ABSTRACT
Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress-response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle-TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle-TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.
Subject(s)
Carrier Proteins/metabolism , Cell Nucleolus/metabolism , DNA Damage , DNA Replication , DNA, Ribosomal/biosynthesis , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Aphidicolin/pharmacology , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Carrier Proteins/genetics , Cell Nucleolus/drug effects , Cell Nucleolus/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/genetics , Genomic Instability , HCT116 Cells , HeLa Cells , Humans , Hydroxyurea/pharmacology , Microscopy, Fluorescence , Nuclear Proteins/genetics , Phosphoproteins/genetics , Protein Binding , Protein Transport , Signal TransductionABSTRACT
Cockayne syndrome (CS) is a segmental premature aging syndrome caused primarily by defects in the CSA or CSB genes. In addition to premature aging, CS patients typically exhibit microcephaly, progressive mental and sensorial retardation and cutaneous photosensitivity. Defects in the CSB gene were initially thought to primarily impair transcription-coupled nucleotide excision repair (TC-NER), predicting a relatively consistent phenotype among CS patients. In contrast, the phenotypes of CS patients are pleiotropic and variable. The latter is consistent with recent work that implicates CSB in multiple cellular systems and pathways, including DNA base excision repair, interstrand cross-link repair, transcription, chromatin remodeling, RNAPII processing, nucleolin regulation, rDNA transcription, redox homeostasis, and mitochondrial function. The discovery of additional functions for CSB could potentially explain the many clinical phenotypes of CSB patients. This review focuses on the diverse roles played by CSB in cellular pathways that enhance genome stability, providing insight into the molecular features of this complex premature aging disease.
Subject(s)
DNA Helicases/physiology , DNA Repair Enzymes/physiology , Poly-ADP-Ribose Binding Proteins/physiology , Chromatin Assembly and Disassembly , DNA Breaks, Double-Stranded , DNA Helicases/chemistry , DNA Repair , DNA Repair Enzymes/chemistry , DNA, Ribosomal/biosynthesis , Gene Expression Regulation , Humans , Mitochondria/genetics , Mitochondria/metabolism , Poly-ADP-Ribose Binding Proteins/chemistry , RNA Polymerase II/metabolism , Transcription, GeneticABSTRACT
Human fibroblasts become senescent after a limited number of replications or by diverse stresses, such as DNA damage. However, replicative and damage induced senescence are indistinguishable in respect to proliferation cessation and expression of senescence markers, senescence-associated ß-galactosidase, p16 and p21. Here, we show that senescence types can be distinguished by reduced levels of 18S, 5.8S and 28S rRNA, in replicative but not induced senescence. We also demonstrate that promoter region of rRNA is hypermethylated in replicative senescence. The findings show that expression level of rRNA or methylation of its promoter can be used to distinguish between senescence types.
Subject(s)
Cellular Senescence , DNA Damage , DNA Methylation , DNA, Ribosomal/biosynthesis , Fibroblasts/metabolism , Gene Expression Regulation , RNA, Ribosomal/biosynthesis , HumansABSTRACT
The transcription of 18S, 5.8S, and 18S rRNA genes (45S rDNA), cotranscriptional processing of pre-rRNA, and assembly of mature rRNA with ribosomal proteins are the linchpins of ribosome biogenesis. In yeast (Saccharomyces cerevisiae) and animal cells, hundreds of pre-rRNA processing factors have been identified and their involvement in ribosome assembly determined. These studies, together with structural analyses, have yielded comprehensive models of the pre-40S and pre-60S ribosome subunits as well as the largest cotranscriptionally assembled preribosome particle: the 90S/small subunit processome. Here, we present the current knowledge of the functional organization of 45S rDNA, pre-rRNA transcription, rRNA processing activities, and ribosome assembly factors in plants, focusing on data from Arabidopsis (Arabidopsis thaliana). Based on yeast and mammalian cell studies, we describe the ribonucleoprotein complexes and RNA-associated activities and discuss how they might specifically affect the production of 40S and 60S subunits. Finally, we review recent findings concerning pre-rRNA processing pathways and a novel mechanism involved in a ribosome stress response in plants.
Subject(s)
DNA, Ribosomal/biosynthesis , Plant Proteins/biosynthesis , RNA Precursors/biosynthesis , Ribosomal Proteins/biosynthesis , Ribosomes/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Cell Nucleolus , DNA, Ribosomal/genetics , Eukaryotic Cells/metabolism , Plant Proteins/genetics , RNA Precursors/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
The heavily transcribed rDNA repeats that give rise to the ribosomal RNA are clustered in a unique chromatin structure, the nucleolus. Due to its highly repetitive nature and transcriptional activity, the nucleolus is considered a hotspot of genomic instability. Breaks in rDNA induce a transient transcriptional shut down to conserve energy and promote rDNA repair; however, how nucleolar chromatin is modified and impacts on rDNA repair is unknown. Here, we uncover that phosphorylation of serine 14 on histone H2B marks transcriptionally inactive nucleolar chromatin in response to DNA damage. We identified that the MST2 kinase localises at the nucleoli and targets phosphorylation of H2BS14p in an ATM-dependent manner. We show that establishment of H2BS14p is necessary for damage-induced rDNA transcriptional shut down and maintenance of genomic integrity. Ablation of MST2 kinase, or upstream activators, results in defective establishment of nucleolar H2BS14p, perturbed DNA damage repair, sensitisation to rDNA damage and increased cell lethality. We highlight the impact of chromatin regulation in the rDNA damage response and targeting of the nucleolus as an emerging cancer therapeutic approach.
Subject(s)
DNA Breaks, Double-Stranded/radiation effects , DNA, Ribosomal/biosynthesis , Histones/metabolism , Nucleolus Organizer Region/genetics , Protein Serine-Threonine Kinases/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Chromatin/genetics , DNA Damage/genetics , DNA Repair/genetics , DNA, Ribosomal/genetics , Humans , Phosphorylation , Serine-Threonine Kinase 3 , Transcription, Genetic/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability.
Subject(s)
DNA Replication , Genomic Instability , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/metabolism , Chromosomes, Fungal/genetics , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , DNA, Ribosomal/biosynthesis , DNA, Ribosomal/genetics , Gene Deletion , Genome, Fungal , Humans , Models, Biological , Repetitive Sequences, Nucleic Acid , Replication Origin , Silent Information Regulator Proteins, Saccharomyces cerevisiae/deficiency , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Sirtuin 2/deficiency , Sirtuin 2/geneticsABSTRACT
Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation.
Subject(s)
Chromosomes, Fungal/enzymology , DNA Helicases/metabolism , DNA, Fungal/biosynthesis , DNA, Ribosomal/biosynthesis , DNA-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , S Phase , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Binding Sites , Chromosomes, Fungal/genetics , DNA Helicases/genetics , DNA Polymerase I/metabolism , DNA, Fungal/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/genetics , Gene Dosage , Intracellular Signaling Peptides and Proteins/genetics , Models, Molecular , Multiprotein Complexes , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity RelationshipABSTRACT
H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.
Subject(s)
DNA, Ribosomal/genetics , Histones/genetics , Spermatogenesis/genetics , Transcription, Genetic , Amino Acid Sequence/genetics , Animals , Cell Nucleolus/genetics , Chromatin/genetics , DNA, Ribosomal/biosynthesis , Gene Expression Regulation, Developmental , Germ Cells/metabolism , Histones/biosynthesis , Male , Mice , Mouse Embryonic Stem Cells/metabolism , Sequence Homology, Amino Acid , Spermatids/growth & development , Spermatids/metabolism , Spermatocytes/growth & development , Spermatocytes/metabolism , Testis/growth & development , Testis/metabolismABSTRACT
The central serotonergic system is implicated differentially in the pathogenesis of depression and schizophrenia. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of forebrain limbic structures disturbed in both disorders. The study was carried out on paraffin-embedded brains from 27 depressed (15 major depressive disorder, MDD and 12 bipolar disorder, BD) and 17 schizophrenia (9 residual and 8 paranoid) patients and 28 matched controls without mental disorders. The transcriptional activity of ribosomal DNA (rDNA) in DRN neurons was evaluated by the AgNOR silver staining method. A significant effect of diagnosis on rDNA activity was found in the cumulative analysis of all DRN subnuclei. Further analysis revealed an increase in this activity in residual (but not paranoid) schizophrenia compared to depressed (both MDD and BD) patients. The effect was most probably neither confounded by suicide nor related to antidepressant and antipsychotic medication. Our findings suggest that increased activity of rDNA in DRN neurons is a distinct phenomenon in residual schizophrenia, related presumably to differentially disturbed inputs to the DRN and/or their local transformation compared with depressive episodes in patients with affective disorders.
Subject(s)
Bipolar Disorder/metabolism , DNA, Ribosomal/biosynthesis , Depressive Disorder, Major/metabolism , Dorsal Raphe Nucleus/metabolism , Schizophrenia/metabolism , Transcription, Genetic/physiology , Adult , Aged , Bipolar Disorder/diagnosis , Bipolar Disorder/epidemiology , Cross-Sectional Studies , Depressive Disorder, Major/diagnosis , Depressive Disorder, Major/epidemiology , Dorsal Raphe Nucleus/pathology , Female , Humans , Male , Middle Aged , Mood Disorders/diagnosis , Mood Disorders/epidemiology , Mood Disorders/metabolism , Neurons/metabolism , Neurons/pathology , Schizophrenia/diagnosisABSTRACT
The nucleolus is an important nuclear structure where transcription of ribosomal DNA (rDNA) takes place. During mitotic division, the nucleolus passes through different processes that inactivate rDNA transcription; in meiosis, its reassembly takes place during telophase II. The objective of this study was to identify the activity patterns and localization of nucleolar organizer regions (NORs) during meiotic division in fish species of the family Curimatidae. For this analysis, the meiotic division in five curimatid species was studied using silver nitrate impregnation, fluorescent in situ hybridization (FISH), and base-specific fluorochrome staining. Silver nitrate staining indicated the presence of a nucleolus in interphase nuclei, one chromosome pair in the spermatogonial metaphases, and one bivalent at the pachytene stage. No Ag-NORs were identified for cells at the diplotene, diakinesis, metaphase I, or metaphase II stages; however, FISH confirmed the presence of Ag-NORs in the nuclei, in spermatogonia, and at the pachytene phase. FISH identified this region during the other stages of meiosis, as did fluorochrome CMA3 staining, which revealed fluorescent marks corresponding to NORs during all stages of meiosis analyzed. The gene activity and localization of this ribosomal sequence during the different stages involved will also be discussed.
Subject(s)
Cell Nucleolus/genetics , Characiformes/genetics , Meiosis/genetics , Nucleolus Organizer Region/genetics , Animals , DNA, Ribosomal/biosynthesis , DNA, Ribosomal/genetics , In Situ Hybridization, Fluorescence , Male , Silver Staining , Spermatogenesis/geneticsABSTRACT
Eco1 is the acetyltransferase that establishes sister-chromatid cohesion during DNA replication. A budding yeast strain with an eco1 mutation that genocopies Roberts syndrome has reduced ribosomal DNA (rDNA) transcription and a transcriptional signature of starvation. We show that deleting FOB1--a gene that encodes a replication fork-blocking protein specific for the rDNA region--rescues rRNA production and partially rescues transcription genome-wide. Further studies show that deletion of FOB1 corrects the genome-wide replication defects, nucleolar structure, and rDNA segregation that occur in the eco1 mutant. Our study highlights that the presence of cohesin at the rDNA locus has a central role in controlling global DNA replication and gene expression.
Subject(s)
Acetyltransferases/genetics , DNA Replication/genetics , DNA, Ribosomal/biosynthesis , DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA, Fungal/genetics , DNA, Ribosomal/genetics , Gene Deletion , Mutation , RNA, Ribosomal/genetics , Ribosomes/metabolism , Transcription, Genetic/genetics , CohesinsABSTRACT
Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.
Subject(s)
DNA Replication , DNA Topoisomerases, Type I/metabolism , DNA, Ribosomal/biosynthesis , DNA Breaks, Double-Stranded , DNA Cleavage , DNA, Ribosomal/metabolism , DNA-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Stability , RecQ Helicases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
It has been shown that SIRT7 regulates rDNA transcription and that reduced SIRT7 levels inhibit tumor growth. This anti-tumor effect could be due to reduced Pol I activity and perturbed ribosome biogenesis. In this study, using pulse labeling with RNA and amino acid analogs, we found that SIRT7 knockdown efficiently suppressed both RNA and protein synthesis. Surprisingly, SIRT7 knockdown preferentially inhibited protein synthesis over rDNA transcription, whereas the levels of both were reduced to similar extents following Pol I knockdown. Using an affinity purification mass spectrometry approach and functional analyses of the resulting SIRT7 interactome, we identified and validated SIRT7 interactions with proteins involved in ribosomal biogenesis. Indeed, SIRT7 co-fractionated with monoribosomes within a sucrose gradient. Using reciprocal isolations, we determined that SIRT7 interacts specifically with mTOR and GTF3C1, a component of the Pol III transcription factor TFIIIC2 complex. Further studies found that SIRT7 knockdown triggered an increase in the levels of LC3B-II, an autophagosome marker, suggesting a link between SIRT7 and the mTOR pathway. Additionally, we provide several lines of evidence that SIRT7 plays a role in modulating Pol III function. Immunoaffinity purification of SIRT7-GFP from a nuclear fraction demonstrated specific SIRT7 interaction with five out of six components of the TFIIIC2 complex, but not with the TFIIIA or TFIIIB complex, the former of which is required for Pol III-dependent transcription of tRNA genes. ChIP assays showed SIRT7 localization to the Pol III targeting genes, and SIRT7 knockdown triggered a reduction in tRNA levels. Taken together, these data suggest that SIRT7 may regulate Pol III transcription through mTOR and the TFIIIC2 complex. We propose that SIRT7 is involved in multiple pathways involved in ribosome biogenesis, and we hypothesize that its down-regulation may contribute to an antitumor effect, partly through the inhibition of protein synthesis.
Subject(s)
DNA, Ribosomal/biosynthesis , Protein Biosynthesis/genetics , Ribosomes/genetics , Sirtuins/genetics , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Gene Knockdown Techniques , Humans , Mass Spectrometry , Neoplasms/genetics , Neoplasms/pathology , RNA/biosynthesis , Ribosomes/metabolism , Sirtuins/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factors, TFIII/metabolismABSTRACT
Cockayne syndrome (CS) is a rare genetic disorder characterized by a variety of growth and developmental defects, photosensitivity, cachectic dwarfism, hearing loss, skeletal abnormalities, progressive neurological degeneration, and premature aging. CS arises due to mutations in the CSA and CSB genes. Both gene products are required for the transcription-coupled (TC) branch of the nucleotide excision repair (NER) pathway, however, the severe phenotype of CS patients is hard to reconcile with a sole defect in TC-NER. Studies using cells from patients and mouse models have shown that the CSB protein is involved in a variety of cellular pathways and plays a major role in the cellular response to stress. CSB has been shown to regulate processes such as the transcriptional recovery after DNA damage, the p53 transcriptional response, the response to hypoxia, the response to insulin-like growth factor-1 (IGF-1), transactivation of nuclear receptors, transcription of housekeeping genes and the transcription of rDNA. Some of these processes are also affected in combined XP/CS patients. These new advances in the function(s) of CSB shed light onto the etiology of the clinical features observed in CS patients and could potentially open therapeutic avenues for these patients in the future. Moreover, the study of CS could further our knowledge of the aging process.
Subject(s)
Cockayne Syndrome/metabolism , DNA Damage , DNA Helicases/metabolism , DNA Repair Enzymes/metabolism , DNA Repair , Transcription, Genetic , Animals , Cell Hypoxia/drug effects , Cockayne Syndrome/genetics , Cockayne Syndrome/pathology , DNA Helicases/genetics , DNA Repair Enzymes/genetics , DNA, Ribosomal/biosynthesis , DNA, Ribosomal/genetics , DNA-Binding Proteins , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Mice , Poly-ADP-Ribose Binding Proteins , Proteins/genetics , Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.
Subject(s)
DNA Polymerase I/biosynthesis , Gene Expression Regulation/genetics , Muscle, Skeletal/pathology , Stress, Mechanical , Weight-Bearing/physiology , Animals , DNA Polymerase I/genetics , DNA, Ribosomal/biosynthesis , Hypertrophy/genetics , Hypertrophy/pathology , Hypertrophy/physiopathology , Mice , Mice, Inbred C57BL , Muscle, Skeletal/physiopathologyABSTRACT
Major eukaryotic genomic elements, including the ribosomal DNA (rDNA), are composed of repeated sequences with well-defined copy numbers that must be maintained by regulated recombination. Although mechanisms that instigate rDNA recombination have been identified, none are directional and they therefore cannot explain precise repeat number control. Here, we show that yeast lacking histone chaperone Asf1 undergo reproducible rDNA repeat expansions. These expansions do not require the replication fork blocking protein Fob1 and are therefore independent of known rDNA expansion mechanisms. We propose the existence of a regulated rDNA repeat gain pathway that becomes constitutively active in asf1Δ mutants. Cells lacking ASF1 accumulate rDNA repeats with high fidelity in a processive manner across multiple cell divisions. The mechanism of repeat gain is dependent on highly repetitive sequence but, surprisingly, is independent of the homologous recombination proteins Rad52, Rad51 and Rad59. The expansion mechanism is compromised by mutations that decrease the processivity of DNA replication, which leads to progressive loss of rDNA repeats. Our data suggest that a novel mode of break-induced replication occurs in repetitive DNA that is dependent on high homology but does not require the canonical homologous recombination machinery.
Subject(s)
DNA Repeat Expansion , DNA, Fungal/chemistry , DNA, Ribosomal/chemistry , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/genetics , DNA Replication , DNA, Fungal/biosynthesis , DNA, Ribosomal/biosynthesis , DNA-Directed DNA Polymerase/genetics , Gene Deletion , Molecular Chaperones/genetics , Mutation , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.
Subject(s)
Chromatin/metabolism , DNA, Ribosomal/metabolism , Ribonuclease III/metabolism , Animals , Cell Cycle/genetics , Chromosomes, Mammalian/metabolism , DNA, Ribosomal/biosynthesis , DNA, Ribosomal/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epigenesis, Genetic/genetics , Gene Silencing , HeLa Cells , Humans , Mice , Promoter Regions, Genetic/genetics , Substrate Specificity , Tandem Repeat Sequences/genetics , Transcription, GeneticABSTRACT
Human MTG16a (CBFA2T3), a chromatin repressor with nucleolar localization, was described to act as a suppressor of breast tumourigenesis. Here we show that MTG16a is a novel ribosomal gene repressor, which can counteract MYC-driven activation of ribosomal RNA (rRNA) transcription. We also show that either knocking down MTG16a by RNA interference, or sequestering MTG16a outside the nucleolus of human breast epithelial cells, hampers acinar morphogenesis concomitant with up-regulation of rRNA synthesis and increased ribogenesis. This is the first demonstration that loss of MTG16a function in the nucleolus of breast epithelial cells can induce morphological and molecular changes typical of breast cancer initiation.
Subject(s)
Breast/embryology , Breast/metabolism , DNA, Ribosomal/metabolism , Morphogenesis , Repressor Proteins/deficiency , Ribosomes/metabolism , Tumor Suppressor Proteins/deficiency , Breast/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Nucleolus/metabolism , DNA, Ribosomal/biosynthesis , Down-Regulation/genetics , Epithelial Cells/metabolism , Female , Fibroblasts/metabolism , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HeLa Cells , Humans , Models, Biological , Nucleolus Organizer Region/genetics , Protein Transport , Proto-Oncogene Proteins c-myc/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Up-Regulation/geneticsABSTRACT
In eukaryotes, the ribosomal DNA (rDNA) consists of long tandem repeat arrays. These repeated genes are unstable because homologous recombination between them results in copy number loss. To maintain high copy numbers, yeast has an amplification system that works through a pathway involving the replication fork barrier site and unequal sister chromatid recombination. In this study, we show that an active replication origin is essential for amplification, and the amplification rate correlates with origin activity. Moreover, origin activity affects the levels of extrachromosomal rDNA circles (ERC) that are thought to promote aging. Surprisingly, we found that reduction in ERC level results in shorter life span. We instead show that life span correlates with rDNA stability, which is preferentially reduced in mother cells, and that episomes can induce rDNA instability. These data support a model in which rDNA instability itself is a cause of aging in yeast.
Subject(s)
Cell Division/genetics , DNA Replication , DNA, Ribosomal/biosynthesis , Gene Amplification , Gene Expression Regulation, Fungal , Genomic Instability , Replication Origin , Saccharomyces cerevisiae/genetics , Aging/genetics , DNA, Circular/metabolism , DNA-Binding Proteins/metabolism , Kinetics , Mutation , Plasmids/metabolism , Recombination, Genetic , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sister Chromatid ExchangeABSTRACT
Bi-directionality is a common feature observed for genomic replication for all three phylogenetic kingdoms: Eubacteria, Archaea, and Eukaryotes. A consequence of bi-directional replication, where the two replication forks initiated at an origin move away from each other, is that the replication termination will occur at positions away from the origin sequence(s). The replication termination processes are therefore physically and mechanistically dissociated from the replication initiation. The replication machinery is a highly processive complex that in short time copies huge numbers of bases while competing for the DNA substrate with histones, transcription factors, and other DNA-binding proteins. Importantly, the replication machinery generally wins out; meanwhile, when converging forks meet termination occurs, thus preventing over-replication and genetic instability. Very different scenarios for the replication termination processes have been described for the three phylogenetic kingdoms. In eubacterial genomes replication termination is site specific, while in archaea and eukaryotes termination is thought to occur randomly within zones where converging replication forks meet. However, a few site-specific replication barrier elements that mediate replication termination have been described in eukaryotes. This review gives an overview about what is known about replication termination, with a focus on these natural site-specific replication termination sites.