HJURP uses distinct CENP-A surfaces to recognize and to stabilize CENP-A/histone H4 for centromere assembly

Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that whereas surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Furthermore, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.     

The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.     

Dual recognition of CENP-A nucleosomes is required for centromere assembly

Centromeres contain specialized nucleosomes in which histone H3 is replaced by the histone variant centromere protein A (CENP-A). CENP-A nucleosomes are thought to act as an epigenetic mark that specifies centromere identity. We previously identified CENP-N as a CENP-A nucleosome-specific binding protein. Here, we show that CENP-C also binds directly and specifically to CENP-A nucleosomes. Nucleosome binding by CENP-C required the extreme C terminus of CENP-A and did not compete with CENP-N binding, which suggests that CENP-C and CENP-N recognize distinct structural elements of CENP-A nucleosomes. A mutation that disrupted CENP-C binding to CENP-A nucleosomes in vitro caused defects in CENP-C targeting to centromeres. Moreover, depletion of CENP-C with siRNA resulted in the mislocalization of all other nonhistone CENPs examined, including CENP-K, CENP-H, CENP-I, and CENP-T, and led to a partial reduction in centromeric CENP-A. We propose that CENP-C binds directly to CENP-A chromatin and, together with CENP-N, provides the foundation upon which other centromere and kinetochore proteins are assembled.     

HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore

Centromeres of higher eukaryotes are epigenetically marked by the centromere-specific CENP-A nucleosome. New CENP-A recruitment requires the CENP-A histone chaperone HJURP. In this paper, we show that a LacI (Lac repressor) fusion of HJURP drove the stable recruitment of CENP-A to a LacO (Lac operon) array at a noncentromeric locus. Ectopically targeted CENP-A chromatin at the LacO array was sufficient to direct the assembly of a functional centromere as indicated by the recruitment of the constitutive centromere-associated network proteins, the microtubule-binding protein NDC80, and the formation of stable kinetochore–microtubule attachments. An amino-terminal fragment of HJURP was able to assemble CENP-A nucleosomes in vitro, demonstrating that HJURP is a chromatin assembly factor. Furthermore, HJURP recruitment to endogenous centromeres required the Mis18 complex. Together, these data suggest that the role of the Mis18 complex in CENP-A deposition is to recruit HJURP and that the CENP-A nucleosome assembly activity of HJURP is responsible for centromeric chromatin assembly to maintain the epigenetic mark.     

Xenopus CENP-A assembly into chromatin requires base excision repair proteins

CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA. Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.     

Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.     

Dimerization of the CENP‐A assembly factor HJURP is required for centromeric nucleosome deposition

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein‐A (CENP‐A). The deposition of new centromeric nucleosomes requires the CENP‐A‐specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3‐like domain of HJURP binds a single CENP‐A–histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP‐A nucleosome. How an octameric CENP‐A nucleosome forms from individual CENP‐A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C‐terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP‐A is deposited. Dimerization of HJURP is essential for the deposition of new CENP‐A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP‐A binding. These data provide a mechanism whereby the CENP‐A pre‐nucleosomal complex achieves assembly of the octameric CENP‐A nucleosome through the dimerization of the CENP‐A chaperone HJURP.     

Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity.     

Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence

Naive T cells encountering their cognate antigen become activated and acquire the ability to proliferate in response to cytokines. Stat5 is an essential component in this response. We demonstrate that Stat5 cannot access DNA in naive T cells and acquires this ability only after T-cell receptor (TCR) engagement. The transition is not associated with changes in DNA methylation or global histone modification but rather chromatin decondensation. Condensation occurs during thymocyte development and proper condensation is dependent on kleisin-β of the condensin II complex. Our findings suggest that this unique chromatin condensation, which can affect interpretations of chromatin accessibility assays, is required for proper T-cell development and maintenance of the quiescent state. This mechanism ensures that cytokine driven proliferation can only occur in the context of TCR stimulation.     

Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres

Centromeres contain specialized chromatin that includes the centromere-specific histone H3 variant, spCENP-A/Cnp1. Here we report identification of five fission yeast centromere proteins, Mis14-18. Mis14 is recruited to kinetochores independently of CENP-A, and, conversely, CENP-A does not require Mis14 to associate with centromeres. In contrast, Mis15, Mis16 (strong similarity with human RbAp48 and RbAp46), Mis17, and Mis18 are all part of the CENP-A recruitment pathway. Mis15 and Mis17 form an evolutionarily conserved complex that also includes Mis6. Mis16 and Mis18 form a complex and maintain the deacetylated state of histones specifically in the central core of centromeres. Mis16 and Mis18 are the most upstream factors in kinetochore assembly as they can associate with kinetochores in all kinetochore mutants except for mis18 and mis16, respectively. RNAi knockdown in human cells shows that Mis16 function is conserved as RbAp48 and RbAp46 are both required for localization of human CENP-A.     

Cdc6 is required for meiotic spindle assembly in Xenopus oocytes

During the maturation of Xenopus oocytes, Cdc6 expression is necessary to establish replication competence to support early embryonic DNA replication. However, Cdc6 is expressed before the completion of MI at a time when its function as a replication factor is not required, suggesting additional roles for Cdc6 in meiosis. Confocal immunofluorescence microscopy revealed that Cdc6 protein was distributed around the spindle precursor at the time of germinal vesicle breakdown (GVBD) and localized to the margin of the nascent spindle early in prometaphase. Cdc6 subsequently localized to spindle poles in late prometaphase, where it remained until metaphase arrest. Microinjection of antisense oligonucleotides specific for Cdc6 mRNA disrupted spindle assembly, resulting in defects, including delayed spindle assembly, misoriented and unattached anaphase spindles, monasters, multiple spindles, microtubule aggregates associated with condensed chromosomes, or the absence of recognizable spindle-like structures, depending on the level of residual Cdc6 expression. Furthermore, Cdc6 co-localized with γ-tubulin in centrosomes during interphase in all somatic cells analyzed and associated with spindle poles in mitotic COS cells. Our data suggest a role for Cdc6 in spindle formation in addition to its role as a DNA replication factor.Key words: Cdc6, spindle assembly, Xenopus, oocytes, pre-RC proteins     

Cdc6 is required for meiotic spindle assembly in Xenopus oocytes

During the maturation of Xenopus oocytes, Cdc6 expression is necessary to establish replication competence to support early embryonic DNA replication. However, Cdc6 is expressed before the completion of MI, at a time when its function as a replication factor is not required, suggesting additional roles for Cdc6 in meiosis. Confocal immunofluorescence microscopy revealed that Cdc6 protein was distributed around the spindle precursor at the time of germinal vesicle breakdown (GVBD), and localized to the margin of the nascent spindle early in prometaphase. Cdc6 subsequently localized to spindle poles in late prometaphase, where it remained until metaphase arrest. Microinjection of antisense oligonucleotides specific for Cdc6 mRNA disrupted spindle assembly, resulting in defects including delayed spindle assembly, misoriented and unattached anaphase spindles, monasters, multiple spindles, microtubule aggregates associated with condensed chromosomes, or the absence of recognizable spindle-like structures, depending on the level of residual Cdc6 expression. Furthermore, Cdc6 co-localized with γ-tubulin in centrosomes during interphase in all somatic cells analyzed, and associated with spindle poles in mitotic COS cells. Our data suggest a role for Cdc6 in spindle formation in addition to its role as a DNA replication factor.     

Meiotic condensin is required for proper chromosome compaction, SC assembly, and resolution of recombination-dependent chromosome linkages

Condensin is an evolutionarily conserved protein complex that helps mediate chromosome condensation and segregation in mitotic cells. Here, we show that condensin has two activities that contribute to meiotic chromosome condensation in Saccharomyces cerevisiae. One activity, common to mitosis, helps mediate axial length compaction. A second activity promotes chromosome individualization with the help of Red1 and Hop1, two meiotic specific components of axial elements. Like Red1 and Hop1, condensin is also required for efficient homologue pairing and proper processing of double strand breaks. Consistent with these functional links condensin is necessary for proper chromosomal localization of Red1 and Hop1 and the subsequent assembly of the synaptonemal complex. Finally, condensin has a Red1/Hop1-independent role in the resolution of recombination-dependent linkages between homologues in meiosis I. The existence of distinct meiotic activities of condensin (axial compaction, individualization, and resolution of recombination-dependent links) provides an important framework to understand condensin's role in both meiotic and mitotic chromosome structure and function.     

Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis

The cardiovascular development is the elaborate process, and despite the extensive studies, the mechanisms underlying endothelial, hematopoietic, and cardiac developments, as well as the interrelation between these processes, are not fully understood. In this study, we demonstrated that Xenopus apelin and Xmsr play pivotal roles in cardiovascular development. Apelin is a recently identified ligand for an orphan G-protein-coupled receptor APJ and is involved in fluid homeostasis in mammals. Xenopus preproapelin (Xpreproapelin) was isolated and its mRNA localized to the region around the presumptive blood vessels, which are overlapping or adjacent to those expressing Xmsr, the Xenopus homologue of APJ. Overexpression of Xpreproapelin disorganized the expression of the endothelial precursor cell marker XlFli and the hematopoietic precursor cell marker SCL at the neurula, whereas embryos injected with morpholino antisense oligonucleotides for Xapelin and Xmsr displayed attenuated expression of Tie2, alpha-globin, XPOX2, and cTnI, markers of endothelium, erythrocytes, myeloid cells, and cardiomyocytes, respectively. XlFli morpholino had similar effects to Xapelin and Xmsr morpholinos on cardiac differentiation, suggesting an unexpected potential relationship between the endothelium and cardiac differentiation. Forced expression of constitutive active G alpha i rescued the phenotypes of Xmsr morpholino-injected embryos, indicating that the i/o type of G protein alpha subunit acts downstream of Xmsr.     

CooC and CooD are required for assembly of CS1 pili

Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple Identical protein subunits encoded by cooA. We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA. Following cooD Is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E. coli K-12 to synthesize CS1 pili. Complementation analysis of mutants cloned in E. coli K-12 and constructed in an ETEC-derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.     

Proteasome synthesis and assembly are required for survival during stationary phase

We examined the alterations in 20S proteasome homeostasis, protein oxidation, and cell viability that occur during the stationary phase or chronological model of yeast aging. Data in this report demonstrate that proteasome subunit expression is increased, proteasome composition is altered, and levels of individual proteasome proteolytic activities are elevated during stationary phase-induced aging in Saccharomyces cerevisiae. Despite such alterations, a progressive loss of proteasome-mediated protein degradation and a significant increase in protein oxidation were observed in cells maintained under stationary phase conditions. Deletion of UMP1, a gene necessary for 20S proteasome biogenesis, had no effect on cellular viability under normal growth conditions, but impaired the ability of cells to survive under stationary phase conditions. During stationary phase, the levels of oxidized protein increased more rapidly and to higher levels in the mutant lacking UMP1 than in the wild-type cells. Taken together, these data implicate a role for proteasome synthesis and altered 20S proteasome composition in maintaining viability during stationary phase, and demonstrate that even with these modifications a gradual loss of proteasome-mediated protein degradation occurs during stationary phase-induced aging. These data also suggest a role for impaired proteasome-mediated protein degradation in increased protein oxidation and cell death observed during the aging of eukaryotic cells.     

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.