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PubMed1999

Maintenance of genomic methylation requires a SWI2/SNF2-like protein.

Altering cytosine methylation by genetic means leads to a variety of developmental defects in mice, plants and fungi. Deregulation of cytosine methylation also has a role in human carcinogenesis. In some cases, these defects have been tied to the inheritance of epigenetic alterations (such as chromatin imprints and DNA methylation patterns) that do not involve changes in DNA sequence. Using a forward genetic screen, we identified a gene (DDM1, decrease in DNA methylation) from the flowering plant Arabidopsis thaliana required to maintain normal cytosine methylation patterns. Additional ddm1 alleles (som4, 5, 6, 7, 8) were isolated in a selection for mutations that relieved transgene silencing (E.J.R., unpublished data). Loss of DDM1 function causes a 70% reduction of genomic cytosine methylation, with most of the immediate hypomethylation occurring in repeated sequences. In contrast, many low-copy sequences initially retain their methylation in ddm1 homozygotes, but lose methylation over time as the mutants are propagated through multiple generations by self-pollination. The progressive effect of ddm1 mutations on low-copy sequence methylation suggests that ddm1 mutations compromise the efficiency of methylation of newly incorporated cytosines after DNA replication. In parallel with the slow decay of methylation during inbreeding, ddm1 mutants accumulate heritable alterations (mutations or stable epialleles) at dispersed sites in the genome that lead to morphological abnormalities. Here we report that DDM1 encodes a SWI2/SNF2-like protein, implicating chromatin remodelling as an important process for maintenance of DNA methylation and genome integrity.

Amino Acid Sequence↗

PubMed1999

Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity.

In higher plants, seed development requires maternal gene activity in the haploid (gametophytic) as well as diploid (sporophytic) tissues of the developing ovule. The Arabidopsis thaliana gene MEDEA (MEA) encodes a SET-domain protein of the Polycomb group that regulates cell proliferation by exerting a gametophytic maternal control during seed development. Seeds derived from female gametocytes (embryo sacs) carrying a mutant mea allele abort and exhibit cell proliferation defects in both the embryo and the endosperm. In this study we show that the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA alleles. Paternally inherited MEA alleles are transcriptionally silent in both the young embryo and endosperm. Mutations at the decrease in DNA methylation1 (ddm1) locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and exhibit some of the abnormalities found in aborting mea seeds. Our results indicate that the maintenance of the genomic imprint at the mea locus requires zygotic DDM1 activity. Because DDM1 encodes a putative chromatin remodeling factor, chromatin structure is likely to be interrelated with genomic imprinting in Arabidopsis.

Alleles↗

PubMed2002

Maintenance of genome stability in Saccharomyces cerevisiae.

Most human cancer cells show signs of genome instability, ranging from elevated mutation rates to gross chromosomal rearrangements and alterations in chromosome number. Little is known about the molecular mechanisms that generate this instability or how it is suppressed in normal cells. Recent studies of the yeast Saccharomyces cerevisiae have begun to uncover the extensive and redundant pathways that keep the rate of genome rearrangements at very low levels. These studies, which we review here, have implicated more than 50 genes in the suppression of genome instability, including genes that function in S-phase checkpoints, recombination pathways, and telomere maintenance. Human homologs of several of these genes have well-established roles as tumor suppressors, consistent with the hypothesis that the mechanisms preserving genome stability in yeast are the same mechanisms that go awry in cancer.

Base Sequence↗

PubMed2006

The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability.

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.

Animals↗

PubMedDate not supplied

Maintaining genetic integrity in aging: a zero sum game.

Aging of somatic cells can be defined as the gradual loss of the information embedded in the global and local properties of complex macromolecular networks. This loss of information may reflect the dynamic interplay between stochastic factors, such as the accumulation of unrepaired somatic damage, and gene-encoded programmatic responses. This would ultimately result in loss of function, impaired response to environmental challenge, and a progressively increased incidence of disease. Here the authors present the case for aging as a continuous battle between maintaining genomic integrity and ensuring sufficient cell functional mass. Focusing on aging of the liver in rodents, evidence is presented that normal aging is associated with a gradual accumulation of random alterations in the DNA of the genome as a consequence of imperfect DNA repair and a decrease in the rate of DNA damage-induced apoptosis. Apoptosis is the cell's genome maintenance mechanism of last resort and an imbalance towards apoptosis can contribute to manifestations of aging-related phenotypes, as exemplified by mouse models of premature aging due to genetic defects in genome maintenance. Prospects to reset the clock in this zero sum game between survival and the maintenance of phenotypic integrity will be discussed.

Aging↗

PubMed2003

Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b.

We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or "maintenance," of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.

Amino Acid Motifs↗

PubMed2006

Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function.

The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.

Cell Cycle Proteins↗

PubMed1997

Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.

Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.

Amino Acid Sequence↗

PubMed2026

Loss of maternal PADI6 disrupts DNA methylation and genomic imprinting maintenance in late preimplantation mouse embryos.

BACKGROUND: The maternal-effect protein PADI6, which is part of the subcortical maternal complex, is involved in proper spindle assembly, organelle distribution, ribosome storage, and cytoplasmic lattice organization in mouse oocytes. In humans, variants of PADI6 are associated with female infertility and multilocus imprinting disturbance in offspring. Recently, it was demonstrated that PADI6 plays a role in the storage and cytoplasmic localization of epigenetic factors, including UHRF1 and DNMT1. Moreover, maternal PADI6 depletion leads to defective epigenetic reprogramming and zygotic genome activation but not to an imprinting defect in two-cell mouse embryos. These findings raise the possibility that imprinting disturbances arise later in development. RESULTS: By employing combined single-blastocyst RNA-seq/BS-seq and immunostaining validation in the embryos derived from Padi6P620A-mutant oocytes, we investigated the role of Padi6 in late preimplantation development. We demonstrated that embryos that overcame the two-cell stage block had a dramatic reduction in UHRF1 and DNMT1 protein levels, a decrease in H3K9me3, and whole-genome hypomethylation, including most imprinted loci and repetitive elements, at the blastocyst stage. Furthermore, these maternal mutant embryos showed deregulation of inner cell mass markers and defective blastocyst implantation, but no effect on trophoblast differentiation. CONCLUSION: Our results demonstrate that maternal PADI6 is a key regulator of the stability of epigenetic factors required to maintain repressive marks in late preimplantation mouse embryos. Its deficiency results in genomic imprinting defects that closely resemble those found in human patients and provide a mechanistic explanation for MLID caused by maternal PADI6 variants. Furthermore, the impairment of blastocyst implantation capacity, likely due to dysregulation of inner cell mass differentiation, provides new mechanistic insights into the control of female fertility and embryo development exerted by PADI6.

DNA Methylation↗

PubMed2006

Nucleotide excision repair disorders and the balance between cancer and aging.

Cancer incidence increases with age and is driven by accumulation of mutations in the DNA. In many so-called premature aging disorders, cancer appears earlier and at elevated rates. These diseases are predominantly caused by genome instability and present with symptoms, including cancer, resembling "segments" of aging and are thus often referred to as "segmental progerias". Two related segmental progerias, Cockayne syndrome (CS) and trichothiodystrophy (TTD), don't fit this pattern. Although caused by defects in genome maintenance via the nucleotide excision DNA repair (NER) pathway and displaying severe progeroid symptoms, CS and TTD patients appear to lack any cancer predisposition. More strikingly, genetic defects in the same NER pathway, and in some cases even within the same gene, XPD, can also give rise to disorders with greatly elevated cancer rates but without progeria (xeroderma pigmentosum). In this review, we will discuss the connection between genome maintenance, aging and cancer in light of a new mouse model of XPD disease.

Aging↗

PubMed2003

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae.

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.

DNA-Binding Proteins↗

PubMed2006

Proteomic analysis of the Kaposi's sarcoma-associated herpesvirus terminal repeat element binding proteins.

Terminal repeat (TR) elements of Kaposi's sarcoma-associated herpesvirus (KSHV), the potential origin sites of KSHV replication, have been demonstrated to play important roles in viral replication and transcription and are most likely also critical for the segregation of the KSHV genome to daughter cells. To search for the cellular proteins potentially involved in KSHV genome maintenance, we performed affinity chromatography analysis, using KSHV TR DNA as the affinity ligand. Proteomic analysis was then carried out to identify the TR-interacting proteins. We identified a total of 123 proteins from both KSHV-positive and -negative cells, among which most were identified exclusively from KSHV-positive cells. These proteins were categorized as proliferation/cell cycle regulatory proteins, proteins involved in spliceosome components, such as heterogeneous nuclear ribonuclear proteins, the DEAD/H family, the switch/sucrose nonfermenting protein family, splicing factors, RNA binding proteins, transcription regulation proteins, replication factors, modifying enzymes, and a number of proteins that could not be broadly categorized. To support the proteomic results, the presence of four candidate proteins, ATR, BRG1, NPM1 and PARP-1, in the elutions was further characterized in this study. The binding and colocalization of these proteins with the TR were verified using chromatin immunoprecipitation and immunofluorescence in situ hybridization analysis. These newly identified TR binding proteins provide a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. This study will facilitate the generation and testing of new hypotheses to further our understanding of the mechanisms involved in KSHV persistence and its associated pathogenesis.

Cell Nucleus↗

PubMed2006

Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options.

BACKGROUND & AIMS: How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. METHODS: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. RESULTS: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. CONCLUSIONS: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Adenocarcinoma↗

PubMed1993

Maintenance of genomic imprinting at the IGF2 locus in hepatoblastoma.

Genomic imprinting is the parental allele specific expression of genes and has recently been shown to occur in humans. Evidence for a role for genomic imprinting in human cancer comes from the finding of preferential retention of paternal alleles in embryonal tumors undergoing loss of heterozygosity, e.g., Wilms' tumor and osteogenic sarcoma. Recent studies have demonstrated imprinting of the insulin-like growth factor II gene at 11p15 in normal individuals, with the paternally inherited allele expressed and the maternal allele silent. It has been shown that normal imprinting is relaxed, and gene expression is biallelic in a majority of Wilms' tumors which retain heterozygosity at this locus. In this study an intragenic ApaI polymorphism is used to examine imprinting of the insulin-like growth factor II gene in hepatoblastoma. Three of 5 tumors studied were heterozygous and hence informative. All cases showed monoallelic expression of the insulin-like growth factor II gene, indicating maintenance of normal imprinting at this locus.

Alleles↗

PubMed2026

Protein persulfidation emerges as a conserved component of the redox response to DNA damage.

Genotoxic stress is frequently accompanied by alterations in cellular redox homeostasis; however, the mechanisms linking redox regulation to the DNA damage response (DDR) remain incompletely understood. Here, we investigated the early redox response to DNA damage induced by methyl methanesulfonate (MMS) in Saccharomyces cerevisiae, focusing on cysteine oxidative post-translational modifications (PTM). We show that activation of the DNA damage response is accompanied by rapid redox changes that occur in the absence of a generalized oxidative stress response. MMS exposure promotes selective remodeling of cysteine oxidative modifications, characterized by decreased free thiols, robust induction of protein persulfidation, and comparatively modest changes in sulfenylation. These alterations are accompanied by increased intracellular hydrogen sulfide levels, supporting the involvement of reactive sulfur species in the cellular response to DNA damage. Proteome-wide analyses revealed that cysteine oxidative modifications preferentially target proteins involved in central metabolism, nucleotide biosynthesis, and genome maintenance. Consistent with these observations, MMS-induced genotoxic stress promotes metabolic adaptation characterized by increased mitochondrial respiration, elevated ATP production, and mitochondrial morphological remodeling, linking bioenergetic adaptation to redox regulation. Importantly, perturbation of intracellular redox balance using N-acetylcysteine compromises survival under DNA-damaging conditions, supporting a functional role for redox signaling during the DDR. Finally, MMS treatment also induces protein persulfidation in mammalian cells. Moreover, exposure to etoposide, a mechanistically distinct genotoxic agent that induces DNA double-strand breaks through topoisomerase II inhibition, showed a similar trend, suggesting that protein persulfidation may not be restricted to alkylation-induced DNA damage. Together our findings identify protein persulfidation as a prominent component of the redox response to DNA damage and provide new insight into the functional interplay between mitochondrial metabolism, cysteine-based redox regulation, and genome maintenance.

Oxidation-Reduction↗

PubMed2003

Src in human carcinogenesis.

The signaling machinery in cells is a complex, multi-factorial network of cross-talking proteins that enables dynamic communication between upstream causal factors and downstream effectors. Non-receptor tyrosine kinases, including Src, are the intermediates of information transfer, controlling pathways as diverse as cell growth, migration, death, and genome maintenance. When expressed as viral genes these proteins are potent carcinogens, yet analogous genetic alterations are rarely observed in human tumors. In seeking to characterize the role of the non-receptor tyrosine kinase Src in neoplasia, arguments can be made that the consequences of mutation, or perturbations in the activity or expression of this protein is a determinative factor in clinical prognosis and pathogenicity. In a variety of tumor types including those derived from the colon and breast, the Src non-receptor tyrosine kinase is either overexpressed or constitutively active in a large percentage of the tumors. Increased expression or activity of Src correlates with the stage and metastatic potential of some neoplasia.

Animals↗

PubMed2005

Yeast Nhp6A/B and mammalian Hmgb1 facilitate the maintenance of genome stability.

Saccharomyces cerevisiae Nhp6A and Nhp6B are chromatin architectural factors that belong to the high-mobility group box (HMGB) superfamily and appear to be functionally related to mammalian Hmgb1. They bind to the minor groove of double-stranded DNA in a non-sequence-specific manner and thereby influence chromatin structure. Previous work has implicated these proteins in a variety of nuclear processes, including chromatin remodeling, DNA replication, transcription, and recombination . Here, we show that Nhp6A/B loss leads to increased genomic instability, hypersensitivity to DNA-damaging agents, and shortened yeast cell life span that is associated with elevated levels of extrachromosomal rDNA circles. Furthermore, we show that hypersensitivity toward UV light does not appear to reflect a decreased capacity for DNA repair but instead correlates with higher levels of UV-induced thymine dimer adducts being formed in cells lacking Nhp6A/B. Likewise, we show that mouse fibroblasts lacking Hmgb1 display higher rates of damage after UV irradiation than wild-type controls and also exhibit pronounced chromosomal instability. Taken together, these data indicate that Nhp6A/B and Hmgb1 protect DNA from damaging agents and thus guard against the generation of genomic aberrations.

Animals↗

PubMed2003

Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability.

The postreplication repair pathway (PRR) is composed of error-free and error-prone sub-pathways that allow bypass of DNA damage-induced replication-blocking lesions. The error-free sub-pathway is also used for bypass of spontaneous DNA damage and functions in cooperation with recombination pathways. In diploid yeast cells, error-free PRR is needed to prevent genomic instability, which is manifest as loss of heterozygosity (LOH) events of increased chromosome loss and recombination. Homologous recombination acts synergistically with the error-free damage avoidance branch of PRR to prevent chromosome loss. The DNA damage checkpoint gene MEC1 acts synergistically with the PRR pathway in maintaining genomic stability. Integration of the PRR pathway with other cellular pathways for preventing genomic instability is discussed. In diploid strains, the most dramatic increase is in the abnormality of chromosome loss when a repair or damage detection pathway is defective.

Crossing Over, Genetic↗