A direct measurement of crossing over in human prophase oocytes

The nature of the relationship between crossing over and failed segregation in human oocytes is of obvious interest. A recent paper by Cheng and colleagues provides important insights into the distribution of crossover events (as marked by MLH1 foci) in human oocytes and raises complex questions regarding discrepancies between direct cytological assessment of exchange and measurement of crossing over by linkage analysis

A Meiotic Tapas Menu

this past fall (September 13-18), the 7th European Meiosis Meeting held in San Lorenzo de El Escorial brought us'Meiosis in Madrid.''This is a sister conference to the Gordon Meiosis series held every other year in the United States, and is an international small-format forum for sharing hot, new results, with an emphasis on unpublished work. At the conference, 21 countries were represented by 167 participants, with a nearly equal split between male and female scientists. The aim of the meeting was to present and promote in-depth discussions about all aspects of meiotic chromosome dynamics, recombination, and segregation. Rather than providing a comprehensive description of the meeting abstracts, this report will briefly describe meeting highlights, guided in large part by feedback from session chairs

Targeting determinants of dosage compensation in Drosophila

The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high-and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs

All Paired Up with No Place to Go: Pairing, Synapsis, and DSB Formation in a Balancer Heterozygote

The multiply inverted X chromosome balancer FM7 strongly suppresses, or eliminates, the occurrence of crossing over when heterozygous with a normal sequence homolog. We have utilized the LacI-GFP: lacO system to visualize the effects of FM7 on meiotic pairing, synapsis, and double-strand break formation in Drosophila oocytes. Surprisingly, the analysis of meiotic pairing and synapsis for three lacO reporter couplets in FM7/X heterozygotes revealed they are paired and synapsed during zygotene/pachytene in 70%–80% of oocytes. Moreover, the regions defined by these lacO couplets undergo double-strand break formation at normal frequency. Thus, even complex aberration heterozygotes usually allow high frequencies of meiotic pairing, synapsis, and double-strand break formation in Drosophila oocytes. However, the frequencies of failed pairing and synapsis were still 1.5- to 2-fold higher than were observed for corresponding regions in oocytes with two normal sequence X chromosomes, and this effect was greatest near a breakpoint. We propose that heterozygosity for breakpoints creates a local alteration in synaptonemal complex structure that is propagated across long regions of the bivalent in a fashion analogous to chiasma interference, which also acts to suppress crossing over

Drosophila brca2 Is Required for Mitotic and Meiotic DNA Repair and Efficient Activation of the Meiotic Recombination Checkpoint

Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal

Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser(487) in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr(172). Surprisingly, the equivalent site on AMPK-α2, Ser(491), is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr(172) phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca(2+) ionophore A23187, effects we show to be dependent on Akt activation and Ser(487) phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr(172) phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation

Dynamics of Wolbachia pipientis gene expression across the Drosophila melanogaster life cycle

Symbiotic interactions between microbes and their multicellular hosts have manifold impacts on molecular, cellular and organismal biology. To identify candidate bacterial genes involved in maintaining endosymbiotic associations with insect hosts, we analyzed genome-wide patterns of gene expression in the alpha-proteobacteria Wolbachia pipientis across the life cycle of Drosophila melanogaster using public data from the modENCODE project that was generated in a Wolbachia-infected version of the ISO1 reference strain. We find that the majority of Wolbachia genes are expressed at detectable levels in D. melanogaster across the entire life cycle, but that only 7.8% of 1195 Wolbachia genes exhibit robust stage- or sex-specific expression differences when studied in the "holo-organism" context. Wolbachia genes that are differentially expressed during development are typically up-regulated after D. melanogaster embryogenesis, and include many bacterial membrane, secretion system and ankyrin-repeat containing proteins. Sex-biased genes are often organised as small operons of uncharacterised genes and are mainly up-regulated in adult males D. melanogaster in an age-dependent manner suggesting a potential role in cytoplasmic incompatibility. Our results indicate that large changes in Wolbachia gene expression across the Drosophila life-cycle are relatively rare when assayed across all host tissues, but that candidate genes to understand host-microbe interaction in facultative endosymbionts can be successfully identified using holo-organism expression profiling. Our work also shows that mining public gene expression data in D. melanogaster provides a rich set of resources to probe the functional basis of the Wolbachia-Drosophila symbiosis and annotate the transcriptional outputs of the Wolbachia genome.Comment: 58 pages, 6 figures, 6 supplemental figures, 4 supplemental files (available at https://github.com/bergmanlab/wolbachia/tree/master/gutzwiller_et_al/arxiv

Multiple-Pathway Analysis of Double-Strand Break Repair Mutations in Drosophila

The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage “decision circuit” in which the DSB is first placed into one of two pools from which a specific pathway is then selected

C. elegans Germ Cells Switch between Distinct Modes of Double-Strand Break Repair During Meiotic Prophase Progression

Chromosome inheritance during sexual reproduction relies on deliberate induction of double-strand DNA breaks (DSBs) and repair of a subset of these breaks as interhomolog crossovers (COs). Here we provide a direct demonstration, based on our analysis of rad-50 mutants, that the meiotic program in Caenorhabditis elegans involves both acquisition and loss of a specialized mode of double-strand break repair (DSBR). In premeiotic germ cells, RAD-50 is not required to load strand-exchange protein RAD-51 at sites of spontaneous or ionizing radiation (IR)-induced DSBs. A specialized meiotic DSBR mode is engaged at the onset of meiotic prophase, coincident with assembly of meiotic chromosome axis structures. This meiotic DSBR mode is characterized both by dependence on RAD-50 for rapid accumulation of RAD-51 at DSB sites and by competence for converting DSBs into interhomolog COs. At the mid-pachytene to late pachytene transition, germ cells undergo an abrupt release from the meiotic DSBR mode, characterized by reversion to RAD-50-independent loading of RAD-51 and loss of competence to convert DSBs into interhomolog COs. This transition in DSBR mode is dependent on MAP kinase-triggered prophase progression and coincides temporally with a major remodeling of chromosome architecture. We propose that at least two developmentally programmed switches in DSBR mode, likely conferred by changes in chromosome architecture, operate in the C. elegans germ line to allow formation of meiotic crossovers without jeopardizing genomic integrity. Our data further suggest that meiotic cohesin component REC-8 may play a role in limiting the activity of SPO-11 in generating meiotic DSBs and that RAD-50 may function in counteracting this inhibition