1. Volume 4, Issue 5, Pages 725-734 (November 1999)
Acetylation of MyoD Directed by PCAF Is Necessary for the Execution of the Muscle Program 
Vittorio Sartorelli, Pier Lorenzo Puri, Yasuo Hamamori, Vasily Ogryzko, Gene Chung, Yoshihiro Nakatani, Jean Y.J. Wang, Larry Kedes 
Molecular Cell 
Volume 4, Issue 5, Pages (November 1999)
DOI: /S (00)

2. Figure 1 MyoD Is Acetylated In Vivo
(A) Cell extracts derived from C2C12 cells cultured in either growth medium (GM) or differentiation medium (DM) for 24 hr transfected with FLAG-MyoD expression vector and pulsed-labeled with [3H]-sodium acetate were immunoprecipitated with an antibody against the FLAG epitope (lanes 1 and 3). The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. The absence of a radioactive signal corresponding to FLAG-MyoD in extracts from nontransfected, but pulsed-labeled, cells (lane 2) or immunoprecipitation conducted with a control anti-Myc antibody (lanes 4 and 5) indicates that the band corresponding to FLAG-MyoD is specific.
(B) Aliquots of the immunoprecipitated material from the experiments represented in (A), lanes 1 and 3, were processed for Western blot analysis with a MyoD monoclonal antibody.
(C) The radioactive signals corresponding to acetylated FLAG-MyoD in GM and in DM for 12, 24, and 36 hr and the corresponding chemiluminescent signals (from Western analysis) of immunoprecipitated MyoD were quantified by densitometry. The values reported in the graph correspond to the radioactive signals (acetylated MyoD) corrected for the corresponding chemiluminescent signals (immunoprecipitated MyoD). The values derive from the results of three independent experiments.
(D) In vivo acetylation assay performed in fibroblasts transfected with either FLAG-MyoD alone (lane 1) or FLAG-MyoD and PCAF (lane 2) expression vectors.
(E) Total extracts employed in (D), as well as an extract derived from nontransfected cells (lane 1), were processed for Western blot analysis with a MyoD monoclonal antibody.
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2 Mapping the Acetylated Region of MyoD
(A) PCAF (4.5 pmol) was incubated with MyoD polypeptides (∼25 pmol) and [3H]-acetyl-CoA (5 nmol) and the reaction processed for filter-binding assay as described in (Mizzen et al. 1996). Bovine serum albumin (BSA) and purified histones serve as negative and positive controls, respectively. A, MyoD aa 1–318; B, MyoD aa 155–298; C, MyoD aa 87–154; D, MyoD aa 1–86. The coordinates of the MyoD polypeptides employed in the experiments described above and location of the lysines (asterisks) are schematically indicated at the bottom. C/H indicates a cysteine–histidine rich region and bHLH refers to the basic helix-loop-helix motif of MyoD. A summary of the acetylation status of the MyoD polypeptides (+ or −) is indicated.
(B) The first N-terminal 108 aa contain the major acetylation lysine determinants of MyoD and their substitution renders MyoD nonacetylatable. PCAF (4.5 pmol) was incubated with different MyoD polypeptides (∼25 pmol) and [14C] acetyl-CoA (5 nmol) for 1 hr at 30°C. The reactions were resolved on a 4%–20% gradient SDS-PAGE, the peptides identified by staining with Coomassie blue (right panel) and processed for autoradiography (left panel, arrows). Note autoacetylation of PCAF. The coordinates of the MyoD polypeptides, location of the lysines (asterisks), and Lys-to-Arg substitutions (lines) are schematically reported at the bottom. A summary of the acetylation status of the MyoD polypeptides (+ or −) is indicated.
(C) MyoD wild type (A), MyoD with Lys-to-Arg substitutions at position 99 and 104 (A m1), or at positions 99, 102, and 104 (A m2) were tested in an in vitro acetylation assay with PCAF and [14C] acetyl-CoA. A schematic representation of the MyoD polypeptides employed is given at the bottom. Lysines are indicated by asterisks and Lys-to-Arg substitutions by lines.
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3 Identification of the Acetylated Lysines
(A) Schematic representation of MyoD and position of the MyoD peptides.
(B) Amino acid sequences and coordinates of the MyoD peptides. The ionization constants at the isoelectric points (pKa) of each MyoD peptide in water at 25°C were calculated by adding the individual amino acids pKa and dividing them by the total amino acid residues. Filter-binding reactions were performed at pH 8.0.
(C) A MyoD peptide containing lysines at positions 99, 102, and 104 is acetylated by PCAF. Approximately 1 μg of each of a series of synthetic peptide fragments of MyoD spanning eight out of nine lysine residues was incubated with [3H]-acetyl-CoA (25 nCi) and PCAF (1 pmol) for 1 hr at 30°C. [3H] incorporation was evaluated in a filter-binding assay.
(D) Amino acid sequence of the MyoD peptides used in (E). Sites where ε-N-acetyllysine was incorporated during peptide synthesis to block PCAF-mediated acetylation are indicated by Ac. The underlined lysines are those available for acetylation by PCAF.
(E) Results of filter binding assay conducted with PCAF and the 96–107 aa MyoD peptide with single or all pairwise ε-N-acetyllysine substitutions. Counts present in the (−) columns derive from autoacetylation of PCAF. Bovine serum albumin (BSA) (2 μg) was employed as a negative control.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4
Substitution of Acetylated Lysines by Nonacetylatable Arginines at Positions 99, 102, and 104 Impairs MyoD Acetylation In Vivo
(A) Vectors for FLAG-MyoD wild type (lane 1) or MyoD bearing Lys-to-Arg substitutions at positions 99, 102, and 104 [FLAG MyoD (RRR), lane 2] were transfected in C2C12 muscle cells and the expressed proteins evaluated for acetylation using the in vivo acetylation assay.
(B) Total cell lysate from cells transfected with either FLAG-MyoD wild type (lane 2) or FLAG MyoD (RRR) (lane 3) were analyzed in Western blot using a FLAG monoclonal antibody. Lane 1 contains cell lysate from nontransfected cells.
(C) Lysines (arrows) acetylated in MyoD are conserved in every member of the murine myogenic bHLH protein family.
(D) Conservation of the acetylated lysines (arrows) from Drosophila to human MyoD.
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5
Nonacetylatable MyoD Is Deficient in Promoting Transactivation and Muscle Conversion
Transient transfections were performed in NIH3T3 fibroblasts (A) and in C2C12 muscle cells (B) using either the synthetic (4RE) or muscle creatine kinase (MCK) enhancer fused to the luciferase reporter gene. One microgram of the reporter construct was transfected with 100 ng of the indicated MyoD expression constructs. Each experiment, in triplicate, was performed 5–10 times. In (B), a titration (25, 50, and 100 ng) was performed using either the MyoD wild type of the MyoD (RRR) expression vector. Luciferase activity was measured after culturing the transfected cells in differentiation medium for 48 hr. (C) C3H10T1/2 fibroblasts were transiently transfected with either MyoD wt (top panels) or MyoD (RRR) (lower panels). Cells were immunostained with a MyoD antiserum (revealed with a Texas red–conjugated secondary antibody [MyoD panels]), and a monoclonal antibody against the sarcomeric MHC (revealed with fluorescein-conjugated secondary antibody, [MHC panels]). In cells expressing MyoD (RRR) (lower panels), only a small subpopulation of MyoD-positive cells are also MHC positive. The left-most panels represent fields observed by phase-contrast microscopy.
Molecular Cell 1999 4, DOI: ( /S (00) )

7. Figure 6
Nonacetylatable MyoD Is Competent for Nuclear Localization, DNA Binding, and E Protein Dimerization
(A) MyoD (RRR) was fused to the green fluorescent protein and the resulting plasmid, MyoD (RRR)-GFP, transfected in NIH3T3 cells. The fusion protein is predominantly nuclear.
(B) MyoD (RRR) interacts with the E box and dimerizes with E12. Gel mobility shift assay was performed with equivalent amounts of either His-MyoD wild type (lane 1) or His-MyoD (RRR) proteins (lane 2) and a radiolabeled oligonucleotide spanning a MyoD-binding site from the MCK enhancer. Dimerization of MyoD was observed when E12 was added to the reaction (lanes 4 and 5). The asterisk in lane 3 indicates a band of unknown identity but possibly deriving from partial E12 protein degradation.
Molecular Cell 1999 4, DOI: ( /S (00) )

8. Figure 7
PCAF-Mediated Acetylation Increases the DNA Binding Activity and Induces a Conformational Change of MyoD
(A) MyoD (25 pmol) and PCAF (8.5 pmol) were incubated with either desulfo-CoA (5 nmol) (unmodified) or with acetyl-CoA (5 nmol) (acetylated) at 30°C for 1 hr. A radiolabeled oligonucleotide containing a MyoD-binding site was added and incubation continued for an additional 30 min. The reactions were analyzed by autoradiography following nondenaturing PAGE. The fold increases of MyoD binding after acetylation were calculated, by densitometry, by comparing the percentage of radiolabeled DNA probe bound at each concentration of acetylated versus unmodified protein.
(B) MyoD (RRR) mutant protein (25 pmol) was incubated with PCAF (8.5 pmol) and either desulfo-CoA (unmodified) or acetyl-CoA (acetylated) and processed as described in (A).
(C) Purified MyoD (lanes 1 and 2) and E12 (lanes 3 and 4) were incubated either alone or in combination (lanes 5 and 6) with either desulfo-CoA (lanes 1, 3, and 5) or acetyl-CoA (lanes 2, 4, and 6) and their DNA binding activity assessed using a radiolabeled oligonucleotide containing a MyoD-binding site. The asterisk indicates a band of unknown identity but possibly deriving from partial E12 protein degradation.
(D) Protease clipping of unmodified and acetylated MyoD. MyoD was incubated with PCAF and either desulfo-CoA (unmodified) or acetyl-CoA (acetylated) and then allowed to interact with a radiolabeled E box containing a MyoD-binding site before being subjected to limited proteolytic digestion with increasing concentrations of endoproteinase Lys-C. The reactions were analyzed on nondenaturing PAGE followed by autoradiography. The extent of MyoD proteolysis was calculated by comparing the percentage of radiolabeled DNA probe bound to acetylated versus unmodified MyoD in the absence (lanes 1 and 4) or at two different concentrations of endoproteinase Lys-C (compare lanes 2 and 5; lanes 3 and 6).
(E) MyoD was treated as described in (D) but incubated with endoproteinase Lys-C before binding to DNA.
Molecular Cell 1999 4, DOI: ( /S (00) )