBP (see Discussion). Br, bromodomain. Curved arrows, substrate acetylation by the HAT; asterisks, acetylated lysines on MyoD.