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SUMO homeostasis promotes error-free chromosome segregation. Quan et al. report the structure of a targeting peptide of the Ulp2 desumoylase bound to yeast Ctf3/CENP-I. Disrupting the interaction produces hyper-sumoylated kinetochores, demonstrating the existence of a conserved and dedicated pathway for the regulation of kinetochore sumoylation., The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle–regulated processes.

Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

During a single human lifetime, nearly one quintillion chromosomes separate from their sisters and transit to their destinations in daughter cells. Unlike DNA replication, chromosome segregation has no template, and, unlike transcription, errors frequently lead to a total loss of cell viability. Rapid progress in recent years has shown how kinetochores enable faithful execution of this process by connecting chromosomal DNA to microtubules. These findings have transformed our idea of kinetochores from cytological features to immense molecular machines and now allow molecular interpretation of many long-appreciated kinetochore functions. In this review we trace kinetochore protein connectivity from chromosomal DNA to microtubules, relating new findings to important points of regulation and function.

SummaryAccurate segregation of genetic material in eukaryotes relies on the kinetochore, a multiprotein complex that connects centromeric DNA with microtubules. In yeast and humans, two proteins—Mif2/CENP-C and Chl4/CNEP-N—interact with specialized centromeric nucleosomes and establish distinct but cross-connecting axes of chromatin-microtubule linkage. Proteins recruited by Chl4/CENP-N include a subset that regulates chromosome transmission fidelity. We show that Chl4 and a conserved member of this subset, Iml3, both from Saccharomyces cerevisiae, form a stable protein complex that interacts with Mif2 and Sgo1. We have determined the structures of an Iml3 homodimer and an Iml3-Chl4 heterodimer, which suggest a mechanism for regulating the assembly of this functional axis of the kinetochore. We propose that at the core centromere, the Chl4-Iml3 complex participates in recruiting factors, such as Sgo1, that influence sister chromatid cohesion and encourage sister kinetochore biorientation.

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surface of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres. DOI: http://dx.doi.org/10.7554/eLife.06057.001, eLife digest DNA replication copies the genetic information contained in a cell's chromosomes. A ring-like protein complex, cohesin, holds together each pair of newly-replicated chromosomes, known as ‘sister chromatids’. When the cell divides, cohesin is cleaved; this allows sister chromatids to separate, so that each daughter cell receives one member of each sister chromatid pair and thereby inherits a full complement of genes. Defects in this process result in severe developmental abnormalities. Moreover, the genes that underlie this process are among the most frequently mutated in cancer. Cohesin is enriched at centromeres: the chromosomal points of attachment to the apparatus (called the ‘mitotic spindle’) that segregates the sister chromatids into the daughter cells. The protein complex that loads cohesin onto chromosomes determines this preferential localization. The two components of the loading complex, Scc2 and Scc4, associate as a 1:1 pair. Hinshaw et al. used a technique called X-ray crystallography to determine the structure of Scc4 bound with a large fragment of Scc2. The result showed that the elongated Scc4 twists around the fragment of Scc2, forming an extended groove. Scc2 snakes through the Scc4 groove and emerges at both ends. Hinshaw et al. then performed a series of experiments in yeast cells to probe how Scc4 determines the location at which cohesin loads onto chromosomes. These experiments revealed that a region on the surface of Scc4 targets both Scc4 and Scc2 to centromeres. The amino-acid sequence of this centromere-targeting patch on the surface of Scc4 is conserved across species. Thus, the mechanism by which Scc4 localizes cohesin to centromeres may be similar in all eukaryotic organisms. DOI: http://dx.doi.org/10.7554/eLife.06057.002

Dengue virus (DENV), a member of the Flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. The increasing prevalence of DENV worldwide heightens the need for an effective vaccine and specific antivirals. Due to the dependence of DENV upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. We screened a library of bioactive lipids and modulators of lipid metabolism and identified 4-hydroxyphenyl retinamide (4-HPR) (fenretinide) as an inhibitor of DENV in cell culture. 4-HPR inhibits the steady-state accumulation of viral genomic RNA and reduces viremia when orally administered in a murine model of DENV infection. The molecular target responsible for this antiviral activity is distinct from other known inhibitors of DENV but appears to affect other members of the Flaviviridae , including the West Nile, Modoc, and hepatitis C viruses. Although long-chain ceramides have been implicated in DENV replication, we demonstrate that DENV is insensitive to the perturbation of long-chain ceramides in mammalian cell culture and that the effect of 4-HPR on dihydroceramide homeostasis is separable from its antiviral activity. Likewise, the induction of reactive oxygen species by 4-HPR is not required for the inhibition of DENV. The inhibition of DENV in vivo by 4-HPR, combined with its well-established safety and tolerability in humans, suggests that it may be repurposed as a pan- Flaviviridae antiviral agent. This work also illustrates the utility of bioactive lipid screens for identifying critical interactions of DENV and other viral pathogens with host lipid biosynthesis, metabolism, and signal transduction.

PFMG5 (Pinctada fucata mantle gene 5) was identified from mantle tissue of the pearl oyster, Pinctada fucada (P. fucada). Here we report that PFMG5 decreased osteoblast differentiation marker alkaline phosphatase (ALP) activity and the transcript levels of osteoblast differentiation specific marker genes in MC3T3-E1 cells. PFMG5 was identified as a new molecule inhibiting osteoblast differentiation.

The spermatogenesis associated 4 gene (Spata4, previously named TSARG2) was demonstrated to participate in spermatogenesis. Here we report that Spata4 is expressed in osteoblasts and that overexpression of Spata4 accelerates osteoblast differentiation and mineralization in MC3T3-E1 cells. We found that Spata4 interacts with p-Erk1/2 in the cytoplasm and that overexpression of Spata4 enhances the phosphorylation of Erk1/2. Intriguingly, we observed that Spata4 increases the transcriptional activity of Runx2, a critical transcription factor regulating osteoblast differentiation. We showed that Spata4-activated Runx2 is through the activation of Erk1/2. Consistent with this observation, we found that overexpression of Spata4 increases the expression of osteoblastic marker genes, including osteocalcin (Ocn), Bmp2, osteopontin (Opn), type 1 collagen, osterix (Osx), and Runx2. We concluded that Spata4 promotes osteoblast differentiation and mineralization through the Erk-activated Runx2 pathway. Our findings provided new evidence that Spata4 plays a role in regulation of osteoblast differentiation. © 2011 American Society for Bone and Mineral Research