The role of non-chromosomal histones in the host defense system

Histone H1 is located at the inter-nucleosome and more correctly at both ends of the double-stranded DNA that protrude from the nucleosome unit. It has long been recognized to be localized only inside the nuclei as a constituent for packaging nucleosome into chromatin. Thus, it could be hardly believed that detatched or solubilized histone H1 plays the role of a host defense molecule. Given the old reports on histone-like basic proteins that show bacteriostatic functions, I herein chose some recent related articles and tried review them. Recent advances in research on the cell death mechanism makes it possible to understand that programmed cell death, (i.e. apoptosis) could serve as a good source of soluble histones. Some forms of them are highly probable to be bacteriostatic.     

Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Ourrecent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator ofspindle checkpoint signaling. However, it is unclear how NEK2 A regulates kinetochore-microtubule attachment in mitosis.Here we show that NEK2A phosphorylates human Sgol and such phosphorylation is essential for faithful chromosomecongression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore ofmitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgol is a substrate of NEK2A and the phos-phorylation sites were mapped to Ser~(14) and Ser~(507) as judged by the incorporation of ~(32)P. Although such phosphorylation isnot required for assembly of HsSgol to the kinetochore, expression of non-phosphorylatable mutant HsSgol perturbedchromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic andmonotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgol in orches-trating dynamic kinetochore-microtubule interaction. We propose that NEK2 A-mediated phosphorylation of human Sgolprovides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.     

Architecture of the budding yeast kinetochore reveals a conserved molecular core

How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.     

Structure and dynamics of nucleosomal DNA

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.     

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.     

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30?bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.     

Distinct roles for histone chaperones in the deposition of Htz1 in chromatin

Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)₂ tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin.     

Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly

The functions of Beclin‐1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K‐III complex. Here, we describe a new role for Beclin‐1 in mitotic chromosome congression that is independent of the PI3K‐III complex and its role in autophagy. Beclin‐1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP‐E, CENP‐F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin‐1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin‐1 interacts directly with Zwint‐1—a component of the KMN (KNL‐1/Mis12/Ndc80) complex—which is essential for kinetochore–microtubule interactions. This suggests that Beclin‐1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.     

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.     

Transcription-wide mapping of dihydrouridine reveals that mRNA dihydrouridylation is required for meiotic chromosome segregation

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Mitotic phosphorylation of PRC1 at Thr470 is required for PRC1 oligomerization and proper central spindle organization

During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with thecentromere. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separatingchromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kine-sins, the chromosomal passenger protein complex, and microtubule bundling protein PRC1. PRC1 is phosphorylated byCdc2 at Thr470 and Thr481 during mitosis. However, the functional relevance of PRC1 phosphorylation at Thr470 hasremained elusive. Here we show that expression of the non-phosphorylatable mutant PRC1~(T470A) but not the phospho-mimi-cking mutant PRC1~(T470E) causes aberrant organization of the central spindle. Immunoprecipitation experiment indicatesthat both PRC1~(T470A) and PRC1~(T470E) mutant proteins associate with wild-type PRC1, suggesting that phosphorylationof Thr470 does not alter PRC1 self-association. In addition, in vitro co-sedimentation experiment showed that PRC1binds to microtubule independent of the phosphorylation state of Thr470. Gel-filtration experiment suggested that phos-phorylation of Thr470 promotes oligomerization of PRC1. Given the fact that prevention of the Thr470 phosphorylationinhibits PRC1 oligomerization in vitro and causes an aberrant organization of central spindle in vivo, we propose thatthis phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriatetime in the cell cycle.     

Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation

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Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

Structured summary

MINT-7713125: IN (uniprotkb:P04585) and IN (uniprotkb:P04585) bind (MI:0407) by X-ray crystallography (MI:0114)     

Structural Basis for Recognition of Ubiquitylated Nucleosome by Dot1L Methyltransferase

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Evolutionary analysis of HIV-1 protease inhibitors: Methods for design of inhibitors that evade resistance

Drug-resistant strains are rapidly selected during AIDS therapy because of the high rate of mutation in HIV. In this report, we present an evolutionary simulation method for analysis of viral mutation and its use for optimization of HIV-1 protease drugs to improve their robustness in the face of resistance mutation. We first present an analysis of the range of resistant mutants that produce viable viruses by using a volume-based viral fitness model. Then, we analyze how this range of mutant proteases allows development of resistance to an optimal inhibitor previously designed by computational coevolution techniques. Finally, we evaluate the resistance patterns of commercially available drugs, and we discuss how resistance might be overcome by optimizing the size of specific side-chains of these inhibitors.     

Crystal structure of a fully glycosylated HIV‐1 gp120 core reveals a stabilizing role for the glycan at Asn262

The crystal structure of a fully glycosylated HIV‐1 gp120 core in complex with CD4 receptor and Fab 17b at 4.5‐Å resolution reveals 9 of the 15 N‐linked glycans of core gp120 to be partially ordered. The glycan at position Asn262 had the most extensive and well‐ordered electron density, and a GlcNAc₂Man₇ was modeled. The GlcNAc stem of this glycan is largely buried in a cleft in gp120, suggesting a role in gp120 folding and stability. Its arms interact with the stems of neighboring glycans from the oligomannose patch, which is a major target for broadly neutralizing antibodies. Proteins 2015; 83:590–596. © 2014 Wiley Periodicals, Inc.     

Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging

Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome‐wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild‐type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3–3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson–Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild‐type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.