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1.
Stem Cell Reports ; 8(4): 1062-1075, 2017 04 11.
Article in English | MEDLINE | ID: mdl-28330622

ABSTRACT

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation.


Subject(s)
Cell Differentiation , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Mouse Embryonic Stem Cells/cytology , Transcription Factors/genetics , Amino Acid Sequence , Animals , Cell Line , Cell Polarity , Cell Proliferation , DNA-Binding Proteins/analysis , DNA-Binding Proteins/metabolism , Endoderm/cytology , Endoderm/embryology , Endoderm/metabolism , Mice , Mouse Embryonic Stem Cells/metabolism , Octamer Transcription Factor-3/metabolism , Protein Interaction Maps , Protein Isoforms/analysis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , Proteolysis , Transcription Factors/analysis , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism
2.
Mol Biol Cell ; 25(20): 3105-18, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25143403

ABSTRACT

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-ß-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.


Subject(s)
Cell Cycle/physiology , Cellular Senescence/physiology , Tetraploidy , Animals , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Fibroblasts/metabolism , Humans , Mitosis , RNA, Small Interfering/genetics , Rats , Tumor Suppressor Protein p53/metabolism
3.
Proc Natl Acad Sci U S A ; 107(9): 4159-64, 2010 Mar 02.
Article in English | MEDLINE | ID: mdl-20142474

ABSTRACT

Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing.


Subject(s)
Centromere , DNA Damage , Spindle Apparatus , Animals , Cells, Cultured , DNA Repair , Histones/metabolism , Mice , Mutation , Phosphorylation
4.
Chromosoma ; 118(5): 617-32, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19557426

ABSTRACT

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3-/- spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.


Subject(s)
DNA-Binding Proteins/metabolism , Histones/genetics , Meiosis/physiology , Synaptonemal Complex/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Lysine/metabolism , Male , Methylation , Mice , Spermatocytes/metabolism , Transcription Factors/genetics
5.
Plant Mol Biol ; 52(6): 1169-80, 2003 Aug.
Article in English | MEDLINE | ID: mdl-14682616

ABSTRACT

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.


Subject(s)
Indoleacetic Acids/metabolism , Lotus/metabolism , Plant Roots/metabolism , Alphaproteobacteria/growth & development , Biological Transport , Glucuronidase/genetics , Glucuronidase/metabolism , Green Fluorescent Proteins , Indoleacetic Acids/pharmacology , Lipopolysaccharides/pharmacology , Lotus/genetics , Lotus/growth & development , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Phthalimides/pharmacology , Plant Roots/genetics , Plant Roots/microbiology , Plants, Genetically Modified , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Response Elements/genetics , Rhizobium/growth & development , Symbiosis
6.
Mol Plant Microbe Interact ; 16(2): 159-68, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12575750

ABSTRACT

Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values. Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness. The insertion was mapped to the 5' end of atvA, encoding a product with high sequence identity to the agro-bacterial AcvB virulence protein. Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance. A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix+ but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function. atvA is the second gene in an operon that is transcriptionally upregulated by acid shock. The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon. lpiA-like genes are found in several alpha, beta, and gamma Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes. The latter proteins are involved in resistance to cationic antimicrobial peptides. A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.


Subject(s)
Adaptation, Physiological/genetics , Bacterial Proteins/genetics , Operon/genetics , Plant Roots/microbiology , Rhizobium/genetics , Virulence Factors , Adaptation, Physiological/physiology , Bacterial Proteins/metabolism , DNA Transposable Elements/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Insertional , Mutation , Open Reading Frames/genetics , Phaseolus/microbiology , Restriction Mapping , Symbiosis/genetics
7.
Carbohydr Res ; 337(13): 1193-202, 2002 Jul 16.
Article in English | MEDLINE | ID: mdl-12110194

ABSTRACT

The novel lipochitin oligosaccharide (LCOs) structures produced by Rhizobium etli KIM5s were characterized using a nanoHPLC reverse-phase system coupled to an ion-trap mass spectrometer. This technique was shown to be more sensitive for structural elucidation of LCOs than previously used mass spectrometric methods. The structures of the LCOs of R. etli KIM5s, the majority containing six monosaccharide residues, differed from those synthesized by all other rhizobia analyzed to date. In addition, novel structures in which the chitin backbone was deacetylated at one or more GlcNAc moieties were found as minor compounds. The difference in host range of this strain compared to that of other known bean microsymbionts is discussed.


Subject(s)
Chitin/chemistry , Lipopolysaccharides/chemistry , Rhizobium/chemistry , Acetylation , Carbohydrate Sequence , Chitin/isolation & purification , Chromatography, High Pressure Liquid , Lipopolysaccharides/isolation & purification , Lotus/microbiology , Mass Spectrometry , Molecular Sequence Data , Phenotype , Rhizobium/growth & development
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