Programming sites of meiotic crossovers using Spo11 fusion proteins.

Meiotic recombination shapes the genetic diversity transmitted upon sexual reproduction. However, its non-random distribution along the chromosomes constrains the landscape of potential genetic combinations. For a variety of purposes, it is desirable to expand the natural repertoire by changing the distribution of crossovers in a wide range of eukaryotes. Toward this end, we report the local stimulation of meiotic recombination at a number of chromosomal sites by tethering the natural Spo11 protein to various DNA-binding modules: full-length DNA binding proteins, zinc fingers (ZFs), transcription activator-like effector (TALE) modules, and the CRISPR-Cas9 system. In the yeast Saccharomyces cerevisiae, each strategy is able to stimulate crossover frequencies in naturally recombination-cold regions. The binding and cleavage efficiency of the targeting Spo11 fusions (TSF) are variable, being dependent on the chromosomal regions and potential competition with endogenous factors. TSF-mediated genome interrogation distinguishes naturally recombination-cold regions that are flexible and can be warmed-up (gene promoters and coding sequences), from those that remain refractory (gene terminators and centromeres). These results describe new generic experimental strategies to increase the genetic diversity of gametes, which should prove useful in plant breeding and other applications.

Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae.

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.

Radiation induction of delayed recombination in Schizosaccharomyces pombe.

Ionizing radiation is known to induce delayed chromosome and gene mutations in the descendants of the irradiated tissue culture cells. Molecular mechanisms of such delayed mutations are yet to be elucidated, since high genomic complexity of mammalian cells makes it difficult to analyze. We now tested radiation induction of delayed recombination in the fission yeast Schizosaccharomyces pombe by monitoring the frequency of homologous recombination after X-irradiation. A reporter with 200 bp tandem repeats went through spontaneous recombination at a frequency of 1.0 x 10(-4), and the frequency increased dose-dependently to around 10 x 10(-4) at 500 Gy of X-irradiation. Although the repair of initial DNA damage was thought to be completed before the restart of cell division cycle, the elevation of the recombination frequency persisted for 8-10 cell generations after irradiation (delayed recombination). The delayed recombination suggests that descendants of the irradiated cells keep a memory of the initial DNA damage which upregulates recombination machinery for 8-10 generations even in the absence of DNA double-strand breaks (DSBs). Since radical scavengers were ineffective in inhibiting the delayed recombination, a memory by continuous production of DNA damaging agents such as reactive oxygen species (ROS) was excluded. Recombination was induced in trans in a reporter on chromosome III by a DNA DSB at a site on chromosome I, suggesting the untargeted nature of delayed recombination. Interestingly, Rad22 foci persisted in the X-irradiated population in parallel with the elevation of the recombination frequency. These results suggest that the epigenetic damage memory induced by DNA DSB upregulates untargeted and delayed recombination in S. pombe.

p53-dependent S-phase damage checkpoint and pronuclear cross talk in mouse zygotes with X-irradiated sperm.

One difficulty in analyzing the damage response is that the effect of damage itself and that of cellular response are hard to distinguish in irradiated cells. In mouse zygotes, damage can be introduced by irradiated sperm, while damage response can be studied in the unirradiated maternal pronucleus. We have analyzed the p53-dependent damage responses in irradiated-sperm mouse zygotes and found that a p53-responsive reporter was efficiently activated in the female pronucleus. [(3)H]thymidine labeling experiments indicated that irradiated-sperm zygotes were devoid of G(1)/S arrest, but pronuclear DNA synthesis was suppressed equally in male and female pronuclei. p53(-/-) zygotes lacked this suppression, which was corrected by microinjection of glutathione S-transferase-p53 fusion protein. In contrast, p21(-/-) zygotes exhibited the same level of suppression upon fertilization by irradiated sperm. About a half of the 6-Gy-irradiated-sperm zygotes managed to synthesize a full DNA content by prolonging S phase, while the other half failed to do so. Regardless of the DNA content, all the zygotes cleaved to become two-cell-stage embryos. These results revealed the presence of p53-dependent pronuclear cross talk and a novel function of p53 in the S-phase DNA damage checkpoint of mouse zygotes.

Persistent induction of somatic reversions of the pink-eyed unstable mutation in F1 mice born to fathers irradiated at the spermatozoa stage.

Untargeted mutation and delayed mutation are features of radiation-induced genomic instability and have been studied extensively in tissue culture cells. The mouse pink-eyed unstable (p(un)) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts back to the wild type in germ cells as well as in somatic cells. The reversion event can be detected in the retinal pigment epithelium as a cluster of pigmented cells (eye spot). We have investigated the reversion p(um) in F1 mice born to irradiated males. Spermatogonia-stage irradiation did not affect the frequency of the reversion in F1 mice. However, 6 Gy irradiation at the spermatozoa stage resulted in an approximately twofold increase in the number of eye spots in the retinal pigment epithelium of F1 mice. Somatic reversion occurred for the paternally derived p(un) alleles. In addition, the reversion also occurred for the maternally derived, unirradiated p(un) alleles at a frequency equal to that for the paternally derived allele. Detailed analyses of the number of pigmented cells per eye spot indicated that the frequency of reversion was persistently elevated during the proliferation cycle of the cells in the retinal pigment epithelium when the male parents were irradiated at the spermatozoa stage. The present study demonstrates the presence of a long-lasting memory of DNA damage and the persistent up-regulation of recombinogenic activity in the retinal pigment epithelium of the developing fetus.

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm.

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.

Characterization of spontaneous mutation in the delta soxR and SoxS overproducing strains of Escherichia coli.

To examine the role of the soxRS regulon in mutagenesis, we characterized the spontaneous mutations occurring in the endogenous tonB gene in the delta soxR strain and the SoxS overproducing strain of Escherichia coli. Neither the delta soxR strain nor the SoxS overproducing strain led to an enhancement or diminishment of the spontaneous mutation frequency. By DNA sequencing, we determined 50 spontaneous mutants from the delta soxR strains, and found that 36% were both base substitutions and IS insertions, 14% frameshifts and 10% deletions. Among the base substitutions, G:C-->T:A transversions and G:C-->A:T transitions predominated, followed by A:T-->T:A transversions. We determined 54 spontaneous mutants from the SoxS overproducing strains, and found that 37% were IS insertions, 31% base substitutions, 17% frameshifts, 9% deletions and 6% duplications. Among the base substitutions, G:C-->T:A transversions dominated, followed by A:T-->T:A transversions and G:C-->A:T transitions. These results were similar to those from the soxRS+ strains. Thus, it is suggested that the soxRS-regulated genes do not play a significant role in the defense against spontaneous mutagenesis.

BM-ca is a newly defined type I/II anti-CD20 monoclonal antibody with unique biological properties.

Rituximab (chimeric anti-CD20 mAb) is currently used in the treatment of B-NHL and B cell malignancies, alone or in combination with chemotherapy. However, subsets of patients do not initially respond and/or develop resistance to additional treatments. Hence, there is a need to develop more effective anti-CD20 mAbs that may improve clinical response. BM-ca is a novel humanized anti-CD20 mAb that was tested against several B-NHL cell lines and was compared to several anti-CD20 mAbs (Rituximab, ofatumumab, 2H7, B1 and B-Ly1). BM-ca was shown to strongly induce both homotypic cell aggregation and redistribution of CD20 to membrane lipid rafts. BM-ca was also able to induce programmed cell death (apoptosis) without the need for cross-linking and demonstrated potent complement-dependent cytotoxicity (CDC). BM-ca was more cytotoxic than rituximab even in malignant B cells expressing low amounts of membrane CD20. Type I anti-CD20 mAbs typically induce minimal levels of homotypic cell aggregation and apoptosis but strong localization of CD20 to lipid rafts and potent CDC. Type II anti-CD20 mAbs typically exert the reverse activities. Noteworthy, BM-ca exhibits properties that are shared by both type I and type II anti-CD20 mAbs, which may reflect the recognition of a new CD20 epitope and/or exhibit different molecular signaling. Overall, the present data show that BM-ca is a novel anti-CD20 mAb that may be classified as a type I/II. The therapeutics efficacy of BM-ca awaits its use in clinical trials.

Dysregulation of the cell survival/anti-apoptotic NF-kappaB pathway by the novel humanized BM-ca anti-CD20 mAb: implication in chemosensitization.

Treatment of patients with relapsed or refractory low grade follicular B-NHL lymphoma with rituximab (chimeric anti-CD20 mAb) has resulted in approximately 50% response rate. The mechanism underlying the failure of rituximab to affect the remaining 50% of the patients is not clear, though their tumors express CD20. The in vivo effector functions of rituximab include ADCC, CDC and seldom apoptosis. In addition, we have reported that rituximab signals the cells and inhibits several intracellular cell survival pathways that are responsible for the immuno and chemo-sensitizing effects of rituximab on resistant B-NHL cell lines. The objective of this study was to develop novel and fully humanized anti-CD20 monoclonal antibodies with enhanced effector functions and molecular signaling that may potentiate their therapeutic efficacy. Novel humanized anti-CD20 monoclonal antibodies were derived from a chimerized form of murine anti-CD20 1K11791, shown to exert a more potent ADCC, CDC and apoptotic activities compared to rituximab. A representative humanized monoclonal antibody, BM-ca was used to examine its biological effect and molecular signaling using Ramos B-NHL cell line as a model. The studies were also performed in parallel with rituximab treatment for comparison. Ramos cells were treated with various concentrations of BM-ca monoclonal antibody. Inhibition of cell proliferation was observed in a concentration-dependent manner, suggesting cell signal perturbations must have occurred. Compared to untreated cells, treatment with BM-ca inhibited both the constitutively activated NF-kappaB and p38 MAPK pathways, as assessed by inhibition of both phospho-p65 and phospho-IkappaBalpha and phospho-p38, respectively, but not the unphosphorylated forms. BM-ca significantly induced the expression of the metastasis suppressor and immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). These alterations resulted in inhibition of anti-apoptotic gene products and sensitized Ramos cells to apoptosis by CDDP. In comparison with rituximab, BM-ca showed qualitative and quantitative differences in the above analyses. These findings demonstrate that BM-ca triggers CD20 expressing B-NHL cells resulting in a significant alteration of several gene products that regulate cell growth and chemoresistance.

Delayed and stage specific phosphorylation of H2AX during preimplantation development of gamma-irradiated mouse embryos.

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as gamma-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of gamma-H2AX in mouse embryos gamma-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy gamma-irradiation is hindered up to the two cell stage of development. When left in utero for another 24-64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages.