Identification of a signal-responsive nuclear export sequence in class II histone deacetylases.

Whether or not a gene is transcribed is dependent on the packaging state of local chromatin, which is organized in nucleosomes. Chromosomal DNA present in condensed chromatin is generally inaccessible to high-molecular-weight transcriptional machinery and is thus transcriptionally silent. However, acetylation of lysine residues in the tails of nucleosomal histones relaxes chromatin structure and promotes gene expression. This posttranslational modification is catalyzed by histone acetyl transferases (HATs). Histone acetylation also provides a reinforcing mechanism for chromatin relaxation by creating binding sites for bromodomain-containing transcriptional activators, which typically possess HAT activity. The stimulatory effects of HATs on gene transcription are antagonized by histone deacetylases (HDACs) (reviewed in reference 30). Skeletal muscle provides a tractable model system to study the dynamic interplay between HATs and HDACs in the control of cellular differentiation (reviewed in reference 23). During the formation of skeletal muscle, undifferentiated myoblasts irreversibly exit the cell cycle and fuse to form multinucleated myofibers with an organized contractile apparatus. Skeletal myogenesis is controlled by a highly specific transcriptional program, which regulates subordinate genes in either a positive or a negative manner. Members of the MEF2 family of transcription factors serve a central role in governing this program (reviewed in reference 1). MEF2 belongs to the MADS-box superfamily of transcription factors and binds an A/T-rich element present in the regulatory regions of numerous muscle-specific genes. The four MEF2 factors, MEF2A, -B, -C, and -D, share homology in an amino-terminal MADS domain, which mediates DNA binding and dimerization, and an adjacent MEF2 domain, which regulates cofactor interactions. MEF2 can function as either a transcriptional activator or repressor, depending on whether it is physically associated with HATs or HDACs. Binding of the p300 coactivator, which possesses HAT activity, to the MADS-MEF2 domains of MEF2 results in activation of downstream target genes (28). Conversely, binding of class II HDAC4 and -5 to the same region of MEF2 converts the transcription factor into a potent repressor of gene expression (15, 16, 24, 33). Class II HDACs contain a carboxy-terminal catalytic domain and an amino-terminal extension that mediates binding to MEF2 (4, 6, 31). HDAC4 and -5 act as potent repressors of skeletal myogenesis by virtue of their ability to associate with MEF2 (17). However, during myogenesis they dissociate from MEF2 and are exported to the cytoplasm, thereby freeing MEF2 to stimulate muscle gene expression (21). The effect of myogenic signals on HDAC localization can be mimicked by calcium-calmodulin-dependent protein kinase (CaMK) signaling (21). Previously, we showed that CaMK-mediated nuclear export of HDAC5 was dependent on phosphorylation of two serine residues in its amino-terminal extension, Ser-259 and Ser-498 (21). Phosphorylation at these residues creates docking sites for the intracellular chaperone protein 14-3-3 (22). Nuclear export of HDAC5 appears to require 14-3-3 binding, since replacement of Ser-259 and Ser-498 with alanine abolishes not only its association with 14-3-3 but also its nuclear export (21, 22). However, the existence of carboxy-terminal truncation mutants of HDAC5 that retain the ability to bind 14-3-3 but remain in the nucleus despite active CaMK signaling suggests that 14-3-3 binding alone is insufficient to drive HDAC5 out of the nucleus (22). To further define the mechanism that regulates nuclear export of class II HDACs, we analyzed the behavior of a series of HDAC5 mutants. Here we describe a CaMK-responsive nuclear export sequence (NES) present near the extreme carboxy terminus of HDAC5. This regulatory sequence is conserved in HDAC4 and -7 but is absent in class I HDACs and the MEF2-interacting transcription repressor (MITR). Our studies indicate that phosphorylation-dependent binding of 14-3-3 to the amino-terminal extension of HDAC4 and -5 is required to activate the carboxy-terminal NES, culminating in shuttling of these transcriptional repressors to the cytoplasm. These results provide a molecular explanation for the signal-responsiveness of class II HDACs and the MEF2 target genes that are under their control.