A vertebrate family without a functional Hypocretin/Orexin arousal system.

The Hypocretin/Orexin signaling pathway suppresses sleep and promotes arousal, whereas the loss of Hypocretin/Orexin results in narcolepsy, including the involuntary loss of muscle tone (cataplexy).1 Here, we show that the South Asian fish species Chromobotia macracanthus exhibits a sleep-like state during which individuals stop swimming and rest on their side. Strikingly, we discovered that the Hypocretin/Orexin system is pseudogenized in C. macracanthus, but in contrast to Hypocretin-deficient mammals, C. macracanthus does not suffer from sudden behavioral arrests. Similarly, zebrafish mutations in hypocretin/orexin show no evident signs of cataplectic-like episodes. Notably, four additional species in the Botiidae family also lack a functional Hypocretin/Orexin system. These findings identify the first vertebrate family that does not rely on a functional Hypocretin/Orexin system for the regulation of sleep and arousal.

The SKP1-Cullin-F-box E3 ligase ßTrCP and CDK2 cooperate to control STIL abundance and centriole number.

Deregulation of centriole duplication has been implicated in cancer and primary microcephaly. Accordingly, it is important to understand how key centriole duplication factors are regulated. E3 ubiquitin ligases have been implicated in controlling the levels of several duplication factors, including PLK4, STIL and SAS-6, but the precise mechanisms ensuring centriole homeostasis remain to be fully understood. Here, we have combined proteomics approaches with the use of MLN4924, a generic inhibitor of SCF E3 ubiquitin ligases, to monitor changes in the cellular abundance of centriole duplication factors. We identified human STIL as a novel substrate of SCF-ßTrCP. The binding of ßTrCP depends on a DSG motif within STIL, and serine 395 within this motif is phosphorylated in vivo SCF-ßTrCP-mediated degradation of STIL occurs throughout interphase and mutations in the DSG motif causes massive centrosome amplification, attesting to the physiological importance of the pathway. We also uncover a connection between this new pathway and CDK2, whose role in centriole biogenesis remains poorly understood. We show that CDK2 activity protects STIL against SCF-ßTrCP-mediated degradation, indicating that CDK2 and SCF-ßTrCP cooperate via STIL to control centriole biogenesis.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging.

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.

Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524.

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.

Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells.

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.

On the regulation, function, and localization of the DNA-dependent ATPase PICH.

The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression.

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.

Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro.

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.

Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally.

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, alpha-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.

Nuclear pore association confers optimal expression levels for an inducible yeast gene.

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.

A homotrimer-heterotrimer switch in Sir2 structure differentiates rDNA and telomeric silencing.

The budding yeast genome contains transcriptionally repressed domains at mating-type and telomeric loci, and within rDNA repeats. Gene silencing at telomeres requires the Silent information regulators Sir2p, Sir3p, and Sir4p, whereas only the Sir2p histone deacetylase is required for rDNA repression. To understand these silencing mechanisms biochemically, we examined the subunit structure of Sir2p-containing complexes. Sir2p alone forms a stable homotrimer, whereas the SIR complex is a heterotrimer containing one copy of each Sir protein. A point mutation in the Sir2p core domain (sir2(P394L)) compromises selectively rDNA repression. This mutation impairs homotrimerization but allows SIR heterotrimer formation. Surprisingly, when sir2(P394L) is coexpressed with wild-type Sir2p, rDNA repression increases and homotrimers form. Furthermore, coexpression of sir2(P394L) and enzymatically inactive sir2(H364Y) allows crosscomplementation of rDNA repression defects. The correlation of genetic and biochemical complementation argues that Sir2p trimerization is physiologically relevant for rDNA silencing.

Control of Shugoshin function during fission-yeast meiosis.

Meiosis consists of a single round of DNA replication followed by two consecutive nuclear divisions. During the first division (MI), sister kinetochores must orient toward the same pole to favor reductional segregation. Correct chromosome segregation during the second division (MII) requires the retention of centromeric cohesion until anaphase II. The spindle checkpoint protein Bub1 is essential for both processes in fission yeast . When bub1 is deleted, the Shugoshin protein Sgo1 is not recruited to centromeres, cohesin Rec8 does not persist at centromeres, and sister-chromatid cohesion is lost by the end of MI. Deletion of bub1 also affects kinetochore orientation because sister centromeres can move to opposite spindle poles in approximately 30% of MI divisions. We show here that these two functions are separable within the Bub1 protein. The N terminus of Bub1 is necessary and sufficient for Sgo1 targeting to centromeres and the protection of cohesion, whereas the C-terminal kinase domain acts together with Sgo2, the second fission-yeast Shugoshin protein, to promote sister-kinetochore co-orientation during MI. Additional analyses suggest that the protection of centromeric cohesion does not operate when sister kinetochores attach to opposite spindle poles during MI. Sgo1-mediated protection of centromere cohesion might therefore be regulated by the mode of kinetochore attachment.

Multiple roles of Condensins: a complex story.

Condensins are pentameric complexes that were initially described as being involved in the dynamics of chromosomes during mitosis. It has been recently established that two related complexes (Condensin I and Condensin II) contribute to this process. An increasing sum of studies, using different approaches in various organisms, leads to the paradigm that Condensins are required for the correct segregation of replicated chromosomes by cooperating somehow with Topoisomerase II in sister chromatid resolution. Depending on species and/or experimental studies, these complexes also contribute to some aspects of the assembly and compaction of mitotic chromosomes. Recent studies provided evidences that Condensins and related complexes also function in non-mitotic processes such as replication and transcription. Biochemical studies have highlighted mechanistic aspects of Condensin function and initiated a fine functional dissection of core and regulatory subunits. However, the exact contribution of each subunit remains largely elusive as well as the functional interplay between Condensin I and Condensin II.

Expression and functional dynamics of the XCAP-D2 condensin subunit in Xenopus laevis oocytes.

The 13 S condensin complex plays a crucial role in the condensation and segregation of the two sets of chromosomes during mitosis in vivo as well as in cell-free extracts. This complex, conserved from yeast to human, contains a heterodimer of structural maintenance of chromosome (SMC) family proteins and three additional non-SMC subunits. We have investigated the expression of the non-SMC condensin component XCAP-D2 in Xenopus laevis oocytes. When studied during meiotic maturation, XCAP-D2 starts to accumulate at the time of germinal vesicle breakdown and reaches its maximal amount in metaphase II oocytes. This accumulation is specifically blocked by injection of antisense oligonucleotides. XCAP-D2 antisense-injected oocytes progress normally through meiosis until metaphase II. At this stage, however, chromosomes exhibit architecture defaults, and resolution of sister chromatids is impaired. Surprisingly, in mitotic extracts made from XCAP-D2 knocked-down oocytes, sperm chromatin normally condenses into compacted chromosomes, whereas the amounts of both free and chromosome-bound XCAP-D2 are markedly reduced. This apparent discrepancy is discussed in light of current knowledge on chromosome dynamics.

Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis condensin complex in interphase cells.

Cell cycle dynamics and localization of condensins--multiprotein complexes involved in late stages of mitotic chromosome condensation--were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to beta-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF, fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.