Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.     

Substrate Affinity and Specificity of the ScSth1p Bromodomain Are Fine-Tuned for Versatile Histone Recognition

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Distinct roles of the histone chaperones NAP1 and NRP and the chromatin‐remodeling factor INO80 in somatic homologous recombination in Arabidopsis thaliana

Homologous recombination (HR) of nuclear DNA occurs within the context of a highly complex chromatin structure. Despite extensive studies of HR in diverse organisms, mechanisms regulating HR within the chromatin context remain poorly elucidated. Here we investigate the role and interplay of the histone chaperones NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) and NAP1‐RELATED PROTEIN (NRP) and the ATP‐dependent chromatin‐remodeling factor INOSITOL AUXOTROPHY80 (INO80) in regulating somatic HR in Arabidopsis thaliana. We show that simultaneous knockout of the four AtNAP1 genes and the two NRP genes in the sextuple mutant m123456‐1 barely affects normal plant growth and development. Interestingly, compared with the respective AtNAP1 (m123‐1 and m1234‐1) or NRP (m56‐1) loss‐of‐function mutants, the sextuple mutant m123456‐1 displays an enhanced plant hypersensitivity to UV or bleomycin treatments. Using HR reporter constructs, we show that AtNAP1 and NRP act in parallel to synergistically promote somatic HR. Distinctively, the AtINO80 loss‐of‐function mutation (atino80‐5) is epistatic to m56‐1 in plant phenotype and telomere length but hypostatic to m56‐1 in HR determinacy. Further analyses show that expression of HR machinery genes and phosphorylation of H2A.X (γ‐H2A.X) are not impaired in the mutants. Collectively, our study indicates that NRP and AtNAP1 synergistically promote HR upstream of AtINO80‐mediated chromatin remodeling after the formation of γ‐H2A.X foci during DNA damage repair.     

Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin

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Rapid replacement of somatic linker histones with the oocyte-specific linker histone H1foo in nuclear transfer

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.     

Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Not going with the flow: Locomotor activity does not constrain immunity in a wild fish

Immunity is a central component of fitness in wild animals, but its determinants are poorly understood. In particular, the importance of locomotory activity as a constraint on immunity is unresolved. Using a piscine model (Gasterosteus aculeatus), we combined a 25‐month observational time series for a wild lotic habitat with an open flume experiment to determine the influence of locomotor activity (countercurrent swimming) on natural variation in immune function. To maximize the detectability of effects in our flume experiment, we set flow velocity and duration (10 cm/s for 48 hr) just below the point at which exhaustion would ensue. Following this treatment, we measured expression in a set of immune‐associated genes and infectious disease resistance through a standard challenge with an ecologically relevant monogenean infection (Gyrodactylus gasterostei). In the wild, there was a strong association of water flow with the expression of immune‐associated genes, but this association became modest and more complex when adjusted for thermal effects. Our flume experiment, although statistically well‐powered and based on a scenario near the limits of swimming performance in stickleback, detected no countercurrent swimming effect on immune‐associated gene expression or infection resistance. The field association between flow rate and immune expression could thus be due to an indirect effect, and we tentatively advance hypotheses to explain this. This study clarifies the drivers of immune investment in wild vertebrates; although locomotor activity, within the normal natural range, may not directly influence immunocompetence, it may still correlate with other variables that do.     

Chromatin regulation of transcriptional enhancers and cell fate by the Sotos syndrome gene NSD1

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Lisosan G, a powder of grain, does not interfer with the drug metabolizing enzymes and has a protective role on carbon tetrachloride-induced hepatotoxicity

Lisosan G is a powder of grain registered as an alimentary integrator. The treatment of rats for 4 days with 0.5 g Lisosan G/kg had no effect on various drug metabolizing enzymes. Experiments in vitro showed that Lisosan G had radical scavenger activity. A confirmation of the antioxidative property of Lisosan G was also confirmed when it was administered in vivo to carbon tetrachloride (CCl₄)-intoxicated rats. The toxicity caused by CCl₄-treatment of rats was restored to the control levels when the rats were given Lisosan G for 4 days before CCl₄. Lisosan G thus does not interfer with drug metabolizing system but has antioxidant properties and protects against CCl₄-induced hepatotoxicity.     

Effect of trichostatin A on human T cells resembles signaling abnormalities in T cells of patients with systemic lupus erythematosus: a new mechanism for TCR zeta chain deficiency and abnormal signaling

Trichostatin A (TSA) is a potent reversible inhibitor of histone deacetylase, and it has been reported to have variable effects on the expression of a number of genes. In this report, we show that TSA suppresses the expression of the T cell receptor zeta chain gene, whereas, it upregulates the expression if its homologous gene Fc(epsilon) receptor I gamma chain. These effects are associated with decreased intracytoplasmic-free calcium responses and altered tyrosine phosphorylation pattern of cytosolic proteins. Along with these effects, we report that TSA suppresses the expression of the interleukin-2 gene. The effects of TSA on human T cells are predominantly immunosuppressive and reminiscent of the signaling aberrations that have been described in patients with systemic lupus erythematosus.     

细胞因子与抗肿瘤药物体外协同抑制宫颈癌细胞的敏感性试验

目的探讨3种细胞因子单一、协同以及与化疗药物顺铂(DDP)的联合作用杀伤宫颈癌Hela细胞株的影响。方法应用四甲基偶氮唑盐比色法(MTT比色法)、检测与化疗药物DDP单独或联合应用组及药物作用的12 h与24 h的吸光度值(A),计算杀伤率。台盼蓝拒染计算杀伤率对照。结果单独应用3种细胞因子时,IFN-α(干扰素-α)、TNF-α(肿瘤坏死因子)对宫颈癌Hela有细胞毒作用,IL-2对宫颈癌Hela细胞株则细胞毒作用不明显。IFN-α、IL-2、TNF-α可以增强DDP的抑瘤作用,与DDP单独作用差异具有显著性(P<0.01)。IFN-α、IL-2(白细胞介素-2)、TNF-α联合作用具有协同作用,均与单一应用差异具有显著性(P<0.01)。细胞因子均可以增强DDP的抑瘤作用并与其作用的先后及时间具有依赖关系。结论MTT比色法为临床肿瘤化疗的药物敏感性检测提供了简便、快速的方法[1]。不同类型的肿瘤有着不同的药敏谱。IFN-α、TNF-α、IL-2与DDP联合具有协同作用,为宫颈癌患者化疗提供了理想的参考方案。     

Purification of a nuclear protein (Receptor Binding Factor-1) associated with the chromatin acceptor sites for the avian oviduct progesterone receptor

The specific high affinity binding of the avian oviduct progesterone receptor (PR) to target cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0–6.0 [Goldberger and Spelsberg (1988),Biochem. 27, 2103–2109]. Using western immunoblots with anti-RBF-1 polyclonal antibodies to monitor the purification, a 10 kD candidate acceptor protein, termed the Receptor Binding Factor-1 (RBF-1), has been purified to apparent homogeneity. RBF-1 has an amino acid composition consistent with a hydrophobic protein having an acidic pI and a unique N-terminal sequence. Two-dimensional polyacrylamide gel electrophoresis and high-performance capillary electrophoresis support the purity of a protein 10 kD in size, having an acidic pI, but with evidence of several differently charged isoforms. Phosphatase treatment provides evidence that charge heterogeneity may result from variable phosphorylation states. A role of this factor as a candidate acceptor protein in the chromatin acceptor sites for the avian oviduct PR is proposed.     

The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1

ISP-1 inhibits de novo sphingolipid biosynthesis and induces growth defects in both mammals and yeast (Saccharomyces cerevisiae). In our previous study, YPK1/SLI2 was identified as one of multicopy suppressor genes for ISP-1 in yeast. Ypk1 is proposed to be a downstream serine/threonine kinase of the sphingolipid signaling pathway in yeast. Other than resistance against ISP-1, Ypk1 is involved in at least two downstream events, namely cell growth and endocytosis. In this study, the effect of mutants of Ypk1 on these three downstream events was investigated. Among Ypk1 mutants, no 'kinase-dead' mutants complemented the defects in any of these three downstream events in the ypk1 null strain. One of the hydrophobic motif phosphorylation-deficient mutants of Ypk1, Ypk1(T662A) had the moderate kinase activity compared with the wild-type Ypk1. Ypk1(T662A) and the wild-type Ypk1 completely restored the slow-growth phenotype and fluid-phase endocytosis defect of the ypk1 null strain. However, unlike the wild-type Ypk1, Ypk1(T662A) lost the ability for the recovery of the ISP-1 resistance in the ypk1 null strain. Furthermore, the expression of Ypk1(T662A) in the wild-type strain showed a dominant-negative effect on the ISP-1-resistance activity. On the other hand, the cell growth revertant of the ypk1 null strain still showed the hypersensitive phenotype to ISP-1. These data suggest that the ISP-1-resistance pathway is under the regulation of the hydrophobic motif phosphorylation and is separated from the other pathways downstream of Ypk1.     

The digalactosyldiacylglycerol (DGDG) synthase DGD1 is inserted into the outer envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on direct interaction with monogalactosyldiacylglycerol synthase for DGDG biosynthesis.

Galactolipids make up the bulk of chloroplast lipids. Therefore, the genes involved in the synthesis of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) play a critical role in chloroplast development. In this study, we analyzed the subcellular localization of the Arabidopsis DGDG synthase DGD1, which was recently identified by complementation of the Arabidopsis dgd1 mutant. In vitro import experiments demonstrated that DGD1 was targeted to the chloroplast outer envelope in an ATP-independent manner. DGD1 could not be extracted from the membranes by high salt or alkali, suggesting that it is an integral membrane protein. Uptake experiments with truncated versions of DGD1 indicated that the information for targeting and insertion into the outer envelope resides in the N-terminal half of DGD1, but not in the first 33 amino acids. DGD1 apparently does not contain a cleavable signal peptide. Antibodies to Arabidopsis DGD1 detected a 90-kDa protein localized to the chloroplast envelopes of both pea and Arabidopsis. Transformation of DGD1 constructs into cyanobacteria resulted in the expression of active DGDG synthase and demonstrated that DGDG synthesis depends on MGDG lipid, but does not require direct interaction with the plant MGDG synthase.