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1.
Nat Immunol ; 15(12): 1171-80, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25344725

ABSTRACT

Mature lymphoid cells express the transcription repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells ('myeloid genes'). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2(hi) CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs.


Subject(s)
B-Lymphocytes/cytology , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Differentiation/physiology , Precursor Cells, B-Lymphoid/cytology , Trans-Activators/metabolism , Animals , B-Lymphocytes/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Cell Lineage , Cell Separation , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Flow Cytometry , Gene Expression Regulation/physiology , Lymphoid Progenitor Cells/cytology , Lymphoid Progenitor Cells/metabolism , Lymphopoiesis/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Precursor Cells, B-Lymphoid/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/genetics
2.
EMBO J ; 40(18): e108345, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34337769

ABSTRACT

PIWI-interacting RNAs (piRNAs) are germline-specific small RNAs that form effector complexes with PIWI proteins (Piwi-piRNA complexes) and play critical roles for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation and increases histone H3K9 methylation (H3K9me3) and histone H1 deposition at these loci, with nuclear RNA export factor variant Nxf2 serving as a co-factor. Using ChEP and DamID-seq, we now uncover a Piwi/Nxf2-dependent target association with nuclear lamins. Hi-C analysis of Piwi or Nxf2-depleted cells reveals decreased intra-TAD and increased inter-TAD interactions in regions harboring Piwi-piRNA target TEs. Using a forced tethering system, we analyze the functional effects of Piwi-piRNA/Nxf2-mediated recruitment of piRNA target regions to the nuclear periphery. Removal of active histone marks is followed by transcriptional silencing, chromatin conformational changes, and H3K9me3 and H1 association. Our data show that the Piwi-piRNA pathway can induce stepwise changes in nuclear architecture and chromatin state at target loci for transcriptional silencing.


Subject(s)
Argonaute Proteins/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Drosophila Proteins/metabolism , Gene Expression Regulation , Genetic Loci , RNA, Small Interfering/metabolism , Animals , Chromatin Assembly and Disassembly , Drosophila melanogaster , Heterochromatin/genetics , Heterochromatin/metabolism , Protein Binding , RNA, Small Interfering/genetics
3.
Genes Cells ; 2024 Sep 08.
Article in English | MEDLINE | ID: mdl-39245559

ABSTRACT

Histone modifications are catalyzed and recognized by specific proteins to regulate dynamic DNA metabolism processes. NSD2 is a histone H3 lysine 36 (H3K36)-specific methyltransferase that is associated with both various transcription regulators and DNA repair factors. Specifically, it has been implicated in the repair of DNA double-strand breaks (DSBs); however, the role of NSD2 during DSB repair remains enigmatic. Here, we show that NSD2 does not accumulate at DSB sites and that it is not further mobilized by DSB formation. Using three different DSB repair reporter systems, which contained the endonuclease site in the active thymidine kinase gene (TK) locus, we demonstrated separate dose-dependent effects of NSD2 on homologous recombination (HR), canonical-non-homologous end joining (c-NHEJ), and non-canonical-NHEJ (non-c-NHEJ). Endogenous NSD2 has a role in repressing non-c-NHEJ, without affecting DSB repair efficiency by HR or total NHEJ. Furthermore, overexpression of NSD2 promotes c-NHEJ repair and suppresses HR repair. Therefore, we propose that NSD2 has functions in chromatin integrity at the active regions during DSB repair.

4.
Clin Sci (Lond) ; 137(2): 163-180, 2023 01 31.
Article in English | MEDLINE | ID: mdl-36598778

ABSTRACT

Cigarette smoking is a major risk factor for atherosclerosis. We previously reported that DNA damage was accumulated in atherosclerotic plaque, and was increased in human mononuclear cells by smoking. As vascular endothelial cells are known to modulate inflammation, we investigated the mechanism by which smoking activates innate immunity in endothelial cells focusing on DNA damage. Furthermore, we sought to characterize the plasma level of cell-free DNA (cfDNA), a result of mitochondrial and/or genomic DNA damage, as a biomarker for atherosclerosis. Cigarette smoke extract (CSE) increased DNA damage in the nucleus and mitochondria in human endothelial cells. Mitochondrial damage induced minority mitochondrial outer membrane permeabilization, which was insufficient for cell death but instead led to nuclear DNA damage. DNA fragments, derived from the nucleus and mitochondria, were accumulated in the cytosol, and caused a persistent increase in IL-6 mRNA expression via the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. cfDNA, quantified with quantitative PCR in culture medium was increased by CSE. Consistent with in vitro results, plasma mitochondrial cfDNA (mt-cfDNA) and nuclear cfDNA (n-cfDNA) were increased in young healthy smokers compared with age-matched nonsmokers. Additionally, both mt-cfDNA and n-cfDNA were significantly increased in patients with atherosclerosis compared with the normal controls. Our multivariate analysis revealed that only mt-cfDNA predicted the risk of atherosclerosis. In conclusion, accumulated cytosolic DNA caused by cigarette smoke and the resultant activation of the cGAS-STING pathway may be a mechanism of atherosclerosis development. The plasma level of mt-cfDNA, possibly as a result of DNA damage, may be a useful biomarker for atherosclerosis.


Subject(s)
Atherosclerosis , Cell-Free Nucleic Acids , Cigarette Smoking , Humans , Atherosclerosis/metabolism , Cell-Free Nucleic Acids/metabolism , DNA, Mitochondrial/metabolism , Endothelial Cells/metabolism , Mitochondria/metabolism , Nucleotidyltransferases/genetics , DNA Damage
5.
Int Heart J ; 63(3): 466-475, 2022.
Article in English | MEDLINE | ID: mdl-35650148

ABSTRACT

Almost 40% of medical radiation exposure is related to cardiac imaging or intervention. However, the biological effects of low-dose radiation from medical imaging remain largely unknown. This study aimed to evaluate the effects of ionized radiation from cardiac catheterization on genomic DNA integrity and inflammatory cytokines in patients and operators.Peripheral mononuclear cells (MNCs) were isolated from patients (n = 51) and operators (n = 35) before and after coronary angiography and/or percutaneous coronary intervention. The expression of γH2AX, a marker for DNA double-strand breaks, was measured by immunofluorescence. Dicentric chromosomes (DICs), a form of chromosome aberrations, were assayed using a fluorescent in situ hybridization technique.In the patient MNCs, the numbers of γH2AX foci and DICs increased after cardiac catheterization by 4.5 ± 9.4-fold and 71 ± 122%, respectively (P < 0.05 for both). The mRNA expressions of interleukin (IL)-1α, IL-1ß, leukemia inhibitory factor, and caspase-1 were significantly increased by radiation exposure from cardiac catheterization. The increase in IL-1ß was significantly correlated with that of γH2AX, but not with the dose area product. In the operators, neither γH2AX foci nor the DIC level was changed, but IL-1ß mRNA was significantly increased. The protein expression of IκBα was significantly decreased in both groups.DNA damage was increased in the MNCs of patients, but not of operators, who underwent cardiac catheterization. Inflammatory cytokines were increased in both the patients and operators, presumably through NF-κB activation. Further efforts to reduce radiation exposure from cardiac catheterization are necessary for both patients and operators.


Subject(s)
Radiation Exposure , Cardiac Catheterization/adverse effects , Cardiac Catheterization/methods , Cytokines , DNA Damage , Humans , In Situ Hybridization, Fluorescence , RNA, Messenger , Radiation Exposure/adverse effects
6.
Microvasc Res ; 134: 104126, 2021 03.
Article in English | MEDLINE | ID: mdl-33373621

ABSTRACT

Bach1 is a known transcriptional repressor of the heme oxygenase-1 (HO-1) gene. The purpose of this study was to determine whether angiogenesis is accelerated by genetic ablation of Bach1 in a mouse ischemic hindlimb model. Hindlimb ischemia was surgically induced in wild-type (WT) mice, Bach1-deficient (Bach1-/-) mice, apolipoprotein E-deficient (ApoE-/-) mice, and Bach1/ApoE double-knockout (Bach1-/-/ApoE-/-) mice. Blood flow recovery after hindlimb ischemia showed significant improvement in Bach1-/- mice compared with that in WT mice. Bach1-/-/ApoE-/- mice showed significantly improved blood flow recovery compared with that in ApoE-/- mice to the level of that in WT mice. Migration of endothelial cells in ApoE-/- mice was significantly decreased compared with that in WT mice. Migration of endothelial cells significantly increased in Bach1-/-/ApoE-/- mice compared with that in ApoE-/- mice to the level of that in WT mice. The expression levels of HO-1, peroxisome proliferator-activated receptor γ co-activator-1α, angiopoietin 1, and fibroblast growth factor 2 in endothelial cells isolated from Bach1-/-/ApoE-/- mice were significantly higher than those in ApoE-/- mice. Oxidative stress assessed by anti-acrolein antibody staining in ischemic tissues and urinary 8-isoPGF2α excretion were significantly increased in ApoE-/- mice compared with those in WT and Bach1-/- mice. Oxidative stress was reduced in Bach1-/-/ApoE-/- mice compared with that in ApoE-/- mice. These findings suggest that genetic ablation of Bach1 plays an important role in ischemia-induced angiogenesis under the condition of increased oxidative stress. Bach1 could be a potential therapeutic target to reduce oxidative stress and potentially improve angiogenesis for patients with peripheral arterial disease.


Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Endothelial Cells/metabolism , Ischemia/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Oxidative Stress , Animals , Apoptosis , Basic-Leucine Zipper Transcription Factors/deficiency , Basic-Leucine Zipper Transcription Factors/genetics , Blood Flow Velocity , Cell Movement , Cells, Cultured , Disease Models, Animal , Endothelial Cells/pathology , Heme Oxygenase-1/metabolism , Hindlimb , Ischemia/genetics , Ischemia/pathology , Ischemia/physiopathology , Male , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout, ApoE , Microvascular Density , Regional Blood Flow , Signal Transduction
7.
Radiology ; 295(2): 439-445, 2020 05.
Article in English | MEDLINE | ID: mdl-32154776

ABSTRACT

Background Although the National Lung Screening Trial reported a significant reduction in lung cancer mortality when low-dose (LD) CT chest examinations are used for a diagnosis, their biologic effects from radiation exposure remain unclear. Purpose To compare LD CT and standard-dose (SD) CT for DNA double-strand breaks and chromosome aberrations (CAs) in peripheral blood lymphocytes. Materials and Methods Between March 2016 and June 2018, 209 participants who were referred to a respiratory surgery department for chest CT studies were prospectively enrolled in this study. Individuals were excluded if they had undergone radiography examinations within the last 3 days or had undergone chemotherapy or radiation therapy. Peripheral blood samples were obtained before and 15 minutes after CT. The number of γ-H2AX foci and unstable CAs in lymphocytes was quantified by immunofluorescent staining of γ-H2AX and by fluorescence in situ hybridization by using peptide nucleic acid probes for centromeres and telomeres, respectively. The Wilcoxon signed rank test was used for statistical analysis. Bonferroni correction was applied for multiple comparisons. Results Of the 209 participants (105 women, 104 men; mean age, 67.0 years ± 11.3 [standard deviation]), 107 underwent chest LD CT and 102 underwent chest SD CT. Sex distribution, age, and body size metrics were similar between the two groups. The median effective dose of LD CT and SD CT was 1.5 and 5.0 mSv, respectively. The number of double-strand breaks and CAs increased after a SD CT examination (γ-H2AX, P < .001; CAs, P = .003); the number of double-strand breaks and CAs before and after LD CT was not different (γ-H2AX, P = .45; CAs, P = .69). Conclusion No effect of low-dose CT on human DNA was detected. In the same setting, DNA double-strand breaks and chromosome aberrations increased after standard-dose CT. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Brenner in this issue.


Subject(s)
Chromosomes/radiation effects , DNA Breaks, Double-Stranded/radiation effects , Radiation Dosage , Radiography, Thoracic/methods , Tomography, X-Ray Computed/methods , Aged , Aged, 80 and over , Female , Humans , Male , Prospective Studies
8.
PLoS Comput Biol ; 15(9): e1007289, 2019 09.
Article in English | MEDLINE | ID: mdl-31509522

ABSTRACT

Higher-order genomic architecture varies according to cell type and changes dramatically during differentiation. One of the remarkable examples of spatial genomic reorganization is the rod photoreceptor cell differentiation in nocturnal mammals. The inverted nuclear architecture found in adult mouse rod cells is formed through the reorganization of the conventional architecture during terminal differentiation. However, the mechanisms underlying these changes remain largely unknown. Here, we found that the dynamic deformation of nuclei via actomyosin-mediated contractility contributes to chromocenter clustering and promotes genomic architecture reorganization during differentiation by conducting an in cellulo experiment coupled with phase-field modeling. Similar patterns of dynamic deformation of the nucleus and a concomitant migration of the nuclear content were also observed in rod cells derived from the developing mouse retina. These results indicate that the common phenomenon of dynamic nuclear deformation, which accompanies dynamic cell behavior, can be a universal mechanism for spatiotemporal genomic reorganization.


Subject(s)
Cell Differentiation/genetics , Cell Nucleus , Chromosome Structures , Animals , Cell Nucleus/genetics , Cell Nucleus/physiology , Chromosome Structures/physiology , Chromosome Structures/ultrastructure , Computational Biology , Male , Mice , Mice, Inbred C57BL , Models, Biological , Retinal Rod Photoreceptor Cells/cytology
9.
Nucleic Acids Res ; 46(19): 10007-10018, 2018 11 02.
Article in English | MEDLINE | ID: mdl-30053102

ABSTRACT

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.


Subject(s)
Histones/chemistry , Neoplasms/genetics , Nucleosomes/genetics , Chromatin/genetics , Histones/genetics , Humans , Mutation , Nucleosomes/chemistry , Protein Folding
10.
Biol Pharm Bull ; 42(4): 531-537, 2019.
Article in English | MEDLINE | ID: mdl-30930412

ABSTRACT

DNA suffers various types of damage even in a normal condition, although they are rapidly repaired by mechanisms called DNA repair. Most progeroid syndromes are caused by genetic defects in specific molecules involved in the DNA repair. DNA damage activates a broad range of signaling pathway that leads to repair, cell cycle arrest, apoptosis and so on, which is called DNA damage response. Recent studies revealed that persistent DNA damage response triggers induction of cell senescence and senescence-associated secretory phenotype (SASP). Here, we review recent advances in the understanding of the molecular mechanisms by which SASP components are regulated, and discuss the possible roles of DNA damage and the DNA damage response, and SASP in the pathogenesis of cardiovascular disease.


Subject(s)
Cardiovascular Diseases , Cellular Senescence , DNA Damage , Animals , Cardiovascular Diseases/genetics , Cardiovascular Diseases/pathology , Genomic Instability , Humans , Inflammation/genetics , Inflammation/pathology
11.
Mol Cell ; 41(5): 554-66, 2011 Mar 04.
Article in English | MEDLINE | ID: mdl-21362551

ABSTRACT

Protein methylation pathways comprise methionine adenosyltransferase (MAT), which produces S-adenosylmethionine (SAM) and SAM-dependent substrate-specific methyltransferases. However, the function of MAT in the nucleus is largely unknown. MafK represses or activates expression of heme oxygenase-1 (HO-1) gene, depending on its heterodimer partners. Proteomics analysis of MafK revealed its interaction with MATIIα, a MAT isozyme. MATIIα was localized in nuclei and found to form a dense network with chromatin-related proteins including Swi/Snf and NuRD complexes. MATIIα was recruited to Maf recognition element (MARE) at HO-1 gene. When MATIIα was knocked down in murine hepatoma cell line, expression of HO-1 was derepressed at both basal and induced levels. The catalytic activity of MATIIα, as well as its interacting factors such as MATIIß, BAF53a, CHD4, and PARP1, was required for HO-1 repression. MATII serves as a transcriptional corepressor of MafK by interacting with chromatin regulators and supplying SAM for methyltransferases.


Subject(s)
Gene Expression Regulation, Neoplastic , Methionine Adenosyltransferase/metabolism , Proto-Oncogene Proteins c-maf/metabolism , Transcription, Genetic , Animals , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Heme Oxygenase-1/biosynthesis , Histones/metabolism , Humans , Isoenzymes/chemistry , Liver Neoplasms/metabolism , Mice , Oncogene Proteins/metabolism , Proteomics/methods
12.
Mol Cell ; 41(5): 515-28, 2011 Mar 04.
Article in English | MEDLINE | ID: mdl-21362548

ABSTRACT

The E3 ubiquitin ligase RNF20 regulates chromatin structure by monoubiquitinating histone H2B in transcription. Here, we show that RNF20 is localized to double-stranded DNA breaks (DSBs) independently of H2AX and is required for the DSB-induced H2B ubiquitination. In addition, RNF20 is required for the methylation of H3K4 at DSBs and the recruitment of the chromatin-remodeling factor SNF2h. Depletion of RNF20, depletion of SNF2h, or expression of the H2B mutant lacking the ubiquitination site (K120R) compromises resection of DNA ends and recruitment of RAD51 and BRCA1. Consequently, cells lacking RNF20 or SNF2h and cells expressing H2B K120R exhibit pronounced defects in homologous recombination repair (HRR) and enhanced sensitivity to radiation. Finally, the function of RNF20 in HRR can be partially bypassed by forced chromatin relaxation. Thus, the RNF20-mediated H2B ubiquitination at DSBs plays a critical role in HRR through chromatin remodeling.


Subject(s)
Chromatin/chemistry , Gene Expression Regulation , Histones/chemistry , Nijmegen Breakage Syndrome/metabolism , Recombination, Genetic , Ubiquitin-Protein Ligases/chemistry , Ubiquitin/chemistry , BRCA1 Protein/chemistry , Cell Line, Tumor , DNA Methylation , DNA Repair , HeLa Cells , Humans , Rad51 Recombinase/chemistry , Time Factors , Transcription, Genetic
13.
Biosci Biotechnol Biochem ; 83(8): 1594-1597, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30898039

ABSTRACT

Rim15p of the yeast Saccharomyces cerevisiae is a Greatwall-family protein kinase that inhibits alcoholic fermentation during sake brewing. To elucidate the roles of Rim15p in barley shochu fermentation, RIM15 was deleted in shochu yeast. The disruptant did not improve ethanol yield, but altered sugar and glycerol contents in the mash, suggesting that Rim15p has a novel function in carbon utilization.


Subject(s)
Carbon/metabolism , Fermentation , Hordeum/metabolism , Protein Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Genes, Fungal
14.
Genes Dev ; 24(4): 333-8, 2010 Feb 15.
Article in English | MEDLINE | ID: mdl-20159953

ABSTRACT

A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.


Subject(s)
DNA Damage/physiology , G1 Phase/physiology , Histone Acetyltransferases/metabolism , Ribonucleotide Reductases/metabolism , Animals , Gene Knockdown Techniques , HeLa Cells , Histone Acetyltransferases/genetics , Humans , Lysine Acetyltransferase 5 , Mice , Trans-Activators
15.
Biochim Biophys Acta ; 1861(11): 1816-1827, 2016 11.
Article in English | MEDLINE | ID: mdl-27616329

ABSTRACT

Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein-RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3'UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.


Subject(s)
AU Rich Elements/genetics , Aging/genetics , ELAV Proteins/metabolism , Leptin/genetics , Lipid-Linked Proteins/metabolism , Obesity/genetics , RNA Stability/genetics , 3' Untranslated Regions/genetics , Animals , Base Sequence , COS Cells , Chlorocebus aethiops , Gene Deletion , Gene Expression Regulation , Leptin/metabolism , Lipid-Linked Proteins/genetics , Male , Metabolome , Mice, Knockout , RNA Processing, Post-Transcriptional/genetics , RNA, Messenger/genetics , Sex-Determining Region Y Protein/genetics , Sex-Determining Region Y Protein/metabolism , Subcellular Fractions/metabolism , Transcription, Genetic
16.
Eur Radiol ; 27(4): 1660-1666, 2017 Apr.
Article in English | MEDLINE | ID: mdl-27510626

ABSTRACT

OBJECTIVES: To investigate whether physical exposure parameters such as the dose index (CTDI), dose length product (DLP), and size-specific dose estimate (SSDE) are predictive of DNA damage. METHODS: In vitro, we scanned a phantom containing blood samples from five volunteers at CTDI 50, 100, and 150 mGy. One sample was not scanned. We also scanned samples in three different-size phantoms at CTDI 100 mGy. In vivo, we enrolled 45 patients and obtained blood samples before and after cardiac CT. The γ-H2AX foci were counted. RESULTS: In vitro, in the control and at CTDI 50, 100, and 150 mGy, the number of γ-H2AX was 0.94 ± 0.24 (standard error, SE), 1.28 ± 0.30, 1.91 ± 0.47, and 2.16 ± 0.20. At SSDE 180, 156, and 135 mGy, it was 2.41 ± 0.20, 1.91 ± 0.47, and 1.42 ± 0.20 foci/cell. The γ-H2AX foci were positively correlated with the radiation dose and negatively correlated with the body size. In vivo, the γ-H2AX foci were significantly increased after CT (from 1.21 ± 0.19 to 1.92 ± 0.22 foci/cell) and correlated with CTDI, DLP, and SSDE. CONCLUSIONS: DNA damage was induced by cardiac CT. There was a correlation between the physical exposure parameters and γ-H2AX. KEY POINTS: • DNA damage was induced by radiation exposure from cardiac CT. • The γ-H2AX foci number was correlated with the CT radiation dose. • Physical exposure parameters reflect the DNA damage by CT radiation exposure.


Subject(s)
DNA Damage , Lymphocytes/radiation effects , Tomography, X-Ray Computed/methods , Adult , Aged , Arrhythmias, Cardiac/diagnostic imaging , Body Size , Female , Histones/analysis , Histones/drug effects , Humans , Male , Middle Aged , Phantoms, Imaging , Radiation Dosage , Radiation Exposure
17.
Biochemistry ; 55(4): 637-46, 2016 Feb 02.
Article in English | MEDLINE | ID: mdl-26757249

ABSTRACT

Linker histones bind to nucleosomes and compact polynucleosomes into a higher-order chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified and may have distinct functions. In this study, we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cell-specific linker histones, respectively, and found that H1T forms less compacted chromatin, as compared to H1.2. An in vitro homologous pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed when H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination.


Subject(s)
DNA Helicases/chemistry , Histones/chemistry , Nuclear Proteins/chemistry , Nucleosomes/chemistry , Rad51 Recombinase/chemistry , Recombination, Genetic , Cell Line , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins , Histones/genetics , Histones/immunology , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism
18.
Cancer Sci ; 107(4): 444-51, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26825989

ABSTRACT

The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR-TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non-small-cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR-TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR-TKIs, we examined the cross-talk of the EGFR pathways with ataxia telangiectasia-mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR-TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/biosynthesis , Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Apoptosis/drug effects , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , Gefitinib , Humans , Morpholines/administration & dosage , Mutation , Protein Kinase Inhibitors/administration & dosage , Pyrones/administration & dosage , Quinazolines/administration & dosage , Signal Transduction/drug effects
19.
Genes Cells ; 20(9): 681-94, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26123175

ABSTRACT

Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.


Subject(s)
Cellular Apoptosis Susceptibility Protein/metabolism , Homologous Recombination , Rad51 Recombinase/metabolism , Recombinational DNA Repair , Cell Line, Tumor , Cell Nucleus/metabolism , Chromosome Aberrations , DNA Breaks, Double-Stranded , Humans
20.
FASEB J ; 29(6): 2514-25, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25733566

ABSTRACT

DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation (IR). RAD51-dependent homologous recombination (HR) is one of the most important pathways in DSB repair and genome integrity maintenance. However, the mechanism of HR regulation by RAD51 remains unclear. To understand the mechanism of RAD51-dependent HR, we searched for interacting partners of RAD51 by a proteomics analysis and identified lamin B1 in human cells. Lamins are nuclear lamina proteins that play important roles in the structural organization of the nucleus and the regulation of chromosome functions. Immunoblotting analyses revealed that siRNA-mediated lamin B1 depletion repressed the DNA damage-dependent increase of RAD51 after IR. The repression was abolished by the proteasome inhibitor MG132, suggesting that lamin B1 stabilizes RAD51 by preventing proteasome-mediated degradation in cells with IR-induced DNA damage. We also showed that lamin B1 depletion repressed RAD51 focus formation and decreased the survival rates after IR. On the basis of these results, we propose that lamin B1 promotes DSB repair and cell survival by maintaining the RAD51 protein levels for HR upon DSB induction after IR.


Subject(s)
DNA Damage , Homologous Recombination , Lamin Type B/metabolism , Recombinational DNA Repair , Cell Cycle/genetics , Cell Cycle/radiation effects , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/genetics , Cell Survival/radiation effects , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Lamin Type B/genetics , Mass Spectrometry/methods , Microscopy, Confocal , Protein Binding , Protein Stability , RNA Interference , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , X-Rays
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