Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
Add more filters










Publication year range
1.
Nat Commun ; 12(1): 3253, 2021 05 31.
Article in English | MEDLINE | ID: mdl-34059674

ABSTRACT

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.


Subject(s)
Cell Self Renewal/genetics , Muscle, Skeletal/injuries , PAX7 Transcription Factor/metabolism , Regeneration/genetics , Satellite Cells, Skeletal Muscle/physiology , Acetylation , Animals , COS Cells , CRISPR-Cas Systems , Cardiotoxins/administration & dosage , Cardiotoxins/toxicity , Cell Differentiation/genetics , Chlorocebus aethiops , Disease Models, Animal , Gene Knockdown Techniques , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Humans , Mice , Mice, Transgenic , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , Mutagenesis , Primary Cell Culture , Promoter Regions, Genetic , Sf9 Cells , Sirtuin 2/genetics , Sirtuin 2/metabolism , Spodoptera , Transcriptional Activation
2.
Cell Stem Cell ; 11(3): 333-45, 2012 Sep 07.
Article in English | MEDLINE | ID: mdl-22863532

ABSTRACT

In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.


Subject(s)
Cell Division/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , PAX7 Transcription Factor/genetics , Protein-Arginine N-Methyltransferases/metabolism , Satellite Cells, Skeletal Muscle/cytology , Transcription, Genetic , Amino Acid Motifs , Amino Acid Sequence , Animals , Arginine/metabolism , DNA/metabolism , HEK293 Cells , Histone-Lysine N-Methyltransferase , Histones/metabolism , Humans , Lysine/metabolism , Methylation , Mice , Molecular Sequence Data , Myogenic Regulatory Factor 5/metabolism , PAX7 Transcription Factor/chemistry , PAX7 Transcription Factor/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Tertiary , Satellite Cells, Skeletal Muscle/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Substrate Specificity
3.
Dev Cell ; 22(6): 1208-20, 2012 Jun 12.
Article in English | MEDLINE | ID: mdl-22609161

ABSTRACT

Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development.


Subject(s)
Amino Acid Motifs/physiology , Homeodomain Proteins/metabolism , Muscle Development/physiology , Muscle, Skeletal/metabolism , PAX7 Transcription Factor/metabolism , Transcription, Genetic , Animals , Cell Differentiation , Cell Proliferation , Gene Expression Profiling , Mice , PAX3 Transcription Factor , Paired Box Transcription Factors/metabolism
4.
PLoS One ; 6(10): e25871, 2011.
Article in English | MEDLINE | ID: mdl-22028794

ABSTRACT

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.


Subject(s)
Embryonic Development , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Cycle/genetics , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Embryonic Stem Cells/metabolism , Female , Male , Mice , Mutation , Phenotype , Protein Stability , Protein Structure, Tertiary , Transcriptome , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics
5.
Nat Cell Biol ; 11(3): 312-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19198599

ABSTRACT

CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins. The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs 13, 14, 15, 16, 17, 18, 19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3.


Subject(s)
Breast Neoplasms/enzymology , Ubiquitin-Protein Ligases/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Transformation, Neoplastic , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Histone Acetyltransferases/metabolism , Humans , Lung Neoplasms/secondary , Mice , Nuclear Receptor Coactivator 3 , Protein Processing, Post-Translational , Trans-Activators/metabolism , Ubiquitination , Xenograft Model Antitumor Assays
6.
Cell Stem Cell ; 3(5): 555-67, 2008 Nov 06.
Article in English | MEDLINE | ID: mdl-18983969

ABSTRACT

The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.


Subject(s)
Embryo Implantation/genetics , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Kruppel-Like Transcription Factors/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Homozygote , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Knockout , Mutation , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/genetics , Transfection
7.
J Cell Biol ; 179(1): 53-63, 2007 Oct 08.
Article in English | MEDLINE | ID: mdl-17923529

ABSTRACT

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.


Subject(s)
Adenosine Triphosphatases/metabolism , Bloom Syndrome/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/physiology , Animals , Bloom Syndrome/genetics , Cell Line , Chickens , Chromosome Aberrations , DNA Helicases/genetics , DNA Helicases/physiology , DNA Repair/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Genetic Predisposition to Disease , Humans , Mutation , RecQ Helicases , Recombination, Genetic , Sister Chromatid Exchange , Ultraviolet Rays
8.
Biochem Biophys Res Commun ; 355(2): 477-82, 2007 Apr 06.
Article in English | MEDLINE | ID: mdl-17303082

ABSTRACT

We investigated the function of the interaction between WRN (Werner syndrome gene product) and Ku70 and between WRN and DNA-PKcs, which are components of the DNA-PKcs/Ku70/Ku80 complex, by generating KU70(-/-)/WRN(-/-) and DNA-PKcs(-/-/-)/WRN(-/-) double-gene knockout chicken DT40 cells. When treated with camptothecin (CPT), an inhibitor of DNA topoisomerase I, WRN(-/-) cells showed higher sensitivity than wild-type cells, whereas KU70(-/-) and DNA-PKcs(-/-/-) cells showed hyper-resistance. Disruption of KU70 or DNA-PKcs suppressed the sensitivity of WRN(-/-) cells to CPT, rendering them as resistant to CPT treatment as KU70(-/-) and DNA-PKcs(-/-/-) cells. On the other hand, CPT sensitivity of BLM(-/-) cells, which are defective in a RecQ helicase similar to WRN, was enhanced by deletion of KU70. The implications for the function of WRN in the non-homologous end-joining pathway of DNA repair involving Ku70 and DNA-PKcs, which may be the cause of lethality in the presence of CPT, will be discussed.


Subject(s)
Camptothecin/pharmacology , RecQ Helicases/physiology , Antigens, Nuclear/metabolism , Blotting, Southern , Cell Line , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Exodeoxyribonucleases , Humans , Ku Autoantigen , RecQ Helicases/genetics , Recombination, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Werner Syndrome Helicase
9.
Mol Cell Biol ; 26(21): 7966-76, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16940184

ABSTRACT

Recent studies have shed light on the ligand-dependent transactivation mechanisms of nuclear receptors (NRs). When the ligand dose is reduced, the transcriptional activity of NRs should be downregulated. Here we show that a ubiquitin-proteasome pathway plays a key role in turning off transcription mediated by estrogen receptor beta (ERbeta). ERbeta shows estrogen-dependent proteolysis, and its degradation is regulated by two regions in the receptor. The N-terminal 37-amino acid-region is necessary for the recruitment of the ubiquitin ligase, i.e., the carboxyl terminus of HSC70-interacting protein (CHIP), to degrade ERbeta. In contrast, the C-terminal F domain protects ligand-unbound ERbeta from proteolysis to abrogate proteasome association. Suppression of CHIP by interfering RNA inhibited this switching off of receptor-mediated transcription when the ligand dose was reduced. Our results suggest that after ligand withdrawal, the active form of the NR is selectively eliminated via ligand-dependent proteolysis to downregulate receptor-mediated transcription.


Subject(s)
Estrogen Receptor beta/metabolism , Estrogens/metabolism , Gene Expression Regulation , Transcription, Genetic , Animals , Cell Line , Estrogen Receptor beta/genetics , Humans , Ligands , Mice , Proteasome Endopeptidase Complex/metabolism , Protein Structure, Tertiary , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
10.
DNA Repair (Amst) ; 5(7): 816-28, 2006 Jul 13.
Article in English | MEDLINE | ID: mdl-16769258

ABSTRACT

Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.


Subject(s)
Carrier Proteins/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Animals , Base Sequence , Binding Sites/genetics , Carrier Proteins/genetics , Cell Line , Cell Proliferation , Chickens , DNA Helicases/deficiency , DNA Helicases/genetics , DNA Primers/genetics , DNA Repair , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Exodeoxyribonucleases , Humans , In Vitro Techniques , Mice , RecQ Helicases , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sister Chromatid Exchange , Two-Hybrid System Techniques , Werner Syndrome/genetics , Werner Syndrome/metabolism , Werner Syndrome Helicase
11.
EMBO J ; 25(5): 1081-92, 2006 Mar 08.
Article in English | MEDLINE | ID: mdl-16498406

ABSTRACT

DNA demethylation plays a critical role in transcriptional regulation in differentiated somatic cells. However, there is no experimental evidence that CpG methylation in a small region of a genome restricts gene expression. Here, we show that the anti-CD3repsilon/CD28 antibody stimulation of human CD4+ T cells induces IL2 expression following epigenetic changes, including active demethylation of a specific CpG site, recruitment of Oct-1, and changes in histone modifications. When the stimulatory signal is withdrawn, Oct-1 remains on the enhancer region as a stable marker of the stimulation, causing the second induction to be faster and stronger. Our observations indicate that Oct-1-binding followed by CpG demethylation are key events in the epigenetic regulation of IL2 expression and may act as a memory of the regulatory event.


Subject(s)
CpG Islands , DNA Methylation , Epigenesis, Genetic , Interleukin-2/genetics , Promoter Regions, Genetic , Breast Neoplasms/metabolism , CD28 Antigens/immunology , CD4-Positive T-Lymphocytes/metabolism , Chromatin Immunoprecipitation , Enhancer Elements, Genetic , Gene Expression Regulation , Histones/metabolism , Humans , Jurkat Cells/metabolism , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism , Tumor Cells, Cultured
12.
EMBO J ; 23(24): 4813-23, 2004 Dec 08.
Article in English | MEDLINE | ID: mdl-15538384

ABSTRACT

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.


Subject(s)
Estrogen Receptor alpha/metabolism , Proteasome Endopeptidase Complex/metabolism , Signal Transduction/physiology , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Animals , Carrier Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , DNA-Binding Proteins , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/genetics , Estrogens/metabolism , Fibroblasts/cytology , Fibroblasts/physiology , HSC70 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/metabolism , Humans , Ligands , Mice , Mice, Knockout , Protein Binding , Protein Conformation , Protein Folding , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors , Ubiquitin-Protein Ligases/genetics
13.
DNA Repair (Amst) ; 3(3): 335-41, 2004 Mar 04.
Article in English | MEDLINE | ID: mdl-15177048

ABSTRACT

Ubc9 is an enzyme involved in the conjugation of small ubiquitin related modifier (SUMO) to target proteins. A Saccharomyces cerevisiae ubc9 temperature sensitive (ts) mutant showed higher sensitivity to various DNA damaging agents such as methylmethanesulfonate (MMS) and UV at a semi-permissive temperature than wild-type cells. The sensitivity of ubc9ts cells was not suppressed by the introduction of a mutated UBC9 gene, UBC9-C93S, whose product is unable to covalently bind to SUMO and consequently fails to conjugate SUMO to target proteins. Diploid ubc9ts cells were more sensitive to various DNA damaging agents than haploid ubc9ts cells suggesting the involvement of homologous recombination in the sensitivity of ubc9ts cells. The frequency of interchromosomal recombination between heteroalleles, his1-1/his1-7 loci, in wild-type cells was remarkably increased upon exposure to MMS or UV. Although the frequency of spontaneous interchromosomal recombination between the heteroalleles in ubc9ts cells was almost the same as that of wild-type cells, no induction of interchromosomal recombination was observed in ubc9ts cells upon exposure to MMS or UV.


Subject(s)
DNA Damage , Recombination, Genetic/drug effects , Recombination, Genetic/radiation effects , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , DNA Primers , Methyl Methanesulfonate/toxicity , Mutation/genetics , Plasmids/genetics , Ploidies , Recombination, Genetic/genetics , Saccharomyces cerevisiae , Temperature , Ubiquitin-Conjugating Enzymes/genetics , Ultraviolet Rays
14.
Mol Cell Biol ; 23(10): 3527-35, 2003 May.
Article in English | MEDLINE | ID: mdl-12724411

ABSTRACT

Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells. Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Animals , Bromodeoxyuridine/pharmacology , Cell Culture Techniques , Cell Division , Chickens , Chromosome Aberrations , DNA/metabolism , Flow Cytometry , Genetic Vectors , Humans , Mitomycin/pharmacology , Models, Genetic , Molecular Sequence Data , Mutation , Phenotype , RecQ Helicases , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/metabolism , Sister Chromatid Exchange , Time Factors , Transfection , Ultraviolet Rays
15.
J Biol Chem ; 278(29): 26704-14, 2003 Jul 18.
Article in English | MEDLINE | ID: mdl-12738788

ABSTRACT

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) alpha and beta, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ERalpha. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ERalpha to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ERalpha was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ERalpha and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERalpha. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Receptors, Estrogen/metabolism , Receptors, Interferon/metabolism , Tamoxifen/analogs & derivatives , 3T3 Cells , Animals , Binding, Competitive , Breast Neoplasms/drug therapy , Cell Division/drug effects , Estrogen Receptor alpha , Female , Humans , In Vitro Techniques , Mice , Neoplasms, Hormone-Dependent/drug therapy , Phthalic Acids/metabolism , Phthalic Acids/pharmacology , Protein Binding , Protein Structure, Tertiary , Receptors, Estrogen/chemistry , Receptors, Estrogen/genetics , Repressor Proteins/metabolism , Tamoxifen/pharmacology , Transcriptional Activation/drug effects , Transfection , Tumor Cells, Cultured , Two-Hybrid System Techniques
16.
Exp Cell Res ; 280(2): 212-21, 2002 Nov 01.
Article in English | MEDLINE | ID: mdl-12413887

ABSTRACT

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.


Subject(s)
Cell Nucleus/metabolism , Cell Survival/physiology , Ligases/genetics , Ligases/metabolism , SUMO-1 Protein/metabolism , Ubiquitin-Conjugating Enzymes , Animals , Anti-Bacterial Agents/metabolism , Apoptosis/physiology , Cell Cycle/physiology , Cell Line , Cell Nucleus/genetics , Chickens , Chromosome Segregation , Flow Cytometry , In Situ Hybridization, Fluorescence , Phenotype , Promoter Regions, Genetic , SUMO-1 Protein/genetics , Tetracycline/metabolism , Transgenes
17.
DNA Repair (Amst) ; 1(8): 671-82, 2002 Aug 06.
Article in English | MEDLINE | ID: mdl-12509289

ABSTRACT

RecQ DNA helicases from many organisms have been indicated to function in the maintenance of genomic stability. In human cells, mutation in the WRN helicase, a RecQ-like DNA helicase, results in the Werner syndrome (WS), a genetic disorder characterized by genomic instability and premature ageing. Similarly, mutation in SGS1, the RECQ homologue in budding yeast, results in genomic instability and accelerated ageing. We previously demonstrated that mouse WRN interacts physically with a novel, highly conserved protein that we named WHIP, and that in budding yeast cells, simultaneous deletion of WHIP/MGS1 and SGS1 results in slow growth and shortened life span. Here we show by using genetic analysis in Saccharomyces cerevisiae that mgs1Delta sgs1Delta cells have increased rates of terminal G2/M arrest, and show elevated rates of spontaneous sister chromatid recombination (SCR) and rDNA array recombination. Finally, we report that complementation of the synthetic relationship between SGS1 and WHIP/MGS1 requires both the helicase and Top3-binding activities of Sgs1, as well as the ATPase activity of Mgs1. Our results suggest that Whip/Mgs1 is implicated in DNA metabolism, and is required for normal growth and cell cycle progression in the absence of Sgs1.


Subject(s)
Adenosine Triphosphatases/genetics , Cell Cycle , DNA Helicases/genetics , Mitosis/physiology , Recombination, Genetic , Saccharomyces cerevisiae/physiology , Sister Chromatid Exchange/genetics , Cell Division/physiology , Cells, Cultured , DNA, Ribosomal/genetics , DNA-Binding Proteins/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Gene Deletion , Mutagenesis , Phenotype , Rad52 DNA Repair and Recombination Protein , RecQ Helicases , Saccharomyces cerevisiae Proteins , Sequence Deletion
SELECTION OF CITATIONS
SEARCH DETAIL
...