MyoD is constitutively acetylated by p300 and PCAF in differentiated muscle cells. Both p300 and PCAF acetylate K99 and K102 (and K104?) at the boundary of DNA-binding domain (109-121) of MyoD. Acetylation of MyoD is required for its DNA-binding and transcriptional abilities in C2 and 10T1/2 cells (Sartorelli, 1999; Polesskaya, 2000).
Interstingly, p300-mediated acetylation sites of GATA4 (K311, K318, K320, and K322) are also near but not within the DNA-binding domain (270-294; 2nd Zn finger) (Takaya, 2008).
Full-length p300 is required for MyoD acetylation; a HAT domain peptide can acetylate histone but not MyoD (Polesskaya, 2001a) because p300 bromodomain but not C/H3 domain physically interacts with MyoD (Polesskaya, 2001b).
Both of GATA4 (80-250; 1st Zn finger) and GATA4 (240-378; 2nd Zn finger) bind to p300 (1587-1817; C/H3) (Dai, 2001). Although MyoD mutants that cannot be acetylated does not associate with p300, which trends are different from GATA4. Both acetylated and non-acetylated GATA4 equally bind to p300 (Takaya, 2008).
Through activating MyoD, p300 induces permanent cell cycle arrest by upregulating p21 in differentiating C2 cells (Puri, 1997). p8 (Nupr1) synergistically acts with p300 for acetylating MyoD and cell cycle arrest (Sambasivan, 2009). Conversely in proliferating C2 cells, class I HDAC1 binds to MyoD and deacetylates it. HDAC1 silences MyoD-dependent transcription of p21 and inhibits myogenic conversion in 10T1/2 cells (Mal, 2001). Interaction of MyoD with HDAC1 in undifferentiated myoblasts also mediates repression of muscle-specific genes. pRb forms complex with HDAC1 and promotes muscle gene expression (Puri, 2001). Although class II HDAC4/5 do not interact directly with MyoD, they interact with MEF2 and suppress the myogenic activity of MyoD (Lu, 2000). Class III HDAC Sirt1 forms complex with PCAF and MyoD. Sirt1 NAD+-dependently deacetylates MyoD and delays muscle differentiation (Fulco, 2003).
Class I HDAC2 with Hopx deacetylates GATA4 expressed in cardiac progenitor cells during heart development. GATA4 hyperacetylation in HDAC2/Hopx double knockout mice causes a marked increase in cardiomyocyte proliferation by upregulating cell cycle genes including cyclin D2 and Cdk4 (Rojas, 2008; Trivedi, 2010). It’s opposite to MyoD. MyoD is constitutively acetylated in differentiated condition, while GATA4 is temporary acetylated in embryonic condition (heart development or disease). This might be a reason of the different pattern of binding to p300 between Ac-MyoD and Ac-GATA4.
p300 knockout mice die during E9-11.5 with defects in heart development (Yao, 1998). Heterozygous mice for p300 HAT mutation (p300+/AS) almost die during E12.5-16.5 due to heart failure; HAT mutation is dominant-negative. p300+/AS embryo show reduced skeletal muscle fibers, diminished or delayed expression of MyoD, Myf5, myogenin, actin, and MHC. Correspondingly, p300AS/AS ES cells fail to activate MyoD and Myf5 transcription efficiently, while Pax3 is expressed (Roth, 2003).
In vitro experiments show non-redundant roles of p300 and PCAF during MyoD-dependent transactivation. At the initial stages, p300 acetylates histone H3 and H4 within the promoter region and then recruits PCAF to MyoD. Once tethered to the promoter, PCAF acetylates MyoD to facilitate the transactivation process. Both p300 and PCAF are required for maximal levels of transactivation (Dilworth, 2004).
p300-dependent nuclear hyperacetylation including histone H3/4 acetylation is observed during hypertrophic responses in cultured cardiomyocytes (Morimoto, 2008).
p300 or PCAF maintains viability of C2 myoblast through mechanisms requiring the HAT activity of PCAF but not of p300. p300 promotes cell survival, which is independent of its acetyltransferase activity and activating MyoD in C2 cells (Kuninger, 2006).
S200 of MyoD is Cdk phosphorylation site. S200A mutation prolongs the half-time of MyoD protein (Wt, 30 min; S200A, 150 min). Phosphorylation of MyoD is required for its rapid degradation by proteasome in 10T1/2 cells (Song, 1998). Cdk7/8/9 and their associated cylclin T1/2 are not serum- or cell cycle-regulated but instead regulate transcriptional elongation by phosphorylating RNA pol II. Cdk9/cyclin T2 phosphorylates MyoD and strengthns MyoD-dependent transcription in C2 cells. MyoD (101-161; bHLH) interacts with Cdk9 (1-128) (Simone, 2002).
GATA4 phosphorylation is involved in hypertrophic responses in cardiomyocytes (Morimoto, 2000). However, it’s still ambiguous whether Cdk9 directly phosphorylates GATA4 (unpublished data). GATA4 (180-255; 1st Zn finger) interacts with Cdk9 (83-129), and p300 (1514-1922; C/H3) binds to Cdk9 (1-82). GATA4- and MyoD-binding sites share the same region og Cdk9. In addition, Cdk9 phosphorylates p300 (1514-1922), which is required for p300 activation, GATA4 acetylation, and eventually cardiac gene expression (Sunagawa, 2010).
MyoD recruits Cdk9/cyclin T2, together with p300 and PCAF, and the chromatin remodeling complex SWI/SNF, on promoters and enhancers of muscle-specific genes, and that this event correlates with the acetylation of hisone tails, remodeling of chromatin, and phosphorylation of the C-terminal domain of the RNA pol II at these elements (Giacinti, 2006).
GATA4 also recruits p300/Cdk9/cyclin T1 on cardiac gene promoters with phosphorylating RNA pol II (Sunagawa, 2010). GATA4 directly interacts with Ini1 which is a component of SWI/SNF complex. Ini1 binds to p300 and inhibits p300-induced GATA4 transactivation (unpublished data).
Cdk9 isoform Cdk9-55 (kDa) is specifically induced in injured myofibers and its activity is strictly required for the completion of muscle regeneration process (Giacinti, 2008). At present, available Cdk9 knockout mouse strain is not exsited.
The mechanism of p300/Cdk9/cyclin-induced activation of MyoD is very similar to that of GATA4. Repressing GATA4 acetylation by inhibiting p300 HAT activity results in decreased cardiac gene expression and pathophysiological changes in cardiomyocytes (Yanazume, 2003; Miyamoto, 2006; Sunagawa, 2010). These suggest that inhibiting p300 would repress MyoD acetylation and serve as MyoD knockdown.
Curcumin, an inhibitor of p300 HAT activity, represses hypertrophic responses in cardiomyocytes by inhibiting GATA4 acetylation and cardiac transcription (Morimoto, 2008; Sunagawa, 2011). If curcumin could inhibit p300-mediated MyoD activation, curcumin administration might serve as well as MyoD knockdown. Supportingly, curcumin alleviates dystrophic muscle pathology in mdx mice (Pan, 2008). THERACURMIN, which is recently developed as an absorptive curcumin dispersed with colloidal nano-particles, can prevent heart failure at lower dose compared with native curcumin (Sasaki, 2011; Sunagawa, 2012). THERACURMIN will be also helpful to perform in vitro experiments.
Dai YS. p300 functions as a coactivator of transcription factor GATA-4. J Biol Chem, 2001.
Dilworth FJ. In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation. Proc Natl Acad Sci USA, 2004.
Giacinti C. MyoD recruits the cdk9/cyclin T2 complex on myogenic-genes regulatory regions. J Cell Physiol, 2006.
Fulco M. Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state. Mol Cell, 2003.
Giacinti C. Cdk9-55: an new player in muscle regeneration. J Cell Physiol, 2008.
Kuninger D. Muscle cell survival mediated by the transcriptional coactivator p300 and PCAF displays different requirements for acetyltransferase activity. Am J Physiol Cell Physiol, 2006.
Lu J. Regulation of skeletal myogenesis by association of the MEF2 transcription factor with class II histone deacetylases. Mol Cell, 2000.
Mal A. A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program. EMBO J, 2001.
Miyamoto S. Histone acetyltransferase activity of p300 is required for the promotion of left ventricular remodeling after myocardial infarction in adult mice in vivo. Circulation, 2006.
Morimoto T. Phosphorylation of GATA-4 is involved in α1-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes. J Biol Chem, 2000.
Morimoto T. The dietary compound curcumin inhibits p300 histone acetyltransferase activity and prevents heart failure in rats. J Clin Invest, 2008.
Pan Y. Curcumin alleviates dystrophic muscle pathology in mdx mice. Mol Cells, 2008.
Polesskaya A. CREB-binding protein/p300 activates MyoD by acetylation. J Biol Chem, 2000.
Polesskaya A. Acetylation of MyoD by p300 requires more than its histone acetyltransferase domain. J Biol Chem, 2001a.
Polesskaya A. Interaction between acetylated MyoD and the bromodomain of CBP and/or p300. Mol Cell Biol, 2001b.
Puri PL. p300 is required for MyoD-dependent cell cycle arrest and muscle-specific gene transcription. EMBO J, 1997.
Puri PL. Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis. Mol Cell, 2001.
Rojas A. GATA4 is a direct transcriptional activator of cyclin D2 and Cdk4 and is required for cardiomyocyte proliferation in anterior heart field-derived myocardium. Mol Cell Biol, 2008.
Roth JF. Differential role of p300 and CBP acetyltransferase during myogenesis: p300 acts upstream of MyoD and Myf5. EMBO J, 2003.
Sambasivan R. The small chromatin-binding protein p8 coordinates the association of anti-proliferative and pro-myogenic proteins at the myogenin promoter. J Cell Sci, 2009.
Sartorelli V. Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program. Mol Cell, 1999.
Sasaki H. Innovative preparation of curcumin for improved oral bioavailability. Biol Pharm Bull, 2011.
Simone C. Activation of MyoD-dependent transcription by cdk9/cyclin T2. Oncogene, 2002.
Song A. Phosphorylation of nuclear MyoD is required for its rapid degradation. Mol Cell Biol, 1998.
Sunagawa Y. Cyclin-dependent kinase-9 is a component of the p300/GATA4 complex required for phenylephrine-induced hypertrophy in cardiomyocytes. J Biol Chem, 2010.
Sunagawa Y. A natural p300-specific histone acetyltransferase inhibitor, curcumin, in addition to angiotensin-converting enzyme inhibitor, exerts beneficial effects of left ventricular systolic function after myocardial infarction in rats. Circ J, 2011.
Sunagawa Y. A novel drug delivery system of oral curcumin markedly improves efficacy of treatment for heart failure after myocardial infarction in rats. Biol Pharm Bull, 2012.
Takaya T. Identification of p300-targeted acetylated residues in GATA4 during hypertrophic responses in cardiac myocytes. J Biol Chem, 2008.
Trivedi CM. Hopx and Hdac2 interact to modulate Gata4 acetylation and embryonic cardiac myocyte proliferation. Dev Cell, 2010.
Yanazume T. Cardiac p300 is involved in myocyte growth with decompensated heart failure. Mol Cell Biol, 2003.
Yao TP. Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell, 1998.
Wang L. GATA-binding protein 4 (GATA-4) and T-cell acute leukemia 1 regulate myogenic differentiation and erythropoietin response via cross-talk with Sirtuin1 (Sirt1). J Biol Chem, 2012.