CFP1 interacts with DNMT1 independently of association with the Setd1 Histone H3K4 methyltransferase complexes

CXXC finger protein 1 (CFP1) is a component of the Setd1A and Setd1B methyltransferase complexes, localizes to euchromatic regions of the genome, and specifically binds unmethylated CpG dinucleotides in DNA. Murine embryos lacking CFP1 exhibit peri-implantation lethality, a developmental time that correlates with global epigenetic reprogramming. CFP1-deficient embryonic stem (ES) cells exhibit a 70% reduction in global cytosine methylation and a 60% decrease in maintenance DNA methyltransferase (DNMT1) activity. DNMT1 protein level is reduced 50% in CFP1-deficient ES cells. Experiments were performed to investigate the role of CFP1 in regulating maintenance cytosine methylation. Coimmunoprecipitation experiments reveal that endogenous DNMT1 and CFP1 interact in vivo. Protein regions required for the interaction between DNMT1 and CFP1 were mapped. Amino acids 169-493 and 970-1617 of DNMT1 are each sufficient for interaction with CFP1. Three regions spanning the CFP1 protein, amino acids 1-123, 103-367, and 361-656, are each sufficient for interaction with DNMT1. Interestingly, a single-point mutation (C375A) within CFP1 that abolishes the interaction with the Setd1A and Setd1B histone H3K4 methyltransferase complexes does not disrupt the interaction between CFP1 and DNMT1. This result indicates that CFP1 intersects the cytosine methylation machinery independently of its association with the Setd1 complexes.     

Overexpression of SPINDLIN1 induces cellular senescence, multinucleation and apoptosis

Human or mouse Spindlin1 is expressed in various tissues and cells, but its biological functions are poorly understood. In this study, we show that human SPINDLIN1 is localized to interphase nucleus and mitotic chromosomes, and its expression in HeLa cells is not regulated in a cell cycle-dependent manner. When SPINDLIN1 is stably overexpressed in HeLa cells, it results in multinucleation of cells, and these multinucleated cells exhibits characteristic features of senescence and apoptosis shown by growth and morphological alterations, beta-galactosidase activity, and Annexin V/7-Aminoactinomycin D staining. Mouse Spindlin1 is highly homologous with human Spindlin1, when overexpressed in NIH3T3 cells, it also induces multinucleation, senescence and apoptosis in murine cells. Our results demonstrate that SPINDLIN1 is an important gene for mammalian mitotic chromosome functions, and disrupted regulation results in abnormal cell division, a mechanism that may be involved in tumorigenesis.     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.     

spindlin1基因的原核表达及多克隆抗体制备

目的:明确人新基因spindlin1的组织表达谱、相互作用蛋白。方法与结果:应用PCR技术获得spindlin1的开放读框,并将其插入原核表达载体pGEX-2T,转化大肠杆菌BL21(DE3),经IPTG诱导后,融合蛋白GST-Spindlin1获得了可溶性表达。用SDS-PAGE胶上的目的条带做抗原制备了多克隆抗体,进行了抗体纯化。结论:spindlin1在OVCar3细胞中可能以二聚体形式存在,这一现象还有待于进一步研究。     

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.     

Par-1 regulates stability of the posterior determinant Oskar by phosphorylation

Par-1 kinase is critical for polarization of the Drosophila melanogaster oocyte and the one-cell Caenorhabditis elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Here we show that Drosophila Par-1 phosphorylates the posterior determinant Oskar (Osk) and demonstrate genetically that Par-1 is required for accumulation of Osk protein. We show in cell-free extracts that Osk protein is intrinsically unstable and that it is stabilized after phosphorylation by Par-1. Our data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk.     

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.     

Identification and characterization of a sphere organelle protein

Sphere organelles are nuclear structures in amphibian oocytes that are easily visible by light microscopy. These structures are up to 10 microns in diameter and have been described morphologically for decades, yet their function remains obscure. The present study defines a protein component of the sphere organelle, named SPH-1, which is recognized by a mAb raised against purified Xenopus laevis oocyte nucleoplasm. SPH-1 is an 80-kD protein which is localized specifically to spheres and is undetectable elsewhere on lampbrush chromosomes or in nucleoli. We show using confocal microscopy that SPH-1 is localized to the cortex of sphere organelles. Furthermore, we have isolated a cDNA that can encode SPH-1. When epitope-tagged forms of SPH-1 are expressed in X. laevis oocytes the protein specifically localizes to spheres, demonstrating that the cloned cDNA encodes the sphere antigen. Comparison of the predicted amino acid sequence with sequence databases shows SPH-1 is related to p80-coilin, a protein associated with coiled bodies; coiled bodies are nuclear structures found in plant and animal cells. The sphere-specific mAb stains X. laevis tissue culture cells in a punctate nuclear pattern, showing that spheres or sphere antigens are present in somatic cells as well as germ cells and suggesting a general and essential function for spheres in all nuclei.     

Bdf1, a yeast chromosomal protein required for sporulation.

The BDF1 gene of Saccharomyces cerevisiae is required for sporulation. Under starvation conditions, most cells from the bdf1 null mutant fail to undergo one or both meiotic divisions, and there is an absolute defect in spore formation. The Bdf1 protein localizes to the nucleus throughout all stages of the mitotic and meiotic cell cycles. Analysis of spread meiotic nuclei reveals that the Bdf1 protein is localized fairly uniformly along chromosomes, except that it is excluded specifically from the nucleolus. A bdf1 null mutant displays a reduced rate of vegetative growth and sensitivity to a DNA-damaging agent. The BDF1 gene encodes a 77-kDa protein that contains two bromodomains, sequence motifs of unknown function. Separation-of-function alleles suggest that only one of the two bromodomains is required for sporulation, whereas both are required for Bdf1 function in vegetative cells. We propose that the Bdf1 protein is a component of chromatin and that the mitotic and meiotic defects of the bdf1 null mutant result from alterations in chromatin structure.     

Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila

uncoordinated (unc) mutants of Drosophila, which lack transduction in ciliated mechanosensory neurons, do not produce motile sperm. Both sensory and spermatogenesis defects are associated with disrupted ciliary structures: mutant sensory neurons have truncated cilia, and sensory neurons and spermatids show defects in axoneme ultrastructure. unc encodes a novel protein with coiled-coil segments and a LisH motif, which is expressed in type I sensory neurons and in the male germline - the only ciliogenic cells in the fly. A functional UNC-GFP fusion protein specifically localizes to both basal bodies in differentiating sensory neurons. In premeiotic spermatocytes it localizes to all four centrioles in early G2, remaining associated with them through meiosis and as they become the basal bodies for the elongating spermatid flagella. UNC is thus specifically required for normal ciliogenesis. Its localization is an early marker for the centriole-basal body transition, a central but enigmatic event in eukaryotic cell differentiation.     

M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis

The human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455-1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 microm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis.     

α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-catenin complex through alpha-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system.