Post-meiotic DNA double-strand breaks are conserved in fission yeast.

In mammals, spermiogenesis is characterized by transient formation of DNA double-strand breaks (DSBs) in the whole population of haploid spermatids. DSB repair in such haploid context may represent a mutational transition. Using a combination of pulsed-field gel electrophoresis and specific labelling of DSBs at 3'OH DNA ends, we showed that post-meiotic, enzyme-induced DSBs are also observed in the synchronizable pat1-114 mutant of Shizosaccharomyces pombe as well as in a wild-type strain, while DNA repair is observed at later stages. This transient DNA fragmentation arises in the whole cell population and is seemingly independent of the caspase apoptotic pathway. Because histones are still present in spores, the transient DSBs do not require a major change in chromatin structure. These observations confirm the highly-conserved nature of the process in eukaryotes and provide a powerful model to study the underlying mechanism and its impact on the genetic landscape and adaptation.

The DNA double-strand "breakome" of mouse spermatids.

De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.

Quantification and genome-wide mapping of DNA double-strand breaks.

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.

Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts.

A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.

Step-specific Sorting of Mouse Spermatids by Flow Cytometry.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

Instability of trinucleotidic repeats during chromatin remodeling in spermatids.

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

Male-driven de novo mutations in haploid germ cells.

At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA end-joining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.

"Breaking news" from spermatids.

During the haploid phase of spermatogenesis, spermatids undergo a complex remodeling of the paternal genome involving the finely orchestrated replacement of histones by the highly-basic protamines. The associated striking change in DNA topology is characterized by a transient surge of both single- and double-stranded DNA breaks in the whole population of spermatids which are repaired before spermiation. These transient DNA breaks are now considered part of the normal differentiation program of these cells. Despite an increasing interest in the study of spermiogenesis in the last decade and the potential threat to the haploid genome, the origin of these DNA breaks still remains elusive. This review briefly outlines the current hypotheses regarding possible mechanisms that may lead to such transient DNA fragmentation including torsional stress, enzyme-induced breaks, apoptosis-like processes or oxidative stress. A better understanding of the origin of these DNA breaks will lead to further investigations on the genetic instability and mutagenic potential induced by the chromatin remodeling.

Lors de la phase haploïde de la spermatogenèse, les spermatides subissent un remodelage complexe du génome paternel impliquant un remplacement finement orchestré des histones par des protamines hautement basiques. Le changement topologique de l'ADN associé est caractérisé par une augmentation transitoire de cassures simple et double brins de l'ADN dans l'entière population des spermatides qui sont réparées avant la spermiation. Ces cassures transitoires de l'ADN sont maintenant considérées comme faisant partie du processus normal de différenciation de ces cellules. Malgré un intérêt croissant dans l'étude de la spermiogenèse ces 10 dernières années et la menace potentielle pour le génome haploïde, l'origine de ces cassures d'ADN reste encore incertaine. Cette revue décrit brièvement les hypothèses actuelles concernant les mécanismes possibles qui pourraient mener à cette fragmentation transitoire de l'ADN incluant le stress torsionnel, les cassures enzymatiques, des processus semblables à l'apoptose et le stress oxidatif. Une meilleure compréhension de l'origine de ces cassures d'ADN mènerait à des études approfondies concernant l'instabilité génétique et le potentiel mutagène induit par le remodelage de la chromatine.

Efficiency of manual scanning in recovering rare cellular events identified by fluorescence in situ hybridization: simulation of the detection of fetal cells in maternal blood.

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1-10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Genome-wide mapping of DNA strand breaks.

Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

Quantification of fetal nucleated cells in maternal blood of pregnant women with a male trisomy 21 fetus using molecular cytogenetic techniques.

BACKGROUND: Prenatal diagnosis of trisomy 21 is based on fetal karyotyping generally obtained using invasive methods. During pregnancy, the circulating fetal cells in maternal blood constitute a potential source for development of a noninvasive prenatal diagnosis. The objective of this study was the identification and quantification of all fetal nucleated cells per unit volume of peripheral blood of pregnant women carrying male fetuses with trisomy 21 using molecular cytogenetic techniques. METHODS: Peripheral blood samples were obtained from 16 women carrying male fetuses with trisomy 21. We used a simple and rapid method of harvesting blood without recourse to any enrichment procedures or cell-separation techniques. To evaluate the potential of this method, 16 specimens were analyzed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) using specific probes to chromosomes X, Y and 21. RESULTS: The number of fetal cells varied between 6 and 32 per mL of maternal blood. This number is 3-5 times higher than that from normal pregnancies. CONCLUSIONS: Our current results are in agreement with the results previously reported by other groups showing that the number of fetal cells in maternal blood in trisomic 21 pregnancies is higher than in normal pregnancies. This high number of fetal cells is regarded as an advantage for the development of a noninvasive prenatal diagnostic test.