Reversible acetylation of alpha-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated alpha-tubulin is tightly controlled and stereotypic. Acetylated alpha-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it associates with microtubules and localizes with the microtubule motor complex containing p150(glued) (ref. 3). In vivo, the overexpression of HDAC6 leads to a global deacetylation of alpha-tubulin, whereas a decrease in HDAC6 increases alpha-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates alpha-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biological processes beyond histone metabolism and gene transcription.

The growing number of proteins controlled by reversible acetylation suggests the existence of a large number of acetyltransferases and deacetylases. Here, we report the identification of a novel class II histone deacetylase, HDAC10. Homology comparison indicates that HDAC10 is most similar to HDAC6. Both contain a unique, putative second catalytic domain not found in other HDACs. In HDAC10, however, this domain is not functional. This tandem organization of two catalytic domains confers resistance to the inhibitors trapoxin B and sodium butyrate, which potently inhibit the deacetylase activity of all other HDAC members. Thus, HDAC10 and HDAC6 share unusual structural and pharmacological characteristics. However, unlike HDAC6, which is normally a cytoplasmic deacetylase, HDAC10 resides in both the nucleus and cytoplasm. In the nucleus, when tethered to a promoter, HDAC10 represses transcription independent of its deacetylase activity, indicating that HDAC10 contains a distinct transcriptional repressor domain. These observations suggest that HDAC10 might uniquely play roles both in the nucleus, as a transcriptional modulator, and in the cytoplasm in an unidentified role. Together, our results identify HDAC10 as a novel deacetylase with distinct structure, pharmacology and localization and further expand the complexity of the HDAC family.It has been known for many years that histone acetylation, a post-translational modification on specific lysine residues, is important for regulating the transcription of many genes (1). It is generally believed that hypo-acetylated histones have an increased charge, allowing chromatin condensation and, consequently, are associated with transcriptionally silent regions. Conversely, hyper-acetylation of histones neutralizes this charge, permitting chromatin decondensation and, thus, is associated with transcriptionally active regions (reviewed by Wade et al. (2)). The acetylation of these lysine residues is catalyzed by histone acetyltransferases, whereas the removal of the acetyl group is carried out by histone deacetylases (HDACs).1 Recent studies, however, have expanded the substrate repertoire of histone acetyltransferases and HDACs, suggesting that the function of acetylation is not limited to histone metabolism or transcription but may regulate a variety of biological processes. Indeed, acetylation has been shown to regulate the DNA binding activity of several transcription factors (3, 4), nuclear import (5), microtubule function (6), 2 and protein-protein interactions (7). Additionally, acetylation is an important modification in the pathway that leads to p53 activation and stabilization (8). These results suggest that acetylation is a critical post-translational modification that, like phosphorylation, has diverse biological effects.Recent studies have identified nine members of the mammalian HDAC family, and they are divided into two classes, originally defined by size and sequence homology to the yeast prototypic HDACs. The class I HDACs, which are about 400 -500 amino ac...

Understanding the generation of neuronal and glial diversity is one of the major goals of developmental neuroscience. The Drosophila CNS midline cells constitute a simple neurogenomic system to study neurogenesis, cell fate acquisition, and neuronal function. Previously, we identified and determined the developmental expression profiles of 224 midline-expressed genes. Here, the expression of 59 transcription factors, signaling proteins, and neural function genes was analyzed using multi-label confocal imaging, and their expression patterns mapped at the single-cell level at multiple stages of CNS development. These maps uniquely identify individual cells and predict potential regulatory events and combinatorial protein interactions that may occur in each midline cell type during their development. Analysis of neural function genes, including those encoding peptide neurotransmitters, neurotransmitter biosynthetic enzymes, transporters, and neurotransmitter receptors, allows functional characterization of each neuronal cell type. This work is essential for a comprehensive genetic analysis of midline cell development that will likely have widespread significance given the high degree of evolutionary conservation of the genes analyzed.

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6⅐PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6⅐PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC⅐phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.Reversible protein phosphorylation is the most prevalent protein modification regulating eukaryotic cell physiology. Identification of numerous acetyltransferases and deacetylases suggests that protein acetylation also controls many physiological events (1). Studies of histone acetylation and phosphorylation show that both protein modifications play key roles in chromatin remodeling and gene transcription (2). The cellular mechanisms that coordinate the acetylation and phosphorylation of histones and other cellular proteins are still poorly understood.The precise orchestration of protein phosphorylation and acetylation is best exemplified during growth factor-stimulated phosphorylation and acetylation of histone H3 (3). Histone H3 is phosphorylated on serine 10 in fibroblasts in response to mitogens or following oncogenic transformation (4). Several kinases (5-8) catalyze the phosphorylation of histone H3 at serine 10 to generate an improved substrate for acetyltransferases that modify the adjacent lysine 14 (9). Conversely, serine 10 phosphorylation inhibits the acetylation of histone H3 at lysine 4 (10). The changes in phosphorylation and acetylation of histone H3 create novel platforms that recruit nuclear factors and/or stabilize pre-existing nuclear complexes required for chromatin remodeling and gene expression (2). How cells orchestrate the ordered phosp...

SUMMARYDopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities.

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