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1.  Plant Nutriomics in China: An Overview 
Annals of Botany  2006;98(3):473-482.
• Background Population and environmental pressure have imposed a great challenge on agriculture in China to explore innovative and effective solutions to its pressing plant nutritional problems. Plant nutriomics is a new frontier in plant biology that can provide innovative solutions for improving plant nutrient efficiency, thus increasing crop productivity through genetic and molecular approaches.
• Scope This review summarizes current efforts and progress in plant nutriomic research in China with examples from several case studies. It also points out potential obstacles and depicts future perspectives in this emerging frontier of plant nutrition.
• Conclusions Although plant nutriomics is still at a conceptual stage, substantial efforts are being made in China aimed at increasing plant nutrient efficiency through a nationwide, co-ordinated project on plant nutriomics. Future studies involving both national and international collaborations are needed to develop nutrient-efficient, stress-tolerant and high-quality crop varieties for both China and elsewhere.
PMCID: PMC2803557  PMID: 16735410
China; genomics; metabolomics; nutrient efficiency; plant nutriomics; proteomics; transcriptomics
2.  Effect of Supplementation with Zinc and Other Micronutrients on Malaria in Tanzanian Children: A Randomised Trial 
PLoS Medicine  2011;8(11):e1001125.
Hans Verhoef and colleagues report findings from a randomized trial conducted among Tanzanian children at high risk for malaria. Children in the trial received either daily oral supplementation with either zinc alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. The investigators did not find evidence from this study that zinc or multi-nutrients protected against malaria episodes.
It is uncertain to what extent oral supplementation with zinc can reduce episodes of malaria in endemic areas. Protection may depend on other nutrients. We measured the effect of supplementation with zinc and other nutrients on malaria rates.
Methods and Findings
In a 2×2 factorial trial, 612 rural Tanzanian children aged 6–60 months in an area with intense malaria transmission and with height-for-age z-score≤−1.5 SD were randomized to receive daily oral supplementation with either zinc alone (10 mg), multi-nutrients without zinc, multi-nutrients with zinc, or placebo. Intervention group was indicated by colour code, but neither participants, researchers, nor field staff knew who received what intervention. Those with Plasmodium infection at baseline were treated with artemether-lumefantrine. The primary outcome, an episode of malaria, was assessed among children reported sick at a primary care clinic, and pre-defined as current Plasmodium infection with an inflammatory response, shown by axillary temperature ≥37.5°C or whole blood C-reactive protein concentration ≥8 mg/L. Nutritional indicators were assessed at baseline and at 251 days (median; 95% reference range: 191–296 days). In the primary intention-to-treat analysis, we adjusted for pre-specified baseline factors, using Cox regression models that accounted for multiple episodes per child. 592 children completed the study. The primary analysis included 1,572 malaria episodes during 526 child-years of observation (median follow-up: 331 days). Malaria incidence in groups receiving zinc, multi-nutrients without zinc, multi-nutrients with zinc and placebo was 2.89/child-year, 2.95/child-year, 3.26/child-year, and 2.87/child-year, respectively. There was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Neither zinc nor multi-nutrients influenced malaria rates (marginal analysis; adjusted HR, 95% CI: 1.04, 0.93–1.18 and 1.10, 0.97–1.24 respectively). The prevalence of zinc deficiency (plasma zinc concentration <9.9 µmol/L) was high at baseline (67% overall; 60% in those without inflammation) and strongly reduced by zinc supplementation.
We found no evidence from this trial that zinc supplementation protected against malaria.
Trial Registration NCT00623857
Please see later in the article for the Editors' Summary.
Editors' Summary
Malaria is a serious global public-health problem. Half of the world's population is at risk of this parasitic disease, which kills a million people (mainly children living in sub-Saharan Africa) every year. Malaria is transmitted to people through the bites of infected night-flying mosquitoes. Soon after entering the human body, the parasite begins to replicate in red blood cells, bursting out every 2–3 days and infecting more red blood cells. The presence of the parasite in the blood stream (parasitemia) causes malaria's characteristic recurring fever and can cause life-threatening organ damage and anemia (insufficient quantity of red blood cells). Malaria transmission can be reduced by using insecticide sprays to control the mosquitoes that spread the parasite and by avoiding mosquito bites by sleeping under insecticide-treated bed nets. Effective treatment with antimalarial drugs can also reduce malaria transmission.
Why Was This Study Done?
One reason why malaria kills so many children in Africa is poverty. Many children in Africa are malnourished, and malnutrition—in particular, insufficient micronutrients in the diet—impairs the immune system, which increases the frequency and severity of many childhood diseases. Micronutrients are vitamins and minerals that everyone needs in small quantities for good health. Zinc is one of the micronutrients that helps to maintain a healthy immune system, but zinc deficiency is very common among African children. Zinc supplementation has been shown to reduce the burden of diarrhea in developing countries, so might it also reduce the burden of malaria? Unfortunately, the existing evidence is confusing—some trials show that zinc supplementation protects against malaria but others show no evidence of protection. One possibility for these conflicting results could be that zinc supplementation alone is not sufficient—supplementation with other micronutrients might be needed for zinc to have an effect. In this randomized trial (a study that compares the effects of different interventions in groups that initially are similar in all characteristics except for intervention), the researchers investigate the effect of supplementation with zinc alone and in combination with other micronutrients on the rate of uncomplicated (mild) malaria among children living in Tanzania.
What Did the Researchers Do and Find?
The researchers enrolled 612 children aged 6–60 months who were living in a rural area of Tanzania with intense malaria transmission and randomly assigned them to receive daily oral supplements containing zinc alone, multi-nutrients (including iron) without zinc, multi-nutrients with zinc, or a placebo (no micronutrients). Nutritional indicators (including zinc concentrations in blood plasma) were assessed at baseline and 6–10 months after starting the intervention. During the study period, there were 1,572 malaria episodes. The incidence of malaria in all four intervention groups was very similar (about three episodes per child-year), and there was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Moreover, none of the supplements had any effect on malaria rates when compared to the placebo, even though the occurrence of zinc deficiency was strongly reduced by zinc supplementation. In a secondary analysis in which they analyzed their data by iron status at baseline, the researchers found that multi-nutrient supplementation increased the overall number of malaria episodes in children with iron deficiency by 41%, whereas multi-nutrient supplementation had no effect on the number of malaria episodes among children who were iron-replete at baseline.
What Do These Findings Mean?
In this study, the researchers found no evidence that zinc supplementation protected against malaria among young children living in Tanzania when given alone or in combination with other multi-nutrients. However, the researchers did find some evidence that multi-nutrient supplementation may increase the risk of malaria in children with iron deficiency. Because this finding came out of a secondary analysis of the data, it needs to be confirmed in a trial specifically designed to assess the effect of multi-nutrient supplements on malaria risk in iron-deficient children. Nevertheless, it is a potentially worrying result because, on the basis of evidence from a single study, the World Health Organization currently recommends that regular iron supplements be given to iron-deficient children in settings where there is adequate access to anti-malarial treatment. This recommendation should be reconsidered, suggest the researchers, and the safety of multi-nutrient mixes that contain iron and that are dispensed in countries affected by malaria should also be carefully evaluated.
Additional Information
Please access these Web sites via the online version of this summary at
Information is available from the World Health Organization on malaria (in several languages), on micronutrients, and on zinc deficiency; the 2010 World Malaria Report provides details of the current global malaria situation
The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish), including a selection of personal stories about malaria
Information is available from the Roll Back Malaria Partnership on the global control of malaria and on malaria in Africa
The Malaria Centre at the UK London School of Hygiene & Tropical Medicine develops tools, techniques, and knowledge about malaria, and has a strong emphasis on teaching, training, and translating research outcomes into practice
The Micronutrient Initiative, the Global Alliance for Improved Nutrition, and the Flour Fortification Initiative are not-for-profit organizations dedicated to ensuring that people in developing countries get the minerals and vitamins they need to survive and thrive
The International Zinc Nutrition Consultative Group (iZiNCG) is a non-profit organization that aims to promote and assist efforts to reduce zinc deficiency worldwide, through advocacy efforts, education, and technical assistance
MedlinePlus provides links to additional information on malaria (in English and Spanish)
PMCID: PMC3222646  PMID: 22131908
3.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
PMCID: PMC1716188  PMID: 17194187
4.  Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents 
PLoS Genetics  2005;1(2):e24.
The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.
Cells have evolved sophisticated ways to respond to DNA damage. This is critical because unrepaired damage can kill cells or cause them to become cancerous. The response to DNA damage has been studied for more than 50 years, and has been found to be extremely complex. The traditional way of understanding this complexity is to divide the process into its component parts with the goal of eventually reconstituting the entire process. In this study, the authors extend classical approaches using genomics—an approach that involves studying all genes in an organism simultaneously. The authors tested 12 distinct compounds (many used in cancer chemotherapy) that damage DNA and uncovered new genes involved in DNA repair. They then grouped the compounds to define how they attack cells. Using this approach, the study found that many similar DNA-damaging agents act in comparable ways to damage DNA, but surprisingly, similar compounds can also act on cells by very different mechanisms. Specifically grouping the findings together and verifying the significant results lends a high degree of confidence in the data. The development of such a reproducible experimental design is important for inspiring future experiments.
PMCID: PMC1189734  PMID: 16121259
5.  Photobiological Implications of Folate Depletion and Repletion in Cultured Human Keratinocytes 
Folate nutrition is critical in humans and a high dietary folate intake is associated with a diminished risk of many types of cancer. Both synthetic folic acid and the most biologically abundant extracellular reduced folate, 5-methyltetrahydrofolate, are degraded under conditions of ultraviolet radiation (UVR) exposure. Skin is a proliferative tissue with increased folate nutrient demands due to a dependence upon continuous epidermal cell proliferation and differentiation to maintain homeostasis. Regions of skin are also chronically exposed to UVR, which penetrates to the actively dividing basal layer of the epidermis, increasing the folate nutrient demands in order to replace folate species degraded by UVR exposure and to supply the folate cofactors required for repair of photo-damaged DNA. Localized folate deficiencies of skin are a likely consequence of UVR exposure. We report here a cultured keratinocyte model of folate deficiency that has been applied to examine possible effects of folate nutritional deficiencies in skin. Utilizing this model, we were able to quantify the concentrations of key intracellular folate species during folate depletion and repletion. We investigated the hypotheses that the genomic instability observed under conditions of folate deficiency in other cell types extends to skin, adversely effecting cellular capacity to handle UVR insult and that optimizing folate levels in skin is beneficial in preventing or repairing the pro-carcinogenic effects of UVR exposure. Folate restriction leads to rapid depletion of intracellular reduced folates resulting in S-phase growth arrest, increased levels of inherent DNA damage, and increased uracil misincorporation into DNA, without a significant losses in overall cellular viability. Folate depleted keratinocytes were sensitized toward UVR induced apoptosis and displayed a diminished capacity to remove DNA breaks resulting from both photo and oxidative DNA damage. Thus, folate deficiency creates a permissive environment for genomic instability, an early event in the process of skin carcinogenesis. The effects of folate restriction, even in severely depleted, growth-arrested keratinocytes, were reversible by repletion with folic acid. Overall, these results indicate that skin health can be positively influenced by optimal folate nutriture.
PMCID: PMC2862485  PMID: 20211567
Folate; Keratinocytes; Skin Biology; Solar Simulated Light; DNA Damage; Cancer
6.  Association of chromosome damage detected as micronuclei with hematological diseases and micronutrient status 
Mutagenesis  2011;26(1):57-62.
Epidemiological studies reveal strong association between micronutrient deficiencies and development of cancer. Since chromosome breaks and abnormal chromosome segregation, identified as micronuclei (MN), are central to malignant transformation, the influence of micronutrient status upon MN frequency has been the subject of intense research. Motivating this effort is the idea that marginal micronutrient deficiencies lead to allocation of scarce cellular resources towards immediate survival at the expense of maintaining genomic integrity, placing the individual at greater risk for degenerative diseases and cancer in old age. The challenge in identifying an association between individual micronutrients and MN frequency stems from the complexity of human diet, simultaneous presence of multiple micronutrient deficiencies, variable genetic susceptibility and methodological difficulties. A unique model for studying MN in humans is provided by a group of haematological diseases, the chronic haemolytic anaemias associated with high reticulocyte count and absence of splenic function. These disorders may prove valuable for assessing the influence of micronutrient status once the effect of abnormal erythropoiesis on MN formation is adequately understood. Eventually, large population-based studies that can account for the baseline variability in MN frequency, lifestyle and genetic factors may be needed to uncover the DNA-damaging effect of poor diet. Understanding the link between micronutrient status and MN frequency will contribute towards determining optimal micronutrient intake to preserve long-term health.
PMCID: PMC3107612  PMID: 21164183
7.  Increased RPA1 Gene Dosage Affects Genomic Stability Potentially Contributing to 17p13.3 Duplication Syndrome 
PLoS Genetics  2011;7(8):e1002247.
A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression.
Author Summary
The widespread use of genomic array technology has lead to the identification of a plethora of novel human genomic disorders. These complex conditions occur as a consequence of structural genomic alterations (deletions, amplifications, complex rearrangements). Understanding the specific consequences of such alterations on gene expression and unanticipated impacts on biochemical pathways represents an important challenge to help untangle the clinical basis of these conditions and ultimately aid in their management. Here, we demonstrate that individuals with specific duplications of 17p13.3 incorporating RPA1 exhibit modest over-expression of RPA1. Unexpectedly, this is associated with elevated levels of genomic instability and sensitivity to DNA damage. RPA1 is a component of the Replication Protein A heterotrimer, a complex that plays fundamental roles in DNA replication, repair, and recombination. Reduced RPA1 levels are associated with impaired DNA damage checkpoint activation, but the cellular impacts of over-expression of this subunit have not previously been described in the context of a genomic disorder. Using model cell and reporter systems, we show that modestly elevated levels of RPA1 can adversely impact on DNA double-strand break–induced homologous recombination resulting in elevated levels of chromosome fusions. This data highlights an unanticipated consequence of copy number variation on genomic stability.
PMCID: PMC3161930  PMID: 21901111
8.  Folate in Skin Cancer Prevention 
Sub-cellular biochemistry  2012;56:181-197.
Skin, the largest, most exposed organ of the body, provides a protective interface between humans and the environment. One of its primary roles is protection against exposure to sunlight, a major source of skin damage where the UV radiation (UVR) component functions as a complete carcinogen. Melanin pigmentation and the evolution of dark skin is an adaptive protective mechanism against high levels of UVR exposure. Recently, the hypothesis that skin pigmentation balances folate preservation and Vitamin D production has emerged. Both micronutrients are essential for reproductive success. Photodegradation of bioactive folates suggests a mechanism for the increased tendency of populations of low melanin pigmentation residing in areas of high UV exposure to develop skin cancers. Folate is proposed as a cancer prevention target for its role in providing precursors for DNA repair and replication, as well as its ability to promote genomic integrity through the generation of methyl groups needed for control of gene expression. The cancer prevention potential of folate has been demonstrated by large-scale epidemiological and nutritional studies indicating that decreased folate status increases the risk of developing certain cancers. While folate deficiency has been extensively documented by analysis of human plasma, folate status within skin has not been widely investigated. Nevertheless, inefficient delivery of micronutrients to skin and photolysis of folate argue that documented folate deficiencies will be present if not exacerbated in skin. Our studies indicate a critical role for folate in skin and the potential to protect sun exposed skin by effective topical delivery as a strategy for cancer prevention.
PMCID: PMC3795437  PMID: 22116700
Cancer prevention; DNA repair; Folate; Folic acid; Skin; Topical delivery strategy; UV light
9.  Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability 
PLoS Biology  2010;8(1):e1000275.
Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer.
Early detection of cancer-associated genomic instability is crucial, particularly in tumour types in which this instability represents the essential underlying mechanism of tumourigenesis. Currently used methods require the presence of already established neoplastic cells because they only detect clonal mutations. In principle, parallel sequencing of single DNA filaments could reveal the early phases of tumour initiation by detecting low-frequency mutations, provided an adequate depth of coverage and an effective control of the experimental error. We applied ultradeep sequencing to estimate the genomic instability of individuals with hereditary non-polyposis colorectal cancer (HNPCC). To overcome the experimental error, we used an ultraconserved region (UCR) of the human genome as an internal control. By comparing the mutability outside and inside the UCR, we observed a tendency of the ultraconserved element to accumulate significantly fewer mutations than the flanking segments in both neoplastic and nonneoplastic HNPCC samples. No difference between the two regions was detectable in cells from healthy donors, indicating that all three HNPCC samples have mutation rates higher than the healthy genome. This is the first, to our knowledge, direct evidence of an intrinsic genomic instability of individuals with heterozygous mutations in mismatch repair genes, and constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability.
Author Summary
In hereditary non-polyposis colorectal cancer (HNPCC), a germline mutation in one allele of a gene responsible for repairing DNA damage predisposes the host to cancer, because subsequent somatic inactivation of the one wild-type allele leads to genomic instability that favours tumourigenesis. Nonneoplastic tissues of HNPCC individuals are believed to repair DNA normally, as they are heterozygous and thus are thought to be genomically stable. However, methods used to date are known to be incapable of detecting very low levels of genome instability. Here, we present a more sensitive procedure based on the resequencing of a HNPCC genomic region using next-generation sequencing technology. With this approach, we show that genomic instability is in fact detectable in nonneoplastic tissues of HNPCC patients compared with healthy donors. This constitutional instability may predispose them to acquiring the second somatic mutation event needed for cancer development.
PMCID: PMC2794366  PMID: 20052272
10.  DNA damage tolerance: a double-edged sword guarding the genome 
Translational cancer research  2013;2(3):107-129.
Preservation of genome integrity is an essential process for cell homeostasis. During the course of life of a single cell, the genome is constantly damaged by endogenous and exogenous agents. To ensure genome stability, cells use a global signaling network, namely the DNA damage response (DDR) to sense and repair DNA damage. DDR senses different types of DNA damage and coordinates a response that includes activation of transcription, cell cycle control, DNA repair pathways, apoptosis, senescence, and cell death. Despite several repair mechanisms that repair different types of DNA lesions, it is likely that the replication machinery would still encounter lesions that are mis-repaired or not repaired. Replication of damaged genome would result in high frequency of fork collapse and genome instability. In this scenario, the cells employ the DNA damage tolerance (DDT) pathway that recruits a specialized low fidelity translesion synthesis (TLS) polymerase to bypass the lesions for repair at a later time point. Thus, DDT is not a repair pathway per se, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and preventing genome instability. Paradoxically, DDT process is also associated with increased mutagenesis, which can in turn drive the cell to cancer development. Thus, DDT process functions as a double-edged sword guarding the genome. In this review, we will discuss the replication stress induced DNA damage-signaling cascade, the stabilization and rescue of stalled replication forks by the DDT pathway and the effect of the DDT pathway on cancer.
PMCID: PMC3779140  PMID: 24058901
DNA damage tolerance (DDT); proliferating cell nuclear antigen (PCNA); replicative DNA polymerase; stalled replication forks; translesion synthesis (TLS); translesion polymerase
11.  CDC5 Inhibits the Hyperphosphorylation of the Checkpoint Kinase Rad53, Leading to Checkpoint Adaptation 
PLoS Biology  2010;8(1):e1000286.
The mechanistic role of the yeast kinase CDC5, in allowing cells to adapt to the presence of irreparable DNA damage and continue to divide, is revealed.
The Saccharomyces cerevisiae polo-like kinase Cdc5 promotes adaptation to the DNA damage checkpoint, in addition to its numerous roles in mitotic progression. The process of adaptation occurs when cells are presented with persistent or irreparable DNA damage and escape the cell-cycle arrest imposed by the DNA damage checkpoint. However, the precise mechanism of adaptation remains unknown. We report here that CDC5 is dose-dependent for adaptation and that its overexpression promotes faster adaptation, indicating that high levels of Cdc5 modulate the ability of the checkpoint to inhibit the downstream cell-cycle machinery. To pinpoint the step in the checkpoint pathway at which Cdc5 acts, we overexpressed CDC5 from the GAL1 promoter in damaged cells and examined key steps in checkpoint activation individually. Cdc5 overproduction appeared to have little effect on the early steps leading to Rad53 activation. The checkpoint sensors, Ddc1 (a member of the 9-1-1 complex) and Ddc2 (a member of the Ddc2/Mec1 complex), properly localized to damage sites. Mec1 appeared to be active, since the Rad9 adaptor retained its Mec1 phosphorylation. Moreover, the damage-induced interaction between phosphorylated Rad9 and Rad53 remained intact. In contrast, Rad53 hyperphosphorylation was significantly reduced, consistent with the observation that cell-cycle arrest is lost during adaptation. Thus, we conclude Cdc5 acts to attenuate the DNA damage checkpoint through loss of Rad53 hyperphosphorylation to allow cells to adapt to DNA damage. Polo-like kinase homologs have been shown to inhibit the ability of Claspin to facilitate the activation of downstream checkpoint kinases, suggesting that this function is conserved in vertebrates.
Author Summary
Cellular surveillance mechanisms, termed checkpoints, have evolved to recognize the presence of DNA damage, halt cell division, and promote repair. The purpose of these checkpoints is to prevent the next generation of cells from inheriting a damaged genome. However, after futile attempts at repair over several hours of growth arrest, yeast cells eventually adapt and continue with cell division despite the presence of persistent DNA lesions. This process of adaptation employs CDC5, a kinase that also has essential roles in promoting cell division in the absence of DNA damage. We found that increasing levels of Cdc5 promote adaptation by suppressing the hyperphosphorylation of the checkpoint kinase Rad53, which in turn suppresses the DNA damage checkpoint and relieves cell division arrest. Intriguingly, overexpression of PLK1, the human homolog of CDC5, has been reported in various tumor types and has been linked to poor prognosis. Therefore, understanding the mechanism of adaptation in yeast may provide valuable insight into the role of PLK1 overexpression in tumor progression. Two related papers, published in PLoS Biology (van Vugt et al., doi:10.1371/journal.pbio.1000287) and PLoS Genetics (Donnianni et al., doi:10.1371/journal.pgen.1000763), similarly investigate the phenomenon of checkpoint adaptation.
PMCID: PMC2811153  PMID: 20126259
12.  Cohesin Is Limiting for the Suppression of DNA Damage–Induced Recombination between Homologous Chromosomes 
PLoS Genetics  2010;6(7):e1001006.
Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G2/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G2/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced recombinants in G2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.
Author Summary
The cellular concentrations of individual proteins are expected to be kept within an optimal range, but protein expression is often stochastic. Some proteins are known to be in limiting amounts, so that even modest reduction can lead to malfunction. Within the network of genes that determine genome stability, proteins that are limiting impose a risk for the cell, because fluctuation in their amounts may start a cascade of genomic alternations that will influence many biochemical pathways either under normal growth conditions or in response to chromosome damage. We sought to identify genes that are limiting for DSB repair by lowering the dosage of key genes from 4 to 1 in tetraploid Saccharomyces cerevisiae strains. We found that the complex that holds sister chromatid cohesion together (cohesin) is limiting in DSB repair. In addition, when it is reduced modestly, recombination between homologous chromosomes is highly increased, suggesting that the risk for loss of hetrozygosity (LOH) is increased too. These results should also be considered in light of increasing evidence that copy number variation can impact cellular function.
PMCID: PMC2895640  PMID: 20617204
13.  Epigenetic reduction of DNA repair in progression to gastrointestinal cancer 
Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.
PMCID: PMC4434036  PMID: 25987950
Epigenetic; DNA damage; DNA repair; DNA repair deficiency disorders; Epimutation; Genomic instability; Germ-line mutation; MicroRNAs; Precancerous conditions; Gastrointestinal cancer
14.  Prevention of Mutation, Cancer, and Other Age-Associated Diseases by Optimizing Micronutrient Intake 
Journal of Nucleic Acids  2010;2010:725071.
I review three of our research efforts which suggest that optimizing micronutrient intake will in turn optimize metabolism, resulting in decreased DNA damage and less cancer as well as other degenerative diseases of aging. (1) Research on delay of the mitochondrial decay of aging, including release of mutagenic oxidants, by supplementing rats with lipoic acid and acetyl carnitine. (2) The triage theory, which posits that modest micronutrient deficiencies (common in much of the population) accelerate molecular aging, including DNA damage, mitochondrial decay, and supportive evidence for the theory, including an in-depth analysis of vitamin K that suggests the importance of achieving optimal micronutrient intake for longevity. (3) The finding that decreased enzyme binding constants (increased Km) for coenzymes (or substrates) can result from protein deformation and loss of function due to an age-related decline in membrane fluidity, or to polymorphisms or mutation. The loss of enzyme function can be compensated by a high dietary intake of any of the B vitamins, which increases the level of the vitamin-derived coenzyme. This dietary remediation illustrates the importance of understanding the effects of age and polymorphisms on optimal micronutrient requirements. Optimizing micronutrient intake could have a major effect on the prevention of cancer and other degenerative diseases of aging.
PMCID: PMC2945683  PMID: 20936173
15.  Cleavage Factor I Links Transcription Termination to DNA Damage Response and Genome Integrity Maintenance in Saccharomyces cerevisiae 
PLoS Genetics  2014;10(3):e1004203.
During transcription, the nascent pre-mRNA undergoes a series of processing steps before being exported to the cytoplasm. The 3′-end processing machinery involves different proteins, this function being crucial to cell growth and viability in eukaryotes. Here, we found that the rna14-1, rna15-1, and hrp1-5 alleles of the cleavage factor I (CFI) cause sensitivity to UV-light in the absence of global genome repair in Saccharomyces cerevisiae. Unexpectedly, CFI mutants were proficient in UV-lesion repair in a transcribed gene. DNA damage checkpoint activation and RNA polymerase II (RNAPII) degradation in response to UV were delayed in CFI-deficient cells, indicating that CFI participates in the DNA damage response (DDR). This is further sustained by the synthetic growth defects observed between rna14-1 and mutants of different repair pathways. Additionally, we found that rna14-1 suffers severe replication progression defects and that a functional G1/S checkpoint becomes essential in avoiding genetic instability in those cells. Thus, CFI function is required to maintain genome integrity and to prevent replication hindrance. These findings reveal a new function for CFI in the DDR and underscore the importance of coordinating transcription termination with replication in the maintenance of genomic stability.
Author Summary
DNA damage occurs constantly in living cells and needs to be recognized and repaired to avoid mutations. DNA repair is particularly relevant for lesions occurring in actively transcribed DNA strands because the RNA polymerase cannot proceed through a damaged site. Stalled RNA polymerases and persisting DNA lesions can lead to genome instability or cell death. Specific mechanisms to repair obstructing DNA lesions are found from bacteria to higher eukaryotes, their malfunction leading to severe genetic syndromes in humans. Termination of transcription comprises cleavage and polyadenylation of the nascent transcript and displacement of the RNA polymerase from its DNA template. These processes, which are crucial for cell viability and growth in eukaryotes, require two major multi-subunit complexes in budding yeast. Here, we found that one of these complexes, Cleavage Factor I (CFI), participates in the cellular response to DNA damage. In addition, we found that CFI dysfunction leads to replication defects, conceivably mediated by stalled RNA polymerases, rendering cell cycle checkpoints mandatory to prevent genomic instability. Our findings emphasize the importance of coordinating transcription termination, DNA damage response and replication in the maintenance of genomic stability suggesting that CFI plays a fundamental function in the coupling of these processes.
PMCID: PMC3945788  PMID: 24603480
16.  Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis 
PLoS Genetics  2013;9(1):e1003259.
Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8−/− mice, we have generated Rnf8−/−p53−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8−/− mice. Similarly, the senescence phenotype of Rnf8−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8−/−p53−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8−/−p53−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8−/− or p53−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.
Author Summary
DNA double-strand breaks are the most dangerous type of DNA lesions that can occur in cells. Failure to repair these breaks quickly and efficiently can result in either cell death or genomic instability and eventually malignant transformation. Rnf8 and p53 are key proteins in the DNA damage response. We have generated mice that are deficient in both Rnf8 and p53 proteins to determine how these two proteins interact. We found that, when p53 is absent, some of the developmental defects caused by Rnf8 deficiency are rescued. However, this is at the expense of increased chromosomal abnormalities caused by the inability of double-mutant cells to restrain DNA synthesis and cell proliferation and by their escape of cell death in the presence of damaged DNA. We showed that loss of both Rnf8 and p53 led to the accelerated development of both hematological and solid tumors. This study has important implications for human cancer, where mutations in the p53 gene are found in more than 50% of all cancers but where the other factors that facilitate cancer development are not well understood. This study suggests cooperation between Rnf8 and p53 in the prevention of cancer.
PMCID: PMC3561120  PMID: 23382699
17.  Human Telomeres Are Hypersensitive to UV-Induced DNA Damage and Refractory to Repair 
PLoS Genetics  2010;6(4):e1000926.
Telomeric repeats preserve genome integrity by stabilizing chromosomes, a function that appears to be important for both cancer and aging. In view of this critical role in genomic integrity, the telomere's own integrity should be of paramount importance to the cell. Ultraviolet light (UV), the preeminent risk factor in skin cancer development, induces mainly cyclobutane pyrimidine dimers (CPD) which are both mutagenic and lethal. The human telomeric repeat unit (5′TTAGGG/CCCTAA3′) is nearly optimal for acquiring UV-induced CPD, which form at dipyrimidine sites. We developed a ChIP–based technique, immunoprecipitation of DNA damage (IPoD), to simultaneously study DNA damage and repair in the telomere and in the coding regions of p53, 28S rDNA, and mitochondrial DNA. We find that human telomeres in vivo are 7-fold hypersensitive to UV-induced DNA damage. In double-stranded oligonucleotides, this hypersensitivity is a property of both telomeric and non-telomeric repeats; in a series of telomeric repeat oligonucleotides, a phase change conferring UV-sensitivity occurs above 4 repeats. Furthermore, CPD removal in the telomere is almost absent, matching the rate in mitochondria known to lack nucleotide excision repair. Cells containing persistent high levels of telomeric CPDs nevertheless proliferate, and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions. Utilizing a lesion-tolerance strategy rather than repair would prevent double-strand breaks at closely-opposed excision repair sites on opposite strands of a damage-hypersensitive repeat.
Author Summary
Telomeres consist of a repeated sequence located at each end of each chromosome. This repeated sequence is required for chromosomal stability and integrity, a function important for both cancer and aging. The DNA sequence of human telomeres is 5–10 kb of a repeated double-strand hexamer (5′TTAGGG/5′CCCTAA). In theory, this sequence is nearly optimal for acquiring UV-induced DNA damage. We developed a novel technique, the immunoprecipitation of DNA damage (IPoD), to study DNA damage induction and repair in the telomere and in coding regions (p53, 28S rDNA, and mitochondrial DNA). We find that human telomeres are hypersensitive to UV-induced DNA photoproducts and that the removal of those DNA photoproducts is almost absent. Cells containing persistent high levels of telomeric DNA damage nevertheless proliferate and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions.
PMCID: PMC2861706  PMID: 20442874
18.  How Chromatin Is Remodelled during DNA Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae 
PLoS Genetics  2011;7(6):e1002124.
Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation–induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin.
Author Summary
Protection against genotoxic insult requires a network of DNA damage responses, including DNA repair. Inherited DNA repair defects cause severe clinical consequences including extreme cancer susceptibility. Despite detailed mechanistic understanding of the core reactions, little is known about the molecular events that initiate and regulate these processes as they occur in chromatin. We study the conserved nucleotide excision repair pathway in Saccharomyces cerevisiae. This pathway removes a broad spectrum of DNA damages including UV radiation–induced damage. Patients with mutations in genes involved in this process suffer dramatically elevated levels of skin and other cancers. Here we demonstrate how a group of genes involved in repair of transcriptionally silent regions of the genome, a process called global genome repair, modifies chromatin structure following UV irradiation to promote efficient removal of DNA damage from the genome. We show that the concerted action of global genome repair genes combine to regulate histone acetyl transferase accessibility to the chromatin in response to UV damage. In this way, global genome repair regulates histone H3 acetylation status, which ultimately regulates chromatin structure promoting efficient DNA repair in the genome. Our results contribute a significant advance in our understanding of how chromatin is processed during DNA repair.
PMCID: PMC3116912  PMID: 21698136
19.  Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae 
PLoS Genetics  2011;7(3):e1001339.
Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.
Author Summary
Expansion of a CAG/CTG trinucleotide repeat is the causative mutation for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and multiple types of spinocerebellar ataxias. Two reasons for the cell death that occurs in these diseases are toxicity of the repeat-containing RNA and of the polyglutamine-containing protein product. Although the expanded repeat can interfere with DNA replication and repair, it was not known whether the presence of the repeat within the DNA causes any additional cellular toxicity. In this study, we show that an expanded CAG/CTG tract placed within the chromosome of the model eukaryote, budding yeast, elicits a cellular response that interferes with cell growth and division. The effect is enhanced when DNA repair pathways, particularly double-strand break repair, are compromised. Moreover, cells experiencing an arrest were more likely to have undergone further repeat expansions. We show that the conserved MRX protein complex locates to the expanded repeat and is required to sense the damage and activate the DNA damage response. Our results suggest that DNA damage at expanded CAG/CTG repeats could contribute to both tissue degeneration and further repeat instability in affected individuals.
PMCID: PMC3060079  PMID: 21437275
20.  Proteins in the Nutrient-Sensing and DNA Damage Checkpoint Pathways Cooperate to Restrain Mitotic Progression following DNA Damage 
PLoS Genetics  2011;7(7):e1002176.
Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA.
Author Summary
Previous studies showed that phosphorylation of a subset of regulatory (R) subunits of the cAMP-dependent protein kinase (PKA) occurred under conditions that down-regulate global PKA activity, including growth on non-fermentable carbon sources. However, the role of the phosphorylation sites has not been elucidated. Addition of glucose to cells growing on a non-fermentable carbon source causes a transient increase of cAMP and PKA activity, which drives cells into S phase. A second peak in cAMP was proposed to restrain mitosis if the daughter cell had not reached an appropriate size. We identified a role for PKA in restraining mitosis following DNA damage. Here we provide evidence of cross-talk between the DNA damage checkpoint and PKA by phosphorylation of the R subunit. The R subunit phosphorylation sites and cAMP are necessary for the role of PKA following DNA damage. We propose that activation of PKA in response to DNA damage occurs in two steps: the phosphorylation of a subset of R subunits, probably to allow localized activation of these complexes, and cAMP to activate PKA. Our work suggests that the checkpoint and nutrient-sensing pathways share a signaling node to restrain mitosis following nutrient-induced rapid transition through the cell cycle and DNA damage.
PMCID: PMC3136438  PMID: 21779180
21.  Alliance of Proteomics and Genomics to Unravel the Specificities of Sahara Bacterium Deinococcus deserti 
PLoS Genetics  2009;5(3):e1000434.
To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.
Author Summary
D. deserti belongs to the Deinococcaceae, a family of bacteria characterized by an exceptional ability to withstand the lethal effects of DNA-damaging agents, including ionizing radiation, UV light, and desiccation. It was isolated from Sahara surface sands, an extreme and nutrient-poor environment, regularly exposed to intense UV radiation, cycles of extreme temperatures, and desiccation. The evolution of organisms that are able to survive acute irradiation doses of 15,000 Gy is difficult to explain given the apparent absence of highly radioactive habitats on Earth over geologic time. Thus, it seems more likely that the natural selection pressure for the evolution of radiation-resistant bacteria was chronic exposure to nonradioactive forms of DNA damage, in particular those promoted by desiccation. Here, we report the first complete genome sequence of a bacterium, D. deserti VCD115, isolated from hot, arid desert surface sand. Accurate genome annotation of its 3,455 genes was guided by extensive proteome analysis in which 1,348 proteins were uncovered after growth in standard conditions. Supplementary genes involved in manganese import, in nutrient import, and in DNA repair were identified and are likely important for survival and adaptation of D. deserti to its hostile environment.
PMCID: PMC2669436  PMID: 19370165
22.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
PMCID: PMC3825654  PMID: 24265601
23.  Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis 
PLoS Genetics  2014;10(10):e1004731.
In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.
Author Summary
DNA replication must be coordinated with cellular physiology to ensure proper genome inheritance. Model bacteria such as the soil-dwelling Bacillus subtilis can achieve a wide range of growth rates in response to nutritional and chemical signals. In order to match the rate of DNA synthesis to the rate of nutrient-mediated cell growth, bacteria regulate the initiation frequency of DNA replication. This control of bacterial DNA replication initiation was first observed over forty years ago, however the molecular basis for this regulation has remained hotly debated. In this paper we test one of the leading models for nutrient-mediated growth rate regulation in bacteria, namely that the abundance of the master DNA replication initiation protein DnaA dictates the frequency of DNA replication events. Critically, our results show that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate regulation of DNA replication initiation in B. subtilis. We then go on to show that there are strong connections between DNA replication and several essential cellular activities, which unexpectedly indicates that there is likely more than one single regulatory pathway involved in coordinating DNA replication with cell physiology. We believe that our work changes thinking regarding this long-standing biological question and reinvigorates the search for the molecular basis of these critical regulatory systems.
PMCID: PMC4207641  PMID: 25340815
24.  Viral Interference with DNA Repair by Targeting of the Single-Stranded DNA Binding Protein RPA 
PLoS Pathogens  2013;9(10):e1003725.
Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.
Author Summary
DNA repair protects genome integrity and unrepaired DNA damage can cause cancer. We have identified a new mechanism by which a tumor virus makes cells hypersensitive to DNA damage. The Large T Antigen (LT) of polyoma virus blocks DNA repair pathways, making cells 100 fold more sensitive to DNA damage. LT does this by targeting replication protein A (RPA). RPA is central to both DNA replication and repair. Ordinarily RPA and then other DNA repair proteins are recruited to sites of DNA damage. LT blocks recruitment of these proteins to damage foci. Current cancer treatment strategies like radiation therapy and chemotherapeutics cause DNA damage to block the growth and spread of cancer. This work suggests a target that might increase the efficacy of such treatment.
PMCID: PMC3812037  PMID: 24204272
25.  Evidence That BRCA1- or BRCA2-Associated Cancers Are Not Inevitable 
Molecular Medicine  2012;18(1):1327-1337.
Inheriting a BRCA1 or BRCA2 gene mutation can cause a deficiency in repairing complex DNA damage. This step leads to genomic instability and probably contributes to an inherited predisposition to breast and ovarian cancer. Complex DNA damage has been viewed as an integral part of DNA replication before cell division. It causes temporary replication blocks, replication fork collapse, chromosome breaks and sister chromatid exchanges (SCEs). Chemical modification of DNA may also occur spontaneously as a byproduct of normal processes. Pathways containing BRCA1 and BRCA2 gene products are essential to repair spontaneous complex DNA damage or to carry out SCEs if repair is not possible. This scenario creates a theoretical limit that effectively means there are spontaneous BRCA1/2-associated cancers that cannot be prevented or delayed. However, much evidence for high rates of spontaneous DNA mutation is based on measuring SCEs by using bromodeoxyuridine (BrdU). Here we find that the routine use of BrdU has probably led to overestimating spontaneous DNA damage and SCEs because BrdU is itself a mutagen. Evidence based on spontaneous chromosome abnormalities and epidemiologic data indicates strong effects from exogenous mutagens and does not support the inevitability of cancer in all BRCA1/2 mutation carriers. We therefore remove a theoretical argument that has limited efforts to develop chemoprevention strategies to delay or prevent cancers in BRCA1/2 mutation carriers.
PMCID: PMC3521784  PMID: 22972572

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