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1.
iScience ; 26(5): 106743, 2023 May 19.
Article in English | MEDLINE | ID: mdl-37197327

ABSTRACT

DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR in vitro but essential for mitotic functions in vivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.

2.
Front Cell Dev Biol ; 11: 987153, 2023.
Article in English | MEDLINE | ID: mdl-36875767

ABSTRACT

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5'-end using a CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.

3.
Int J Mol Sci ; 22(24)2021 Dec 07.
Article in English | MEDLINE | ID: mdl-34947973

ABSTRACT

Serotonin 1A receptors (5-HT1ARs) are implicated in the control of mood, cognition, and memory and in various neuropsychiatric disorders such as depression and anxiety. As such, understanding the regulation of 5-HT1ARs will inform the development of better treatment approaches. We previously demonstrated 5-HT1ARs are SUMOylated by SUMO1 in the rat brain. Agonist stimulation increased SUMOylation and was further enhanced when combined with 17ß-estradiol-3-benzoate (EB), which are treatments that cause the transient and prolonged desensitization of 5-HT1AR signaling, respectively. In the current study, we identified the protein inhibitor of activated STAT (PIAS)xα as the enzyme that facilitates SUMOylation, and SENP2 as the protein that catalyzes the deSUMOylation of 5-HT1ARs. We demonstrated that PIASxα significantly increased in the membrane fraction of rats co-treated with EB and an agonist, compared to either the EB-treated or vehicle-treated groups. The acute treatment with an agonist alone shifted the location of SENP2 from the membrane to the cytoplasmic fraction, but it has little effect on PIASxα. Hence, two separate mechanisms regulate SUMOylation and the activity of 5-HT1ARs by an agonist and EB. The effects of EB on 5-HT1AR SUMOylation and signaling may be related to the higher incidence of mood disorders in women during times with large fluctuations in estrogens. Targeting the SUMOylation of 5-HT1ARs could have important clinical relevance for the therapy for several neuropsychiatric disorders in which 5-HT1ARs are implicated.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Estradiol/analogs & derivatives , Protein Inhibitors of Activated STAT/metabolism , Receptor, Serotonin, 5-HT1A/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cytoplasm/metabolism , Estradiol/administration & dosage , Estradiol/pharmacology , Female , Gene Expression Regulation/drug effects , Rats , Sumoylation/drug effects , Up-Regulation
4.
EMBO J ; 40(18): e107413, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34346517

ABSTRACT

DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.


Subject(s)
DNA Repair , DNA Replication , Nuclear Proteins/metabolism , Signal Transduction , Small Ubiquitin-Related Modifier Proteins/metabolism , Transcription Factors/metabolism , Genomic Instability , Humans , Protein Binding , Protein Processing, Post-Translational , Sumoylation , Ubiquitin/metabolism , Ubiquitination
5.
Nucleic Acids Res ; 49(9): 5003-5016, 2021 05 21.
Article in English | MEDLINE | ID: mdl-33872376

ABSTRACT

DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.


Subject(s)
DNA Ligase ATP/metabolism , DNA/metabolism , X-ray Repair Cross Complementing Protein 1/metabolism , Xenopus Proteins/metabolism , Animals , Cell-Free System , Poly (ADP-Ribose) Polymerase-1/metabolism , Xenopus laevis
6.
EMBO Rep ; 22(6): e50684, 2021 06 04.
Article in English | MEDLINE | ID: mdl-33852194

ABSTRACT

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.


Subject(s)
Ribonucleoproteins, Small Nucleolar , Sumoylation , Cell Cycle Proteins/metabolism , Deubiquitinating Enzymes/genetics , HeLa Cells , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proteomics , Ribonucleoproteins, Small Nucleolar/genetics , Ribonucleoproteins, Small Nucleolar/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Ubiquitin Thiolesterase/genetics
7.
J Biol Chem ; 296: 100164, 2021.
Article in English | MEDLINE | ID: mdl-33293370

ABSTRACT

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.


Subject(s)
Aneuploidy , Aurora Kinase B/genetics , Gene Expression Regulation, Neoplastic , Oncogene Proteins, Fusion/genetics , Protein Processing, Post-Translational , Threonine/metabolism , Alanine/metabolism , Amino Acid Substitution , Anaphase , Aspartic Acid/metabolism , Aurora Kinase B/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , CRISPR-Cas Systems , Cell Line, Tumor , Chromosome Segregation , Gene Editing , Humans , Metaphase , Models, Biological , Mutation , Oncogene Proteins, Fusion/metabolism , Phosphorylation , Prophase , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Signal Transduction , Transgenes
8.
Mol Biol Cell ; 31(23): 2537-2556, 2020 11 01.
Article in English | MEDLINE | ID: mdl-32877270

ABSTRACT

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by the abnormal distribution of genetic information. Polo-like kinase 1-interacting checkpoint helicase (PICH) is a DNA translocase essential for chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by the small ubiquitin-like modifier (SUMO). However, it is unclear how these activities cooperate to resolve chromosome bridges. Here, we show that PICH specifically disperses SUMO2/3 foci on mitotic chromosomes. This PICH function is apparent toward SUMOylated topoisomerase IIα (TopoIIα) after inhibition of TopoIIα by ICRF-193. Conditional depletion of PICH using the auxin-inducible degron (AID) system resulted in the retention of SUMO2/3-modified chromosomal proteins, including TopoIIα, indicating that PICH functions to reduce the association of these proteins with chromosomes. Replacement of PICH with its translocase-deficient mutants led to increased SUMO2/3 foci on chromosomes, suggesting that the reduction of SUMO2/3 foci requires the remodeling activity of PICH. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taking the results together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.


Subject(s)
Chromosome Segregation/physiology , DNA Helicases/metabolism , Centromere/metabolism , Chromosome Segregation/genetics , Chromosomes/metabolism , DNA Helicases/physiology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Humans , Mitosis/genetics , Mitosis/physiology , Sumoylation
9.
J Cell Biol ; 219(1)2020 01 06.
Article in English | MEDLINE | ID: mdl-31712254

ABSTRACT

Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.


Subject(s)
Aurora Kinase B/metabolism , Chromosomes, Human/genetics , DNA Topoisomerases, Type II/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Metaphase , Mitosis , Protein Serine-Threonine Kinases/metabolism , Sumoylation , Aurora Kinase B/genetics , Chromosome Segregation , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Diketopiperazines , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kinetochores , Phosphorylation , Piperazines/pharmacology , Protein Serine-Threonine Kinases/genetics , Topoisomerase II Inhibitors/pharmacology
10.
Oncotarget ; 9(30): 21429-21443, 2018 Apr 20.
Article in English | MEDLINE | ID: mdl-29765550

ABSTRACT

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.

11.
J Bone Miner Metab ; 36(4): 410-419, 2018 Jul.
Article in English | MEDLINE | ID: mdl-28770354

ABSTRACT

Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.


Subject(s)
Chondrogenesis , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , SOX9 Transcription Factor/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , Cell Nucleus/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Gene Knock-In Techniques , Growth Plate/metabolism , HEK293 Cells , Humans , Mice, Inbred C57BL , Protein Binding
12.
Int J Mol Sci ; 18(11)2017 Nov 17.
Article in English | MEDLINE | ID: mdl-29149026

ABSTRACT

DNA Topoisomerase IIα (Topo IIα) is a ubiquitous enzyme in eukaryotes that performs the strand passage reaction where a double helix of DNA is passed through a second double helix. This unique reaction is critical for numerous cellular processes. However, the enzyme also possesses a C-terminal domain (CTD) that is largely dispensable for the strand passage reaction but is nevertheless important for the fidelity of cell division. Recent studies have expanded our understanding of the roles of the Topo IIα CTD, in particular in mitotic mechanisms where the CTD is modified by Small Ubiquitin-like Modifier (SUMO), which in turn provides binding sites for key regulators of mitosis.


Subject(s)
Catalytic Domain/physiology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Aurora Kinase B/metabolism , Centromere/metabolism , Chromatin/metabolism , Cysteine Endopeptidases/metabolism , DNA Topoisomerases, Type II/chemistry , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/physiology
13.
J Biol Chem ; 292(24): 10230-10238, 2017 06 16.
Article in English | MEDLINE | ID: mdl-28455449

ABSTRACT

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.


Subject(s)
Models, Molecular , Protein Inhibitors of Activated STAT/metabolism , Repressor Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Ubiquitins/metabolism , Xenopus Proteins/metabolism , Amino Acid Motifs , Animals , Gene Deletion , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Mutagenesis, Site-Directed , Mutation , Nitrogen Isotopes , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Inhibitors of Activated STAT/chemistry , Protein Inhibitors of Activated STAT/genetics , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/genetics , Ubiquitins/chemistry , Ubiquitins/genetics , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus laevis
15.
J Cell Biol ; 213(6): 665-78, 2016 06 20.
Article in English | MEDLINE | ID: mdl-27325792

ABSTRACT

DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres.


Subject(s)
Antigens, Neoplasm/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Histones/metabolism , Mitosis/physiology , Phosphorylation/physiology , Sumoylation/physiology , Animals , Aurora Kinase B/metabolism , Centromere/metabolism , Centromere/physiology , Chromosome Segregation/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Kinetochores/metabolism , Kinetochores/physiology , Protein Binding/physiology , Protein Serine-Threonine Kinases/metabolism , Threonine/metabolism , Xenopus laevis
16.
Cell Cycle ; 15(16): 2135-2144, 2016 Aug 17.
Article in English | MEDLINE | ID: mdl-27230136

ABSTRACT

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.


Subject(s)
Chromosome Segregation , DNA Helicases/chemistry , DNA Helicases/metabolism , Mitosis , Small Ubiquitin-Related Modifier Proteins/metabolism , Amino Acid Motifs , Anaphase , Animals , Centromere/metabolism , Chromatin/metabolism , HeLa Cells , Humans , Protein Domains , Structure-Activity Relationship , Sumoylation , Xenopus laevis
17.
Cell Cycle ; 14(17): 2777-84, 2015.
Article in English | MEDLINE | ID: mdl-26131587

ABSTRACT

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.


Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Antigens, Neoplasm/biosynthesis , Centromere/metabolism , DNA Topoisomerases, Type II/biosynthesis , DNA-Binding Proteins/biosynthesis , Sumoylation/physiology , Xenopus Proteins/biosynthesis , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Antigens, Neoplasm/genetics , Centromere/chemistry , Centromere/genetics , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Female , Male , Molecular Sequence Data , Xenopus Proteins/analysis , Xenopus Proteins/genetics , Xenopus laevis
18.
Drug Metab Dispos ; 43(9): 1316-25, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26063058

ABSTRACT

Several nuclear receptor (NR) superfamily members are known to be the molecular target of either the small ubiquitin-related modifier (SUMO) or ubiquitin-signaling pathways. However, little is currently known regarding how these two post-translational modifications interact to control NR biology. We show that SUMO and ubiquitin circuitry coordinately modifies the pregnane X receptor (PXR, NR1I2) to play a key role in regulating PXR protein stability, transactivation capacity, and transcriptional repression. The SUMOylation and ubiquitylation of PXR is increased in a ligand- and tumor necrosis factor alpha -: dependent manner in hepatocytes. The SUMO-E3 ligase enzymes protein inhibitor of activated signal transducer and activator of transcription-1 (STAT1) STAT-1 (PIAS1) and protein inhibitor of activated STAT Y (PIASy) drive high levels of PXR SUMOylation. Expression of protein inhibitor of activated stat 1 selectively increases SUMO(3)ylation as well as PXR-mediated induction of cytochrome P450, family 3, subfamily A and the xenobiotic response. The PIASy-mediated SUMO(1)ylation imparts a transcriptionally repressive function by ameliorating interaction of PXR with coactivator protein peroxisome proliferator-activated receptor gamma coactivator-1-alpha. The SUMO modification of PXR is effectively antagonized by the SUMO protease sentrin protease (SENP) 2, whereas SENP3 and SENP6 proteases are highly active in the removal of SUMO2/3 chains. The PIASy-mediated SUMO(1)ylation of PXR inhibits ubiquitin-mediated degradation of this important liver-enriched NR by the 26S proteasome. Our data reveal a working model that delineates the interactive role that these two post-translational modifications play in reconciling PXR-mediated gene activation of the xenobiotic response versus transcriptional repression of the proinflammatory response in hepatocytes. Taken together, our data reveal that the SUMOylation and ubiquitylation of the PXR interface in a fundamental manner directs its biologic function in the liver in response to xenobiotic or inflammatory stress.


Subject(s)
Hepatocytes/metabolism , Receptors, Steroid/metabolism , Animals , Humans , Mice , Mice, Knockout , Pregnane X Receptor , Signal Transduction , Sumoylation , Ubiquitination
19.
J Biol Chem ; 290(6): 3269-76, 2015 Feb 06.
Article in English | MEDLINE | ID: mdl-25564610

ABSTRACT

Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.


Subject(s)
DNA Helicases/metabolism , Mitosis , Sumoylation , Animals , Antigens, Neoplasm/metabolism , Centromere/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Humans , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Inhibitors of Activated STAT/metabolism , Protein Transport , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitins/metabolism , Xenopus
20.
Biochemistry ; 53(27): 4334-45, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24898734

ABSTRACT

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/ß1 = 1.3 ± 0.6 nM, and 1/ß2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA/metabolism , Nucleosomes/metabolism , Nucleotides/metabolism , Transcription Factors/metabolism , Xenopus Proteins/metabolism , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Allosteric Regulation , Animals , Chromatin Assembly and Disassembly , Fluorescence Polarization , Pichia/metabolism , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transcription Factors/genetics , Xenopus Proteins/genetics , Xenopus laevis
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