Functional characterization of an amino-terminal region of HDAC4 that possesses MEF2 binding and transcriptional repressive activity

Like the full-length histone deacetylase (HDAC) 4, its amino terminus (amino acids 1-208) without the carboxyl deacetylase domain is also known to effectively bind and repress myocyte enhancer factor 2 (MEF2). Within this repressive amino terminus, we further show that a stretch of 90 amino acids (119-208) displays MEF2 binding and repressive activity. The same region is also found to associate specifically with HDAC1 which is responsible for the repressive effect. The amino terminus of HDAC4 can associate with the DNA-bound MEF2 in vitro, suggesting that it does not repress MEF2 simply by disrupting the ability of MEF2 to bind DNA. In vivo, MEF2 induces nuclear translocation of both the full-length HDAC4 and HDAC4-(1-208), whereas the nuclear HDAC4 as well as HDAC4-(1-208) in turn specifically sequesters MEF2 to distinct nuclear bodies. In addition, we show that MyoD and HDAC4 functionally antagonize each other to regulate MEF2 activity. Combined with data from others, our data suggest that the full-length HDAC4 can repress MEF2 through multiple independent repressive domains.     

Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC–MEF2 complexes

Histone deacetylase (HDAC) inhibitors are promising new epi‐drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II‐specific HDAC inhibitor MC1568, we separated class I‐ and class II‐dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4–HDAC3–MEF2D complex, and (iii) paradoxically, by inhibiting differentiation‐induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue‐selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2–HDAC complexes in a repressed state. Our results suggest that HDAC class II‐selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.     

Histone deacetylase 4 possesses intrinsic nuclear import and export signals

Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.