1. Regulation of Skeletal Myogenesis by Association of the MEF2 Transcription Factor with Class II Histone Deacetylases 
Jianrong Lu, Timothy A. McKinsey, Chun-Li Zhang, Eric N. Olson 
Molecular Cell 
Volume 6, Issue 2, Pages (August 2000)
DOI: /S (00)

2. Figure 1
HDACs 4 and 5 Inhibit Conversion of 10T1/2 Cells to Skeletal Muscle by MyoD
(A) 10T1/2 cells were transiently transfected with expression vectors for MyoD (0.5 μg) or the indicated HDACs (0.5 μg) as described in Experimental Procedures, and myogenic conversion was scored by staining for MHC expression. A value of 100 for cells transfected with MyoD expression plasmid corresponds to 50 MHC-positive cells per 35 mm dish. Assays were performed three independent times with comparable results.
(B) Schematic diagram of HDACs and deletion mutants used in transfection assays. Amino acids are indicated.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Induced Histone H4 Acetylation at MEF2 Target Sites during Skeletal Myogenesis and Inhibition of C2 Cell Differentiation by HDACs 4 and 5
(A) Soluble chromatin was prepared from cultures of proliferating myoblasts in growth medium (GM) or from multinucleated myotubes in differentiation medium (DM) and immunoprecipitated with an antibody specific for acetylated histone H4 (α-AcH4). Parallel extracts were exposed to normal rabbit serum (nonimmune) to control for nonspecific precipitation of chromatin. Precipitated genomic DNA was analyzed by PCR using primers designed to amplify sequences spanning the MEF2 binding sites in the MCK enhancer and myogenin promoter. Positions of primers and MEF2 sites relative to the transcription initiation sites of each gene are shown. The lower panel shows a DNA input control in which PCR amplification was performed prior to immunoprecipitation to confirm that equivalent amounts of DNA were present in each sample.
(B) C2 cells were stably transfected with HDAC4 and HDAC5 expression vectors, and stable transfectants were isolated following selection in G-418. RNA was isolated from the parental C2 cell line and from representative clones in GM (day 0) or following transfer to DM for the indicated number of days. Transcripts were detected by semiquantitative RT-PCR as described in Experimental Procedures.
(C) HDAC5 transcripts in wild-type C2 cells and in HDAC5-transfected cells (clone #6) were detected by RT-PCR using primers that distinguish the exogenous human HDAC5 and endogenous mouse HDAC5 transcripts. The exogenous transcript is expressed at a level approximately 4-fold higher than the endogenous.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
HDAC5 Selectively Inhibits MyoD-Dependent Promoters that Contain MEF2 Sites
10T1/2 cells were transiently transfected with expression vectors for MyoD, MEF2, or the indicated HDACs, and 4RE-luciferase, MCK-luciferase, or 3× MEF2-luciferase expression plasmids, and luciferase activity was determined as described in Experimental Procedures.
(A) Activation of MCK-luciferase (0.3 μg) by MyoD (0.3 μg) is inhibited by HDAC5 (0.2 μg). “E” stands for E box; “M” stands for MEF2 site. Sites for other enhancer binding factors are not shown.
(B) Activation of 4RE-luciferase (0.3 μg) by MyoD (0.3 μg) is unaffected by the presence of HDACs 4 and 5 (0.2 μg).
(C) Increasing the amount of MyoD overcomes the inhibitory effect of HDAC5 on activation of MCK-luciferase, whereas an amount of MyoD sufficient to fully activate the MCK enhancer in the presence of HDAC5 has no effect on activation of 3× MEF2-luciferase.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Mapping Residues in the MADS/MEF2 Domains of MEF2C Required for Interaction with HDAC
(A) Amino acid sequence of the MADS and MEF2 domains of MEF2C. Mutants tested for interactions with HDACs 4 and 5 are shown below. A dash indicates the wild-type residue at that position, and an X indicates residues that were deleted. The dimerization and DNA binding activity of the mutants was reported previously (Molkentin et al. 1996).
(B) Coimmunoprecipitations of HDAC4 and MEF2C mutants. 293 T cells were transiently transfected with expression vectors for Flag-tagged HDAC4 and the indicated MEF2C mutants, and cell extracts were analyzed by immunoprecipitation and Western blot as described in Experimental Procedures. The top panel shows the results of anti-HDAC4 (Flag) immunoprecipitation followed by anti-MEF2 Western blot, and demonstrates interaction of all MEF2C mutants with HDAC4. The bottom panel shows the results of anti-MEF2 Western blot without anti-HDAC immunoprecipitation, and demonstrates that comparable amounts of each MEF2 were expressed in transfected cells.
(C) Binding of MEF2C mutants to GST-HDAC4 in vitro. MEF2C and various deletion mutants, D-MEF2, and SRF were translated in vitro in the presence of [35S]methionine and incubated with GST-HDAC4 fusion protein containing residues 49–233 of HDAC4 bound to glutathione-agarose beads. Proteins bound to GST-HDAC4 were separated by SDS–PAGE and analyzed by autoradiography (upper panel). As a control, labeled proteins were also incubated with GST alone (middle panel). No labeled proteins were recovered under these conditions. Ten percent of the labeled in vitro translation products used for GST-HDAC interaction assays was run on a separate gel (bottom panel). In the far right lanes, [35S]methionine-labeled HDAC4 (residues 49–233) was incubated with GST-MEF2C (residues 1–86), and bound protein was analyzed as described above.
(D) Alignment of MADS/MEF2 domains of MEF2 factors and SRF. The region corresponding to the HDAC binding domain is conserved among MEF2 factors but divergent in SRF.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5 Simultaneous Binding of MEF2 to DNA and HDACs
(A) Binding of an MEF2-DNA complex to GST-HDAC4. GST-HDAC4 was incubated with [35S]-labeled in vitro translation products that were premixed with a [32P]-labeled MEF2 site as described in Experimental Procedures. Beads were then washed and associated radioactivity was determined.
(B) Modified one-hybrid assay. 10T1/2 cells were transiently transfected with 3× MEF2-luciferase reporter (0.3 μg) and expression plasmids for MEF2C (0.3 μg), HDAC4-VP16 (0.2 μg), and HDAC5-VP16 (0.2 μg) as indicated. HDAC4-VP16 and HDAC5-VP16 were created as described in Experimental Procedures.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6
CaMK and IGF-1 Signaling Overcomes HDAC-Mediated Repression of MyoD Activity
(A) 10T1/2 cells were transiently transfected with expression vectors for MyoD (0.5 μg), HDAC5 (0.3 μg), CaMKI (0.3 μg), and MKK6 (0.3 μg) as indicated, and myogenic conversion was scored by staining for MHC expression as described in Experimental Procedures. A value of 100 for cells transfected with MyoD expression plasmid corresponds to 50 MHC-positive cells per 35 mm dish. Assays were performed three independent times with comparable results. Repression of MyoD activity by HDAC5 was overcome in the presence of CaMKI but not MKK6 alone. Together, CaMKI and MKK6 synergistically stimulated MyoD activity.
(B) L6 myoblasts were transferred from growth medium (GM) to DM in the absence or presence of IGF-1 (50 ng/ml) as indicated. To inhibit CaMK, one set of cultures was also treated with KN62 (5 μM). Following 6 days in DM, cultures were stained with anti-MHC antibody using an immunoperoxidase system. In the presence of IGF-1, there was a dramatic stimulation of MHC expression and myoblast fusion that was blocked by KN62. Under these culture conditions, L6 myoblasts differentiated relatively poorly in the absence of IGF-1. Shown at 5× magnification.
(C) The HDAC4-transfected C2 cell line HDAC4 (#2) (see Figure 2) was cultured in DM in the absence and presence of IGF-1 for 4 days. Cultures were then stained with anti-MHC antibody and a fluorescein-conjugated anti-mouse secondary antibody and Hoechst stain to reveal nuclei. Differentiation was inhibited in cultures in DM, but large differentiated myotubes were observed in the presence of IGF-1. Shown at 40× magnification.
(D) 10T1/2 cells were transiently transfected with expression vectors for MyoD (0.5 μg), HDAC4 (0.3 μg), HDAC5 (0.3 μg), and a dominant-negative mutant of CaMKIV (CaMK-DN; 0.1 μg) as indicated. Twenty-four hours after transfection, cells were transferred to differentiation medium in the absence or presence of IGF-1 (50 ng/ml), and myogenic conversion was scored 5 days later by staining for MHC as described in Experimental Procedures. A value of 100 for cells transfected with MyoD expression plasmid corresponds to 50 MHC-positive cells per 35 mm dish.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7 A Model for the Role of HDACs in Skeletal Myogenesis
(A) Three types of muscle target genes distinguished by their responsiveness to MyoD, MEF2, and HDAC.
(B) Schematic diagram showing the potential roles of MyoD, MEF2, and HDACs in muscle gene expression. In myoblasts, association of HDAC with MEF2 results in repression of muscle genes controlled by MyoD. When myoblasts are triggered to differentiate, myogenic bHLH and MEF2 levels rise, which overcomes repression by HDAC of genes that contain E boxes and MEF2 sites. CaMK signaling, which dissociates MEF2-HDAC complexes, and MAPK signaling, which phosphorylates the MEF2 transcription activation domain, further stimulate muscle gene expression by enhancing MEF2 activity.
Molecular Cell 2000 6, DOI: ( /S (00) )