The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.

TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks.

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identify their conserved interacting domains by structural analysis. We then analyse the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity is strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity is delayed in autosomes. These results suggest that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

PRDM9 Methyltransferase Activity Is Essential for Meiotic DNA Double-Strand Break Formation at Its Binding Sites.

The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA-binding specificity of its zinc-finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9-binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA-binding specificities, we show that each variant generates its own set of H3K4me3 marks independently from the other variant. Altogether, we reveal several basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset is selected for DSB formation.

Motor-driven marginal band coiling promotes cell shape change during platelet activation.

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.

Molecular basis for the regulation of the H3K4 methyltransferase activity of PRDM9.

PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

HDAC6 controls the kinetics of platelet activation.

HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.

The tale of protein lysine acetylation in the cytoplasm.

Reversible posttranslational modification of internal lysines in many cellular or viral proteins is now emerging as part of critical signalling processes controlling a variety of cellular functions beyond chromatin and transcription. This paper aims at demonstrating the role of lysine acetylation in the cytoplasm driving and coordinating key events such as cytoskeleton dynamics, intracellular trafficking, vesicle fusion, metabolism, and stress response.