The enigmatic meiotic dense body and its newly discovered component, SCML1, are dispensable for fertility and gametogenesis in mice.

Meiosis is a critical phase in the life cycle of sexually reproducing organisms. Chromosome numbers are halved during meiosis, which requires meiosis-specific modification of chromosome behaviour. Furthermore, suppression of transposons is particularly important during meiosis to allow the transmission of undamaged genomic information between generations. Correspondingly, specialized genome defence mechanisms and nuclear structures characterize the germ line during meiosis. Survival of mammalian spermatocytes requires that the sex chromosomes form a distinct silenced chromatin domain, called the sex body. An enigmatic spherical DNA-negative structure, called the meiotic dense body, forms in association with the sex body. The dense body contains small non-coding RNAs including microRNAs and PIWI-associated RNAs. These observations gave rise to speculations that the dense body may be involved in sex body formation and or small non-coding RNA functions, e.g. the silencing of transposons. Nevertheless, the function of the dense body has remained mysterious because no protein essential for dense body formation has been reported yet. We discovered that the polycomb-related sex comb on midleg-like 1 (SCML1) is a meiosis-specific protein and is an essential component of the meiotic dense body. Despite abolished dense body formation, Scml1-deficient mice are fertile and proficient in sex body formation, transposon silencing and in timely progression through meiosis and gametogenesis. Thus, we conclude that dense body formation is not an essential component of the gametogenetic program in the mammalian germ line

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.Meiotic cells deliberately break their DNA to allow chromosomes to swap genetic material. Here, authors reveal genetically separable pathways controlling the seeding and growth of chromosome-bound protein condensates responsible for DNA breaks

The role of two sex chromosome associated proteins, SCML1 and ANKRD31, in gametogenesis in mice

Meiosis is a specialized cell division that produces haploid cells (gametes) from diploid progenitors. During meiosis parental chromosomes (homologs) need to pair, synapse and eventually segregate. Faithful chromosome segregation depends on chromosome recombination. In the beginning of prophase I programmed double strand breaks (DSBs) are introduced in meiotic cells by SPO11 enzyme. DSBs are positioned at hotspot sites that are specified by that action of DNA-binding histone methyltransferase PRDM9. Specific enzymes act at the site of breaks to create 5’ single stranded DNA ends. With the assistance of the strand exchange proteins DMC1 and RAD51 these ends invade homologous DNA sequence and DSB repair is initiated. DSB repair can be completed either as a crossover (reciprocal exchange of DNA) or as a non-crossover. Crossover events lead to the formation of chiasmata between homologs and ensure proper segregation during the first meiotic division. An interesting feature in male meiosis is the XY chromosomes. The shared region between sex chromosomes is short and is called pseudoautosomal region (PAR). Due to their large non synapsed region, XY chromosomes need to be transcriptionally silenced. Thus they are covered with the phosphorylated histone variant H2AX (γH2AX) forming the so called sex body. PAR region has higher density of DSBs than autosomes and it had been shown that sex chromosomes undergo delayed homologous pairing. Nevertheless little is known how meiotic recombination is regulated in PAR region of sex chromosomes. In close proximity with sex body it has been found a structure named dense body (DB). There are few reports suggesting that DB contains RNAs/proteins but no DNA. Its role in meiosis was unclear because no structural component had been described. In the present thesis the role of two meiotic expressed genes is described. In our group after performing RNA screens we identified several genes that are highly expressed during meiotic prophase I. Based on the expression profile we selected polycomb-related sex comb on midleg like 1 (Scml1) gene and the ankyrin repeat domain 31 (Ankrd31) to study their role in mammalian meiosis.:List of figures i List of abbreviations ii 1. Introduction 1 1.1 Gametogenesis 1 1.2 Meiotic prophase I 2 1.2.1 Meiotic recombination 4 1.2.2 Regulation of meiotic recombination 7 1.2.2.1 Meiotic recombination hotspots and PRDM9 activity 7 1.2.2.2 Meiotic surveillance mechanisms 8 1.3 Unique properties of XY recombination 9 1.4 Sex chromatin associated structure: The dense body 10 1.5 Aim of the thesis 11 2. Publications 12 3. Discussion 92 4. Summary 98 5. References 102 Acknowledgements 108 Declarations 10

Chromosomal synapsis defects can trigger oocyte apoptosis without elevating numbers of persistent DNA breaks above wild-type levels

Generation of haploid gametes depends on a modified version of homologous recombination in meiosis. Meiotic recombination is initiated by single-stranded DNA (ssDNA) ends originating from programmed DNA double-stranded breaks (DSBs) that are generated by the topoisomerase-related SPO11 enzyme. Meiotic recombination involves chromosomal synapsis, which enhances recombination-mediated DSB repair, and thus, crucially contributes to genome maintenance in meiocytes. Synapsis defects induce oocyte apoptosis ostensibly due to unrepaired DSBs that persist in asynaptic chromosomes. In mice, SPO11-deficient oocytes feature asynapsis, apoptosis and, surprisingly, numerous foci of the ssDNA-binding recombinase RAD51, indicative of DSBs of unknown origin. Hence, asynapsis is suggested to trigger apoptosis due to inefficient DSB repair even in mutants that lack programmed DSBs. By directly detecting ssDNAs, we discovered that RAD51 is an unreliable marker for DSBs in oocytes. Further, SPO11-deficient oocytes have fewer persistent ssDNAs than wild-type oocytes. These observations suggest that oocyte quality is safeguarded in mammals by a synapsis surveillance mechanism that can operate without persistent ssDNAs.Deutsche Forschungsgemeinschaft (DFG) [TO421/3-1/2, TO421/5-1, TO421/6-1/2, TO421/7-1, TO421/8-1/2, TO421/10-1, TO421/11-1, TO421/12-1]; HFSP research grant [RGP0008/2015 to K.R., R.R., F.P., A.T.]; Ministry of Science, Innovation and Universities of Spain (MCIU/AEI/FEDER, EU) [RTI2018-099055-B-I00]; ‘Junta de Castilla y León’ of Spain (FEDER, EU) [CSI259P20 to P.A.S.S.]. Funding for open access charge: DFG, SLUB

Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse.

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse.

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2

Mouse ANKRD31 Regulates Spatiotemporal Patterning of Meiotic Recombination Initiation and Ensures Recombination between X and Y Sex Chromosomes

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