Tso-Pang Yao

HDAC6 is a microtubule-associated deacetylase

Reversible acetylation of α-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated α-tubulin is tightly controlled and stereotypic. Acetylated α-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 p150glued (ref. 3). In vivo, the overexpression of HDAC6 leads to a global deacetylation of α-tubulin, whereas a decrease in HDAC6 increases α-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates α-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 Deacetylase HDAC6 Regulates Aggresome Formation and Cell Viability in Response to Misfolded Protein Stress

The efficient clearance of cytotoxic misfolded protein aggregates is critical for cell survival. Misfolded protein aggregates are transported and removed from the cytoplasm by dynein motors via the microtubule network to a novel organelle termed the aggresome where they are processed. However, the means by which dynein motors recognize misfolded protein cargo, and the cellular factors that regulate aggresome formation, remain unknown. We have discovered that HDAC6, a microtubule-associated deacetylase, is a component of the aggresome. We demonstrate that HDAC6 has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors, thereby acting to recruit misfolded protein cargo to dynein motors for transport to aggresomes. Indeed, cells deficient in HDAC6 fail to clear misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are hypersensitive to the accumulation of misfolded proteins. These findings identify HDAC6 as a crucial player in the cellular management of misfolded protein-induced stress.

HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS

A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. Here we show that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins, is an essential mechanistic link in this compensatory interaction. We found that compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensated for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 was sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Morover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy.

Gene Dosage–Dependent Embryonic Development and Proliferation Defects in Mice Lacking the Transcriptional Integrator p300

The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.

Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation

The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system.

HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.

p300/CBP‐mediated p53 acetylation is commonly induced by p53‐activating agents and inhibited by MDM2

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post‐translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress‐inducing agents including hypoxia, anti‐metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase‐deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP‐mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19ARF, indicating that regulation of acetylation is a central target of the p53–MDM2–p19ARF feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP‐mediated acetylation may be a universal and critical modifi cation for p53 function.

MDM2–HDAC1‐mediated deacetylation of p53 is required for its degradation

The tumor suppressor p53 is stabilized and activated in response to cellular stress through post‐translational modifications including acetylation. p300/CBP‐mediated acetylation of p53 is negatively regulated by MDM2. Here we show that MDM2 can promote p53 deacetylation by recruiting a complex containing HDAC1. The HDAC1 complex binds MDM2 in a p53‐independent manner and deacetylates p53 at all known acetylated lysines in vivo. Ectopic expression of a dominant‐negative HDAC1 mutant restores p53 acetylation in the presence of MDM2, whereas wild‐type HDAC1 and MDM2 deacetylate p53 synergistically. Fibroblasts overexpressing a dominant negative HDAC1 mutant display enhanced DNA damage‐induced p53 acetylation, increased levels of p53 and a more pronounced induction of p21 and MDM2. These results indicate that acetylation promotes p53 stability and function. As the acetylated p53 lysine residues overlap with those that are ubiquitylated, our results suggest that one major function of p53 acetylation is to promote p53 stability by preventing MDM2‐dependent ubiquitylation, while recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.

Distinct roles of the co-activators p300 and CBP in retinoic-acid-induced F9-cell differentiation

The related proteins p300 and CBP (cAMP-response-element-binding protein (CREB)-binding protein)) are transcriptional co-activators that act with other factors to regulate gene expression and play roles in many cell-differentiation and signal transduction pathways. Both proteins have intrinsic histone-acetyltransferase activity, and may act directly on chromatin, of which histone is a component, to facilitate transcription. They are also involved in growth control pathways, as shown by their interaction with the tumour suppressor p53 (refs 13–15) and the viral oncogenes E1A (refs 1, 2, 16) and SV40 T antigen. Here we report functional differences of p300 and CBP in vivo. We examined their roles during retinoic-acid-induced differentiation, cell-cycle exit and programmed cell death (apoptosis) of embryonal carcinoma F9 cells, using hammerhead ribozymes capable of cleaving either p300 or CBP messenger RNAs. F9 cells expressing a p300-specific ribozyme became resistant to retinoic-acid-induced differentiation, whereas cells expressing a CBP-specific ribozyme were unaffected. Similarly, retinoic-acid-induced transcriptional upregulation of the cell-cycle inhibitor p21Cip1 required normal levels of p300, but not CBP, whereas the reverse was true for p27Kip1. In contrast, both ribozymes blocked retinoic-acid-induced apoptosis, indicating that both co-activators are required for this process. Thus, despite their similarities, p300 and CBP have distinct functions during retinoic-acid-induced differentiation of F9 cells.

VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD.

VCP (VCP/p97) is a ubiquitously expressed member of the AAA+-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion, and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion myopathy, Paget’s disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or over-expression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2 –positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.