Regular chromosome segregation during the first meiotic division requires prior pairing of homologous chromosomes into bivalents. During canonical meiosis, linkage between homologous chromosomes is maintained until late metaphase I by chiasmata resulting from meiotic recombination in combination with distal sister chromatid cohesion. Separasemediated elimination of cohesin from chromosome arms at the end of metaphase I permits terminalization of chiasmata and homolog segregation to opposite spindle poles during anaphase I. Interestingly, separase is also required for bivalent splitting during meiosis I in Drosophila males, where homologs are conjoined by an alternative mechanism independent of meiotic recombination and cohesin. Here we report the identification of a novel alternative homolog conjunction protein encoded by the previously uncharacterized gene univalents only (uno). The univalents that are present in uno null mutants at the start of meiosis I, instead of normal bivalents, are segregated randomly. In wild type, UNO protein is detected in dots associated with bivalent chromosomes and most abundantly at the localized pairing site of the sex chromosomes. UNO is cleaved by separase. Expression of a mutant UNO version with a non-functional separase cleavage site restores homolog conjunction in a uno null background. However, separation of bivalents during meiosis I is completely abrogated by this non-cleavable UNO version. Therefore, we propose that homolog separation during Drosophila male meiosis I is triggered by separase-mediated cleavage of UNO.

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.

CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model.

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. Here, we report that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, we suggest that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, we demonstrate that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease.

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors.Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.All rights reserved. No reuse allowed without permission.