Class IIa Histone Deacetylases Are Potent Regulators of MEF2

Members of the histone deacetylase (HDAC) family have emerged as critical downstream targets of CaMKII in response to neural activity. Specifically, the class IIa HDAC subfamily, which includes HDAC4, HDAC5, HDAC7 and HDAC9, has been intimately linked to MEF2 activity. All class IIa HDACs bind and inhibit MEF2 transcriptional activity (Sparrow et al. 1999; Lu et al. 2000a, b; Miska et al. 1999; Wang et al. 1999; Lemercier et al. 2000). They share a common domain structure: an N-terminus coiled-coil domain and a C-terminus deacetylase domain (Fig. 2). The N-terminus binds MEF2, transcriptional corepressors, and SUMO-conjugating enzyme Ubc9 (Verdin et al. 2003; Zhang et al. 2002a). Most studies regarding class IIa HDACs in skeletal muscle development has revolved around HDAC4 and HDAC5. HDAC4 and HDAC5 are highly homologous, sharing 51% identity and 63% similarity in their amino acid sequence (Grozinger et al. 1999). Both are potent inhibitors of MEF2-dependent transcription. Indeed, overexpression of HDAC4 or -5 prevents cultured myocytes from differentiating into myotubes (Lu et al. 2000a). However, this antidifferentiation activity probably does not reflect the physiological function of HDAC4 or -5 in skeletal muscle.

HDACs have been well characterized as transcriptional corepressors. It is widely accepted that recruitment of HDACs by DNA-binding transcription factors leads to

Fig. 2 The domain structure of class lia HDAC members. Serine residues subject to phosphorylation (P) and 14-3-3 binding are marked. The domains responsible for binding CtBP, MEF2, Ubc9, and HP1 are color coded. NLS Nuclear localization signal; NES Nuclear export signal

histone deacetylation at the promoter region, resulting in transcription repression. This simple model, however, does not apply to class Ila HDACs, as the deacetylase domain is dispensable for repressing MEF2 activity (Zhang et al. 2001a). The conserved N-terminus is all that is required for class Ila HDACs to bind and repress MEF2 activity. In fact, Fischle et al. first reported that HDAC4 lacks intrinsic histone deacetylase activity (Fischle et al. 2002), a conclusion subsequently supported by the structural analysis of the catalytic domain of HDAC4 (Lahm et al. 2007; Bottomley et al. 2008). When ectopically expressed in cells, HDAC4 and -5 were found to associate with a class I deacetylase, HDAC3 (Grozinger et al. 1999). An extensive examination of the HDAC4-HDAC3 interaction has led to a conclusion that HDAC3, and not HDAC4, is responsible for the histone deacetylase activity assigned to purified HDAC4 (Fischle et al. 2002). The detailed nature of the HDAC4-HDAC3 complex remains to be established but it requires N-CoR/SMRT, related transcriptional cofactors that can stimulate HDAC3 deacetylase activity by a chaperone-like activity (Guenther et al. 2001). HDAC3 and N-CoR/SMRT exist in a native multiprotein corepressor complex that is responsible for the transcrip-tional repressive activity of unliganded retinoic acid receptor and thyroid hormone receptor (Yoon et al. 2003; Wen et al. 2000). Thus, it is possible that HDAC4 interacts with the HDAC3/N-CoR/SMRT complex to repress MEF2 transcription activity. However, it should be noted that endogenous HDAC4 was not reported in the HDAC3/N-CoR/SMRT complex (Yoon et al. 2003; Wen et al. 2000). Conversely, HDAC3 was not found in the native HDAC4 complex (E. Seto, unpublished observation). Whether HDAC3 is an obligatory cofactor for HDAC4 and other class IIa HDAC to repress MEF2 transcriptional activity remains to be established.

Two additional mechanisms could account for the deacetylase-independent activity of class IIa HDACs. In a yeast two hybrid study, HDAC4 and the N-terminus splice variant of HDAC9, MEF2-interacting transcription repressor (MITR), were found to interact with HP1, a heterochromatin-associated protein involved in transcriptional silencing. HP1 binds lysine 9-methylated histone H3, which typically marks transcriptional repressive region of the chromatin (Zhang et al. 2002a). Indeed, histones (H3) in chromatin flanking myogenic genes are highly methylated at K9 in undifferentiated, proliferating myoblasts, but become demethylated upon myocyte differentiation (Zhang et al. 2002a). The recruitment of HP1 by class IIa HDAC could silence MEF2-dependent transcription.

The conserved N-terminus of HDAC4 also binds Ubc9, an E2-conjugating enzyme for small ubiquitin-like molecule, SUMO (Gregoire and Yang 2005). By simultaneously interacting with Ubc9 and MEF2, HDAC4 promotes MEF2 sumoy-lation (Gregoire and Yang 2005; Zhao et al. 2005). In a reconstituted in vitro system, recombinant HDAC4 can directly stimulate MEF2 sumoylation (Zhao et al. 2005). HDAC4 therefore acts as SUMO E3 ligase for MEF2, whose sumoy-lation results in a loss of transcription activity. MEF2 is sumoylated at lysine 424, one of the lysine residues subject to acetylation by the transcriptional coactivator p300. The coupled HDAC4-dependent deacetylation and sumoylation at K424 unravels an efficient mechanism to inhibit MEF2 transcriptional activity. Interestingly, HDAC4 does not deacetylate MEF2 directly (Zhao et al. 2005); rather it recruits SIRT1, a member of the sirtuin deacetylase family, to catalyze MEF2 deacetylation (Zhao et al. 2005). As SIRT1 is regulated by nutrient and metabolic status, this finding suggests that HDAC4 and SIRT1 might work in conjunction to regulate MEF2 activity in skeletal muscle in response to nutrient availability and change in metabolic demands. In addition to SIRT1, Gregoire et al. showed that HDAC3 is able to bind and deacetylate MEF2 (Gregoire et al. 2007). It is also possible that a potential complex of HDAC4 and HDAC3 might work in tandem to control MEF2 acetylation and sumoylation.

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