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1.
Cell Rep ; 42(6): 112568, 2023 06 27.
Article in English | MEDLINE | ID: mdl-37243594

ABSTRACT

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.


Subject(s)
Centromere , RNA-Binding Protein EWS , Humans , Autoantigens/metabolism , Centromere/metabolism , Centromere Protein A/genetics , Chromosomal Proteins, Non-Histone/metabolism , RNA , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , Sarcoma, Ewing
2.
Histochem Cell Biol ; 159(2): 199-208, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36129568

ABSTRACT

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) make up the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyx mori were inserted into a plasmid, transformed into Escherichia coli, and purified. We then produced antibodies against the SNAP-25, VAMP2, and Syntaxin 1A of Bombyx mori of rabbits and rats, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of VAMP2 was restricted to neurons in the pars intercerebralis (PI), dorsolateral protocerebrum (DL), and central complex (CX) of the brain. SNAP-25 was restricted to neurons in the PI and the CX of the brain. Syntaxin 1A was restricted to neurons in the PI and DL of the brain. VAMP2 co-localized with SNAP-25 in the CX, and with Syntaxin 1A in the PI and DL. VAMP2, SNAP-25, and Syntaxin 1A are present in the CA. Bombyxin-immunohistochemical reactivities (IRs) of brain and CA overlapped with VAMP2-, SNAP-25, and Syntaxin 1A-IRs. VAMP2 and Syntaxin 1A are present in the prothoracicotropic hormone (PTTH)-secretory neurons of the brain.


Subject(s)
Bombyx , SNARE Proteins , Rats , Rabbits , Animals , SNARE Proteins/metabolism , Bombyx/metabolism , Syntaxin 1/chemistry , Syntaxin 1/metabolism , Corpora Allata/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Brain/metabolism
3.
J Vis Exp ; (160)2020 06 10.
Article in English | MEDLINE | ID: mdl-32597847

ABSTRACT

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle. The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.


Subject(s)
Bacterial Proteins/metabolism , Centromere Protein A/metabolism , Luminescent Proteins/metabolism , Mass Spectrometry , Recombinant Fusion Proteins/metabolism , Ubiquitination , Cell Survival , Centromere , HEK293 Cells , Humans , Mutant Proteins/metabolism , Peptides/metabolism
4.
Dev Cell ; 50(6): 683-689.e6, 2019 09 23.
Article in English | MEDLINE | ID: mdl-31550462

ABSTRACT

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability. In the present study, we found that EYFP tagging induces additional ubiquitylation of EYFP-CENP-A K124R, which allows the mutant protein to bind to HJURP. Using a previously developed conditional CENP-A knockout system and our CENP-A K124R knockin mutant created by the CRISPR-Cas9 system, we show that the Flag-tagged or untagged CENP-A K124R mutant is lethal. This lethality is rescued by monoubiquitin fusion, indicating that CENP-A ubiquitylation is essential for viability.


Subject(s)
Centromere Protein A/metabolism , Ubiquitination , Cell Survival , Female , Humans , Mutant Proteins/metabolism , Peptides/metabolism , Protein Binding
5.
Molecules ; 24(3)2019 Jan 22.
Article in English | MEDLINE | ID: mdl-30678315

ABSTRACT

The centromere plays an essential role in accurate chromosome segregation, and the chromosomal location of the centromere is determined by the presence of a histone H3 variant, centromere protein A (CENP-A), in centromeric nucleosomes. However, the precise mechanisms of deposition, maintenance, and inheritance of CENP-A at centromeres are unclear. We have reported that CENP-A deposition requires ubiquitylation of CENP-A lysine 124 mediated by the E3 ligase activity of Cullin 4A (CUL4A)-RING-box protein 1 (RBX1)-COP9 signalsome complex subunit 8 (COPS8). We have proposed a model of inheritance for CENP-A ubiquitylation, through dimerization between rounds of cell divisions, that maintains the position of centromeres.


Subject(s)
Centromere Protein A/metabolism , Centromere/metabolism , Centromere/genetics , Epigenesis, Genetic , HeLa Cells , Humans , Models, Biological , Protein Binding , Ubiquitination
6.
Biopharm Drug Dispos ; 39(8): 371-377, 2018 Sep.
Article in English | MEDLINE | ID: mdl-30098040

ABSTRACT

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and ten mutants were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6ß-hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6ß-hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1.


Subject(s)
Cytochrome P-450 CYP2C9/metabolism , Steroids/metabolism , Cytochrome P-450 CYP2C9/genetics , Escherichia coli/genetics , Hydroxylation , Polymorphism, Single Nucleotide , Recombinant Proteins/metabolism , Steroid Hydroxylases/metabolism
7.
Cell Cycle ; 16(18): 1683-1694, 2017 Sep 17.
Article in English | MEDLINE | ID: mdl-28816574

ABSTRACT

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A-containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. However, the mechanism that controls the E3 ligase activity of the CUL4A-RBX1-COPS8 complex remains obscure. We have discovered that the SGT1-HSP90 complex is required for recognition of CENP-A by COPS8. Thus, the SGT1-HSP90 complex contributes to the E3 ligase activity of the CUL4A complex that is necessary for CENP-A ubiquitylation and CENP-A deposition at the centromere.


Subject(s)
Cell Cycle Proteins/metabolism , Centromere Protein A/metabolism , Centromere/metabolism , HSP90 Heat-Shock Proteins/metabolism , COP9 Signalosome Complex/metabolism , Cullin Proteins/metabolism , HeLa Cells , Humans , Kinetochores/metabolism , Models, Biological , Protein Transport , Proteins/metabolism , Ubiquitination
9.
Mol Cell Oncol ; 3(4): e1188226, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27652331

ABSTRACT

CENP-A (Centromere protein A) is a histone H3 variant that epigenetically determines the centromere position, but the mechanism of its centromere inheritance is obscure. We propose that CENP-A ubiquitylation, which is inherited through dimerization between rounds of cell division, is a candidate for the epigenetic mark of centromere identity.

10.
Cell Rep ; 15(1): 61-76, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-27052173

ABSTRACT

The presence of chromatin containing the histone H3 variant CENP-A dictates the location of the centromere in a DNA sequence-independent manner. But the mechanism by which centromere inheritance occurs is largely unknown. We previously reported that CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. Here, we show that pre-existing ubiquitylated CENP-A is necessary for recruitment of newly synthesized CENP-A to the centromere and that CENP-A ubiquitylation is inherited between cell divisions. In vivo and in vitro analyses using dimerization mutants and dimerization domain fusion mutants revealed that the inheritance of CENP-A ubiquitylation requires CENP-A dimerization. Therefore, we propose models in which CENP-A ubiquitylation is inherited and, through dimerization, determines centromere location. Consistent with this model is our finding that overexpression of a monoubiquitin-fused CENP-A mutant induces neocentromeres at noncentromeric regions of chromosomes.


Subject(s)
Autoantigens/metabolism , Cell Division , Chromosomal Proteins, Non-Histone/metabolism , Protein Multimerization , Ubiquitination , Autoantigens/genetics , Centromere/metabolism , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , HeLa Cells , Humans , Protein Binding
11.
Dev Cell ; 32(5): 589-603, 2015 Mar 09.
Article in English | MEDLINE | ID: mdl-25727006

ABSTRACT

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere. Addition of monoubiquitin is sufficient to restore CENP-A K124R to centromeres and the interaction with HJURP, indicating that "signaling" ubiquitylation is required for CENP-A loading at centromeres. The CUL4A-RBX1 complex is required for loading newly synthesized CENP-A and maintaining preassembled CENP-A at centromeres. Thus, CENP-A K124R ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere.


Subject(s)
Autoantigens/metabolism , Carrier Proteins/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cullin Proteins/metabolism , Proteins/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Autoantigens/genetics , Blotting, Western , COP9 Signalosome Complex , Carrier Proteins/genetics , Cells, Cultured , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , Cullin Proteins/genetics , Fluorescent Antibody Technique , HeLa Cells , Histones/metabolism , Humans , Immunoenzyme Techniques , Luciferases/metabolism , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Molecular Sequence Data , Nucleosomes/metabolism , Protein Binding , Proteins/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Ubiquitination
12.
Proc Natl Acad Sci U S A ; 111(4): 1628-33, 2014 Jan 28.
Article in English | MEDLINE | ID: mdl-24425774

ABSTRACT

The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect. Because the spindle checkpoint pathway is highly conserved between yeast and humans, we performed a synthetic genetic array analysis in yeast, which revealed that Mad2 overexpression induced lethality in 13 gene deletions. Among the human homologs of candidate genes, knockdown of PPP2R1A, a gene encoding a constant regulatory subunit of protein phosphatase 2, significantly inhibited the growth of Mad2-overexpressing tumor cells. PPP2R1A inhibition induced Mad2 phosphorylation and suppressed Mad2 protein levels. Depletion of PPP2R1A inhibited colony formation of Mad2-overexpressing HeLa cells but not of unphosphorylated Mad2 mutant-overexpressing cells, suggesting that the lethality induced by PP2A depletion in Mad2-overexpressing cells is dependent on Mad2 phosphorylation. Also, the PP2A inhibitor cantharidin induced Mad2 phosphorylation and inhibited the growth of Mad2-overexpressing cancer cells. Aurora B knockdown inhibited Mad2 phosphorylation in mitosis, resulting in the blocking of PPP2R1A inhibition-induced cell death. Taken together, our results strongly suggest that PP2A is a good therapeutic target in Mad2-overexpressing tumors.


Subject(s)
Mad2 Proteins/metabolism , Neoplasms/metabolism , Protein Phosphatase 2/antagonists & inhibitors , Base Sequence , Cantharidin/pharmacology , DNA Primers , Enzyme Inhibitors/pharmacology , Gene Knockdown Techniques , HeLa Cells , Humans , Mad2 Proteins/genetics , Neoplasms/enzymology , Neoplasms/pathology , Protein Phosphatase 2/genetics , RNA, Small Interfering
13.
Development ; 140(10): 2149-59, 2013 May.
Article in English | MEDLINE | ID: mdl-23578927

ABSTRACT

The anaphase inhibitor securin plays a crucial role in regulating the timing of sister chromatid separation during mitosis. When sister chromatid pairs become bioriented, the E3 ligase anaphase promoting complex/cyclosome (APC/C) ubiquitylates securin for proteolysis, triggering sister chromatid separation. Securin is also implicated in regulating meiotic progression. Securin protein levels change sharply during cell cycle progression, enabling its timely action. To understand the mechanism underlying the tightly regulated dynamics of securin, we analyzed the subcellular localization of the securin IFY-1 during C. elegans development. IFY-1 was highly expressed in the cytoplasm of germ cells. The cytoplasmic level of IFY-1 declined immediately following meiosis I division and remained low during meiosis II and following mitoses. We identified a C. elegans homolog of another type of E3 ligase, UBE3C, designated ETC-1, as a regulator of the cytoplasmic IFY-1 level. RNAi-mediated depletion of ETC-1 stabilized IFY-1 and CYB-1 (cyclin B1) in post-meiosis I embryos. ETC-1 knockdown in a reduced APC function background caused an embryonic lethal phenotype. In vitro, ETC-1 ubiquitylates IFY-1 and CYB-1 in the presence of the E2 enzyme UBC-18, which functions in pharyngeal development. Genetic analysis revealed that UBC-18 plays a distinct role together with ETC-1 in regulating the cytoplasmic level of IFY-1 during meiosis. Our study reports a novel mechanism, mediated by ETC-1, that co-operates with APC/C to maintain the meiotic arrest required for proper cell cycle timing during reproduction.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/cytology , Carrier Proteins/metabolism , Cyclin B1/metabolism , Gene Expression Regulation, Developmental , Meiosis/physiology , Ubiquitin-Protein Ligases/metabolism , Alleles , Anaphase , Animals , Caenorhabditis elegans Proteins/chemistry , Carrier Proteins/chemistry , Carrier Proteins/physiology , Cytoplasm/metabolism , Immunoprecipitation , Mass Spectrometry , Mitosis , RNA Interference , Ubiquitin/metabolism
14.
Front Biosci (Landmark Ed) ; 14(4): 1529-57, 2009 01 01.
Article in English | MEDLINE | ID: mdl-19273145

ABSTRACT

In contrast to many eukaryotic organisms in which kinetochores are assembled on localized centromeres of monocentric chromosomes, Caenorhabditis elegans has diffuse kinetochores, termed holo-kinetochores, which are assembled along the entire length of the mitotic chromosome. Despite this cytologically distinct chromosomal architecture, holo-kinetochores of C. elegans and kinetochores of other eukaryotes share structurally and functionally conserved properties. The amphitelic attachment of sister kinetochores to microtubules can be achieved by proper chromosomal organization, which relies on spatiotemporally orchestrated functions of conserved protein complexes such as the cohesin, condensin, and chromosomal passenger complexes during mitosis and meiosis in C. elegans. Moreover, the structure of spindle assembly checkpoint components and their safeguard function are also well conserved in C. elegans. Extensive efforts in the last few years to elucidate the molecular mechanisms of the C. elegans spindle assembly checkpoint have revealed its unique features. In this review, I will focus on the conservation and diversity of proteins that are required to maintain chromosome transmission fidelity during mitosis and meiosis in C. elegans.


Subject(s)
Caenorhabditis elegans/genetics , Chromosomes , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Kinetochores/ultrastructure , Meiosis , Mitosis , Spindle Apparatus
15.
Cell Cycle ; 8(3): 338-44, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-19177000

ABSTRACT

The spindle assembly checkpoint (SAC) monitors the microtubule attachment status of the kinetochore and arrests cells before anaphase until all pairs of sister kinetochores achieve bipolar attachment of microtubules, thereby ensuring faithful chromosome transmission. The evolutionarily conserved coiled-coil protein MAD1 has been implicated in the SAC signaling pathway. MAD1 forms a complex with another SAC component MAD2 and specifically localizes to unattached kinetochores to facilitate efficient binding of MAD2 to its target, CDC20, the mitotic substrate-specific activator of the anaphase promoting complex or cyclosome (APC/C). Thus, MAD1 connects 2 sequential events in the SAC signaling pathway-recognition of unattached kinetochores and inhibition of APC/C activity. However, the molecular mechanisms by which it specifically localizes to unattached kinetochores are largely unknown. Studies in multicellular organisms have revealed the role of MAD1 in development and tumor suppression, but the precise time at which MAD1 activity is required is unknown. Investigation of cellular and organismic functions of MAD1 in the simple multicellular organism C. elegans identified functional interactors of MAD1 in both kinetochore-oriented SAC signaling and kinetochore-independent cell cycle regulation. Studying the function of SAC components in C. elegans provides a new molecular insight into the SAC-regulated cell cycle progression in a context of a multicellular organism.


Subject(s)
Caenorhabditis elegans , Cell Cycle Proteins/metabolism , Genes, cdc , Spindle Apparatus/metabolism , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Cycle Proteins/genetics , Humans , Kinetochores/metabolism , Multiprotein Complexes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Signal Transduction/physiology
16.
J Cell Biol ; 183(2): 187-94, 2008 Oct 20.
Article in English | MEDLINE | ID: mdl-18936247

ABSTRACT

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD-ZW10-ZWILCH complex. In two-cell-stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Cell Polarity , Centrosome/metabolism , Chromosome Segregation , Embryo Loss , Embryo, Nonmammalian/cytology , Genes, Helminth , Meiosis , Protein Binding , Protein Transport , RNA Interference , Spindle Apparatus/metabolism , Suppression, Genetic
17.
EMBO J ; 27(7): 1085-96, 2008 Apr 09.
Article in English | MEDLINE | ID: mdl-18309291

ABSTRACT

When newly hatched Caenorhabditis elegans larvae are starved, their primordial germ cells (PGCs) arrest in the post-S phase. This starvation-induced PGC arrest is mediated by the DAF-18/PTEN-AKT-1/PKB nutrient-sensing pathway. Here, we report that the conserved spindle assembly checkpoint (SAC) component MDF-1/MAD1 is required for the PGC arrest. We identified 2 Akt kinase phosphorylation sites on MDF-1. Expression of a non-phosphorylatable mutant MDF-1 partially suppressed the defect in the starvation-induced PGC arrest in L1 larvae lacking DAF-18, suggesting that MDF-1 regulates germ cell proliferation as a downstream target of AKT-1, thereby demonstrating a functional link between cell-cycle regulation by the SAC components and nutrient sensing by DAF-18-AKT-1 during post-embryonic development. The phosphorylation status of MDF-1 affects its binding to another SAC component, MDF-2/MAD2. The loss of MDF-2 or another SAC component also caused inappropriate germ cell proliferation, but the defect was less severe than that caused by mdf-1 hemizygosity, suggesting that MDF-1 causes the PGC arrest by two mechanisms, one involving MDF-2 and another that is independent of other SAC components.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/cytology , Cell Cycle Proteins/genetics , Food , Germ Cells/cytology , Signal Transduction , Spindle Apparatus/metabolism , Alleles , Animals , Apoptosis , Caenorhabditis elegans Proteins/metabolism , Cell Proliferation , Food Deprivation , Germ Cells/enzymology , Heterozygote , Homozygote , Larva , Models, Biological , Mutation/genetics , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Threonine/metabolism
18.
Genetics ; 175(4): 1665-79, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17237515

ABSTRACT

The spindle assembly checkpoint (SAC) governs the timing of metaphase-to-anaphase transition and is essential for genome stability. The Caenorhabditis elegans mutant strain gk2 carries a deletion within the mdf-1/MAD1 gene that results in death of the homozygous strain after two or three generations. Here we describe 11 suppressors of the mdf-1(gk2) lethality, 10 identified in an ethyl methanesulfonate (EMS) mutagenesis screen and 1 isolated using the dog-1(gk10) (deletions of guanine-rich DNA) mutator strain. Using time-lapse imaging of early embryonic cells and germline mitotic division, we demonstrate that there are two classes of suppressors. Eight suppressors compensate for the loss of the checkpoint by delaying mitotic progression, which coincides with securin (IFY-1/Pds1) accumulation; three suppressors have normal IFY-1/Pds1 levels and normal anaphase onset. Furthermore, in the class of suppressors with delayed mitotic progression, we have identified four alleles of known suppressors emb-30/APC4 and fzy-1/CDC20, which are components of the anaphase-promoting complex/cyclosome (APC/C). In addition, we have identified another APC/C component capable of bypassing the checkpoint requirement that has not previously been described in C. elegans. The such-1/APC5-like mutation, h1960, significantly delays anaphase onset both in germline and in early embryonic cells.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Cell Cycle Proteins/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Female , Genes, Helminth , Genes, Lethal , Infertility, Male/genetics , Male , Mitosis/genetics , Molecular Sequence Data , Mutation , Phenotype , Sequence Homology, Amino Acid , Suppression, Genetic
19.
J Cell Biol ; 173(2): 195-206, 2006 Apr 24.
Article in English | MEDLINE | ID: mdl-16618811

ABSTRACT

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.


Subject(s)
Cell Cycle Proteins/physiology , DNA Damage , DNA Repair , DNA/metabolism , Genome , Animals , BRCA1 Protein/physiology , Cell Line , Cells, Cultured , Checkpoint Kinase 1 , Chromatin/physiology , Chromosomal Proteins, Non-Histone/physiology , DNA/radiation effects , Humans , Lasers , Nuclear Proteins/physiology , Phosphorylation , Protein Kinases/physiology
20.
Genes Dev ; 19(24): 3043-54, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16357220

ABSTRACT

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50(S/S) mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50(S/S) mice and found that Rad50(S/S) cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11(ATLD1) and Nbs1(Delta)(B) alleles conferred similar rescue of Rad50(S/S)-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM-Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex-ATM pathway. Conversely, the Rad50(S) mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50(S) allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50(S/S) Atm(-/-) mice.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cell Cycle Proteins/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Hematopoietic Stem Cells/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , ATP-Binding Cassette Transporters/genetics , Acid Anhydride Hydrolases , Alleles , Animals , Apoptosis , Ataxia Telangiectasia Mutated Proteins , Cells, Cultured , Checkpoint Kinase 2 , DNA Repair Enzymes , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation , Hematopoiesis , Hematopoietic Stem Cells/cytology , MRE11 Homologue Protein , Male , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/deficiency
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