Replication independent DNA double-strand break retention may prevent genomic instability

<p>Abstract</p> <p>Background</p> <p>Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability.</p> <p>Results</p> <p>We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51.</p> <p>Conclusions</p> <p>Methylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.</p

LINE-1 Methylation Patterns as a Predictor of Postmolar Gestational Trophoblastic Neoplasia

Objective. To study the potential of long interspersed element-1 (LINE-1) methylation change in the prediction of postmolar gestational trophoblastic neoplasia (GTN). Methods. The LINE-1 methylation pattern from first trimester placenta, hydatidiform mole, and malignant trophoblast specimens were compared. Then, hydatidiform mole patients from 11999 to 2010 were classified into the following 2 groups: a remission group and a group that developed postmolar GTN. Specimens were prepared for a methylation study. The methylation levels and percentages of LINE-1 loci were evaluated for their sensitivity, specificity, and accuracy for the prediction of postmolar GTN. Results. First, 12 placentas, 38 moles, and 19 malignant trophoblast specimens were compared. The hydatidiform mole group had the highest LINE-1 methylation level ( = 0.003) and the u C u C of LINE-1 increased in the malignant trophoblast group ( ≤ 0.001). One hundred forty-five hydatidiform mole patients were classified as 103 remission and 42 postmolar GTN patients. The % m C u C and % u C m C of LINE-1 showed the lowest value for distinguishing between the two groups ( &lt; 0.001). The combination of the pretreatment -hCG level (≥100,000 mIU/mL) with the % m C u C and % u C m C, sensitivity, specificity, PPV, NPV, and accuracy modified the levels to 60.0%, 92.2%, 77.4%, 83.8%, and 82.3%, respectively. Conclusions. A reduction in the partial methylation of LINE-1 occurs early before the clinical appearance of malignant transformation. The % m C u C and % u C m C of LINE-1s may be promising markers for monitoring hydatidiform moles before progression to GTN

LINE-1 Methylation Patterns as a Predictor of Postmolar Gestational Trophoblastic Neoplasia

Objective. To study the potential of long interspersed element-1 (LINE-1) methylation change in the prediction of postmolar gestational trophoblastic neoplasia (GTN). Methods. The LINE-1 methylation pattern from first trimester placenta, hydatidiform mole, and malignant trophoblast specimens were compared. Then, hydatidiform mole patients from 11999 to 2010 were classified into the following 2 groups: a remission group and a group that developed postmolar GTN. Specimens were prepared for a methylation study. The methylation levels and percentages of LINE-1 loci were evaluated for their sensitivity, specificity, and accuracy for the prediction of postmolar GTN. Results. First, 12 placentas, 38 moles, and 19 malignant trophoblast specimens were compared. The hydatidiform mole group had the highest LINE-1 methylation level (p = 0.003) and the uCuC of LINE-1 increased in the malignant trophoblast group (p ≤ 0.001). One hundred forty-five hydatidiform mole patients were classified as 103 remission and 42 postmolar GTN patients. The %mCuC and %uCmC of LINE-1 showed the lowest p value for distinguishing between the two groups (p < 0.001). The combination of the pretreatment β-hCG level (≥100,000 mIU/mL) with the %mCuC and %uCmC, sensitivity, specificity, PPV, NPV, and accuracy modified the levels to 60.0%, 92.2%, 77.4%, 83.8%, and 82.3%, respectively. Conclusions. A reduction in the partial methylation of LINE-1 occurs early before the clinical appearance of malignant transformation. The %mCuC and %uCmC of LINE-1s may be promising markers for monitoring hydatidiform moles before progression to GTN

Higher Alu methylation levels in catch-up growth in twenty-year-old offsprings.

Alu elements and long interspersed element-1 (LINE-1 or L1) are two major human intersperse repetitive sequences. Lower Alu methylation, but not LINE-1, has been observed in blood cells of people in old age, and in menopausal women having lower bone mass and osteoporosis. Nevertheless, Alu methylation levels also vary among young individuals. Here, we explored phenotypes at birth that are associated with Alu methylation levels in young people. In 2010, 249 twenty-years-old volunteers whose mothers had participated in a study association between birth weight (BW) and nutrition during pregnancy in 1990, were invited to take part in our present study. In this study, the LINE-1 and Alu methylation levels and patterns were measured in peripheral mononuclear cells and correlated with various nutritional parameters during intrauterine and postnatal period of offspring. This included the amount of maternal intake during pregnancy, the mother's weight gain during pregnancy, birth weight, birth length, and the rate of weight gain in the first year of life. Catch-up growth (CUG) was defined when weight during the first year was >0.67 of the standard score, according to WHO data. No association with LINE-1 methylation was identified. The mean level of Alu methylation in the CUG group was significantly higher than those non-CUG (39.61% and 33.66 % respectively, P < 0.0001). The positive correlation between the history of CUG in the first year and higher Alu methylation indicates the role of Alu methylation, not only in aging cells, but also in the human growth process. Moreover, here is the first study that demonstrated the association between a phenotype during the newborn period and intersperse repetitive sequences methylation during young adulthood

RIND-EDSBs and chromatin regulators.

<p>(A) The levels of RIND-EDSBs were measured in G0 cells of yeast strains lacking histone deacetylase genes, <i>SIR2, RPD3</i>, and <i>HDA1</i>. The level was decreased in the <i>sir2</i>Δ strain, while it was increased in <i>rpd3</i>Δ strain. (B) No significant change in the level of RIND-EDSBs was observed in yeast strains lacking the silent information regulator genes, <i>SIR1</i>, <i>SIR3</i>, or <i>SIR4</i>. (C) In contrast, deletions of <i>HTZ1</i> and <i>SWR1</i>, genes required for the prevention of heterochromatin spreading, led to significantly increased levels of RIND EDSBs. The values from 9 independent experiments are shown as box plots, with the boxes representing the interquartile ranges (25^th to 75^th percentile) and the median lines representing the 50^th percentile. The whiskers represent the minimum and the maximum values. <i>**P</i><0.001 (Mann-Whitney test).</p

RIND-EDSBs levels using HMW DNA preparation and intranuclear ligation protocols.

<p>(A, B) The levels of RIND-EDSBs were measured in G0 cells of WT, <i>mec1</i>Δ, <i>yku70</i>Δ, <i>nhp6a</i>Δ strains using HMW DNA (A) and intranuclear ligation (B) protocols. (C, D) The levels of RIND-EDSBs in controls and TSA-treated WT and <i>mec1</i>Δ strains using HMW DNA (C) and intranuclear ligation (D) protocols. Bar graphs represent average values and error bars represent standard deviation of triplicate experiments.</p

EDSBs were detected in different DNA preparations including HMW DNA (cell→gel), liquid DNA (cell→liquid), and liquid DNA extracted from in-gel HMW DNA (cell→gel→liquid).

<p>(A) The levels of EDSBs from different DNA preparation methods. (B) Subtracted DSBs levels between liquid DNA and other methods. When comparing cell→gel→liquid with cell→liquid, adding in gel preparation step did not increase the number of DSBs significantly. The average levels of EDSBs from 9 independent experiments are shown as histograms with error bars representing SEM.</p

Levels of RIND-EDSBs in yeast strains with mutations in DSB repair pathways.

<p>The levels of RIND-EDSBs were significantly increased in G0 cells of <i>mec1</i>Δ, <i>tel1</i>Δ, <i>mre11</i>Δ, <i>yku70</i>Δ, <i>yku80</i>Δ, and <i>rad51</i>Δ but not in <i>nej1</i>Δ strains. The levels of EDSBs from 9 independent experiments are shown as box plots, with the boxes representing the interquartile ranges (25^th to 75^th percentile) and the median lines representing the 50^th percentile. The whiskers represent the minimum and the maximum values. <i>**P</i><0.001 (Mann-Whitney test).</p

Levels of RIND-EDSBs in yeast strains with deletions of genes encoding proteins with the High-Mobility Group B (HMGB) domain.

<p>The levels of EDSBs were significantly decreased in G0 cells of yeast strains lacking <i>NHP6A</i>, <i>IXR1</i>, <i>ROX1</i>, and <i>HMO1</i>, suggesting that they play an important role in the production or retention of RIND-EDSBs. Nevertheless, the levels of RIND-EDSBs in <i>nhp6b</i>Δ, <i>nhp10</i>Δ, and <i>abf2</i>Δ strains were unchanged. The levels of EDSBs from 9 independent experiments are shown as box plots, with the boxes representing the interquartile ranges (25^th to 75^th percentile) and the median lines representing the 50^th percentile. The whiskers represent the minimum and the maximum values. <i>**P</i><0.001 (Mann-Whitney test).</p

RIND-EDSBs in strains with mutations in genes encoding topoisomerases, their partners, and endonucleases.

<p>Deletions of genes encoding topoisomerases, their partners, and endonucleases did not reduce the levels of RIND-EDSBs in G0 cells. On the contrary, the levels of RIND-EDSBs were increased in G0 cells of <i>top3</i>Δ, <i>rad27</i>Δ, and <i>sae2</i>Δ strains. The values from 9 independent experiments are shown as box plots, with the boxes representing the interquartile ranges (25^th to 75^th percentile) and the median lines representing the 50^th percentile. The whiskers represent the minimum and the maximum values. <i>*P</i><0.05, <i>**P</i><0.001(Mann-Whitney test).</p