Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, ␣-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.Protein acetylation/deacetylation is involved in the regulation of protein structure and function, and therefore has potentially important roles in most cellular processes. In particular, the impact of histone N-terminal acetylation on chromatin organization and gene expression has been well documented (15). Acetylation and deacetylation of histone tails or of other proteins are catalyzed by histone acetyltransferases and histone deacetylases (HDACs), respectively. In mammals, there are 18 HDACs identified so far that can be grouped into three classes (reviewed in references 35, 36, and 39). In cells, most, if not all, class I and II HDACs are part of high-molecular-weight complexes that typically contain several HDAC polypeptides and are recruited to DNA via their interactions with sequence-specific or nonspecific DNA-binding proteins.HDAC 6 (HDAC6) was first identified through its homology to the Saccharomyces cerevisiae histone deacetylase HDA1 (9, 34). Like other class II HDACs, HDAC6 is mainly localized in the cytoplasm, but it can also shuttle between the nucleus and cytoplasm (33). This process is regulated by an N-terminally located nuclear export signal and possibly other uncharacterized mechanisms. HDAC6 has not been found in any class I or II HDAC-containing repressor complexes, which suggests it may have a unique regulation and possibly substrates different from those of other HDACs. However, it was shown biochemically and in genome-wide two-hybrid experiments to associate with the class III deacetylase SirT2 (22,26). Interestingly, HDAC6 contains two hdac catalyti...

The organization of the nucleus into subcompartments creates microenvironments that are thought to facilitate distinct nuclear functions. In budding yeast, regions of silent chromatin, such as those at telomeres and mating-type loci, cluster at the nuclear envelope creating zones that favour gene repression. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently owing to association of the gene with nuclear pore complexes. Here we report that transcriptional activation of a subtelomeric gene, HXK1 (hexokinase isoenzyme 1), by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3' untranslated region (UTR), which is essential for efficient messenger RNA processing and export, consistent with an accompanying report. However, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Notably, when we interfered with HXK1 localization by either antagonizing or promoting association with the pore, transcript levels were reduced or enhanced, respectively. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.

Histone deacetylases (HDACs) regulate gene expression by deacetylating histones and also modulate the acetylation of a number of nonhistone proteins, thus impinging on various cellular processes. Here, we analyzed the major class I enzymes HDAC1 and HDAC2 in primary mouse fibroblasts and in the B-cell lineage. Fibroblasts lacking both enzymes fail to proliferate in culture and exhibit a strong cell cycle block in the G1 phase that is associated with up-regulation of the CDK inhibitors p21 WAF1/CIP1 and p57 Kip2 and of the corresponding mRNAs. This regulation is direct, as in wild-type cells HDAC1 and HDAC2 are bound to the promoter regions of the p21 and p57 genes. Furthermore, analysis of the transcriptome and of histone modifications in mutant cells demonstrated that HDAC1 and HDAC2 have only partly overlapping roles. Next, we eliminated HDAC1 and HDAC2 in the B cells of conditionally targeted mice. We found that B-cell development strictly requires the presence of at least one of these enzymes: When both enzymes are ablated, B-cell development is blocked at an early stage, and the rare remaining pre-B cells show a block in G1 accompanied by the induction of apoptosis. In contrast, elimination of HDAC1 and HDAC2 in mature resting B cells has no negative impact, unless these cells are induced to proliferate. These results indicate that HDAC1 and HDAC2, by normally repressing the expression of p21 and p57, regulate the G1-to-S-phase transition of the cell cycle.[Keywords: Histone deacetylases; cell cycle control; p21; p57; B-cell development] Supplemental material is available at http://www.genesdev.org.

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from the bimC family (Le Guellec, R., Paris, J., Couturier, A., Roghi, C., and Philippe, M. (1991) Mol. Cell. Biol. 11, 3395-3408) as well as pEg2, an Aurora-related serine/threonine kinase (Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Doré e, M., Philippe, M., and Prigent, C. (1998) J. Cell Sci. 111, 557-572). Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation. Here, we report that in Xenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active when bound to the beads and phosphorylate XlEg5 present in the precipitate. Twodimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo first confirmed that XlEg5 was phosphorylated by p34 cdc2 and next revealed that in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain serine residue that was phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is to our knowledge the first in vivo substrate ever reported for an Aurora-related kinase.

Equal mitotic chromosome segregation is critical for genome integrity and is monitored by the spindle assembly checkpoint (SAC). We have previously shown that the consensus phosphorylation motif of the essential SAC kinase Monopolar spindle 1 (Mps1) is very similar to that of Polo-like kinase 1 (Plk1). This prompted us to ask whether human Plk1 cooperates with Mps1 in SAC signaling. Here, we demonstrate that Plk1 promotes checkpoint signaling at kinetochores through the phosphorylation of at least two Mps1 substrates, including KNL-1 and Mps1 itself. As a result, Plk1 activity enhances Mps1 catalytic activity as well as the recruitment of the SAC components Mad1:C-Mad2 and Bub3:BubR1 to kinetochores. We conclude that Plk1 strengthens the robustness of SAC establishment at the onset of mitosis and supports SAC maintenance during prolonged mitotic arrest.

The kinase Bub1 functions in the spindle assembly checkpoint (SAC) and in chromosome congression, but the role of its catalytic activity remains controversial. Here, we use two novel Bub1 inhibitors, BAY-320 and BAY-524, to demonstrate potent Bub1 kinase inhibition both in vitro and in intact cells. Then, we compared the cellular phenotypes of Bub1 kinase inhibition in HeLa and RPE1 cells with those of protein depletion, indicative of catalytic or scaffolding functions, respectively. Bub1 inhibition affected chromosome association of Shugoshin and the chromosomal passenger complex (CPC), without abolishing global Aurora B function. Consequently, inhibition of Bub1 kinase impaired chromosome arm resolution but exerted only minor effects on mitotic progression or SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of Paclitaxel, impairing both chromosome segregation and cell proliferation. These findings are relevant to our understanding of Bub1 kinase function and the prospects of targeting Bub1 for therapeutic applications.DOI: http://dx.doi.org/10.7554/eLife.12187.001

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.