Homologous recombination, sister chromatid cohesion, and chromosome condensation in mammalian meiosis

In the life cycle of sexually reproducing eukaryotes, haploid and diploid generations of cells alternate. Two types of cell division occur in such a life cycle: mitosis and meiosis. They are compared in chapter 1 . Haploid and diploid cells can multiply by mitoses. A mother cell undergoing mitosis produces two daughter cells each with the same chromosome complement and combination of alleles as the mother cell. Only diploid cells can undergo meiosis. A cell undergoing meiosis produces four haploid cells with different combinations of alleles. Meiosis thus contributes to genetic diversity and is responsible for the transition from the diploid to the haploid phase in the life cycle. Meiosis has probably evolved from mitosis by modifications of the cell cycle, chromosome behavior and recombinational repair.The cell cycle has been modified because in meiosis two nuclear divisions (meiosis I and II) follow a single round of DNA-replication (S-phase), whereas in mitosis, one S-phase is followed by one division.Chromosome behavior has been modified in several respects. In meiotic prophase (prophase I), homologous chromosomes pair and associate by a zipper like structure, the synaptonemal complex (SC). SCs consist of two axial elements (AEs), which each support the two sister chromatids of one homolog. During the course of prophase I, numerous transverse filaments connect the axial elements of the two homologs along their entire length, a process called synapsis. Within the context of SCs, homologous recombination (HR) takes place between the paired chromosomes. At meiosis I, homologous chromosomes disjoin, which brings about the reduction in ploidy level (from diploid to haploid); at meiosis II, the sister chromatids separate, like in mitosis. Proper chromosome segregation in the two successive meiotic divisons is ensured by a modification in the regulation of sister chromatid cohesion. In mitosis, cohesion is released at once all along the chromosomes at the metaphase to anaphase transition. In meiosis, in contrast, cohesion is lost in two steps: chromosome arm cohesion is lost at meiosis I, and centromeric cohesion at meiosis II.Meiotic HR has probably evolved from mitotic homologous recombinational repair (HRR), and this has also been accompanied by several modifications: (1) in contrast to mitotic HRR, meiotic HR is actively induced by the cell; (2) meiotic HR occurs at a 100- to 1000-fold higher frequency than mitotic HRR; and (3) meiotic HR prefers a non-sister chromatid of the homologous chromosome as template, whereas mitotic HRR prefers the sister chromatid. In this thesis I focus on these modifications that the mitotic cell cycle must have undergone to yield a reliable meiotic cycle in which chromosomes are properly duplicated and segregated, and retain their integrity.In chapter 2 , we describe the immunolocalization of two proteins that are involved in the early steps of meiotic recombination, Rad50 and Mre11, in spermatocytes of mouse and rat. We found a similar localization of the two proteins in spermatocytes, which we expected, because Rad50 and Mre11 make part of the same protein complex. In early prophase I (pre-leptotene until early zygotene) Rad50 and Mre11 were present throughout the nucleus. In slightly later stages (mid and late zygotene), the two proteins concentrated in distinct domains, around segments of AEs that were not yet connected by transverse filaments. In the next stage (pachytene), both proteins disappeared from the nucleus except from the pair of sex chromosomes (XY-bivalent), where they remained until the end of diplotene. Unlike other proteins involved in meiotic recombination, Rad50 and Mre11 did not associate visibly with the AEs. We propose that Mre11 and Rad50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the Rad50 and Mre-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place.In mitotic cells, Mre11 and Rad50 are involved in two major pathways of double-strand DNA-break repair (DSB repair), namely nonhomologous endjoining (NHEJ) and homologous recombinational repair (HRR). NHEJ connects DNA-ends irrespective of their sequence; it is an error-prone DSB repair mechanism, which predominates in mitotic cells. In contrast, mitotic HRR and meiotic HR repair DSBs accurately. In meiosis, DSBs should be repaired by HR, not by NHEJ. We wondered what determines the choice between the two repair pathways. Therefore we analysed the interactions engaged by Mre11 in mammalian somatic and meiotic cells ( chapter 3 ). We found a physical interaction between Mre11 and Ku70, a protein that is essential for NHEJ, but not for HR or HRR. Mre11 depended on Ku70 for the formation of subnuclear foci that are assembled upon irradiation of mitotic cells, and that are supposed to represent DNA repair complexes. Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis. In the mouse testis, Mre11 and Ku70 colocalised in nuclei of somatic cells and in the XY bivalent of pachytene spermatocytes. However, in early meiotic prophase, when meiotic recombination is initiated, Mre11 was abundant (like Rad50, see Chapter 2 ), while Ku70 was not detectable. We propose that Ku70 acts as a switch between the two DSB repair pathways. If present, Ku70 would destine DSBs for NHEJ by binding to DNA-ends and attracting other factors for NHEJ, including Mre11. If absent, the DNA-ends and Mre11 can participate in the meiotic HR pathway.In chapters 4 to 6, we focus on meiotic adaptations of sister chromatid cohesion. Cohesion is ensured by a four-protein complex, cohesin. In chapter 4 we analyse the localization of two components of the cohesin complex, Smc1 and Smc3, relative to the AEs of SCs by immunofluorescence. For the recogition of AEs, we used antibodies against two AE-components, Scp2 and Scp3. Smc1 and Smc3 localized in a beaded structure along the AEs. Furthermore, we found that Scp2 and Scp3 co-immuneprecipitated with Smc3 from testis extracts. We also showed interaction between Smc1 and Scp3, using immunoaffinity chromatography. Together, these data suggest interactions between cohesin components (cohesins) and AE-components in vivo.In chapter 5, we describe the identification of a meiosis-specific variant of Smc1, Smc1b. Smc1bis highly homologous to Smc1 (further designated as Smc1a) except for a unique C-terminal, basic, DNA binding motif. Smc1bis specifically expressed in the testis and co-immuneprecipitates with Smc3 from testis nuclear extracts. Immunolocalization of Smc1bin sections of rat testis revealed that Smc1bis localized along the AEs throughout prophase I. In spread spermatocyte nuclei, we found that Smc1b, like Scp3, is present along AEs from leptotene until diakinesis, when AEs disassemble. Smc1bremained present in the centromeric region until metaphase II, together with Scp3, and disappeared at the onset of anaphase II. This localization pattern of Smc1bis consistent with a role of the protein in maintaining sister chromatid cohesion between centromeres until anaphase II. Possibly, a meiosis-specific isoform of Smc1awas needed in mammals for the modified regulation of cohesion in meiosis, and/or Smc1bwas required for the assembly of protein complexes involved in meiotic HR.In chapter 6 , we describe the localization of another meiosis-specific cohesin, Rec8, relative to known AE-components. Rec8 appeared shortly before premeiotic S-phase in the nucleus and formed AE-like structures (Rec8-AEs) in the absence of Smc1b, Smc3, Scp2 and Scp3. In the subsequent stage of prophase I (leptotene) the Smcs and Scps appeared, and localized along the Rec8 AEs. Initially, they formed dots along Rec8-AEs, which later extended and fused until they lined Rec-AEs along their length. Rec8 persisted along the AEs throughout prophase I. In metaphase I, Smc1b, Smc3, Scp2 and Scp3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, Rec8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We conclude that the first steps in AE assembly do not require Smc1b, Smc3, Scp2 and Scp3. We propose that Rec8 provides a basis for AE-formation. Furthermore, Smc1b, Smc3, Scp2 and Scp3 cannot provide cohesion during the earliest stages of meiotic prophase, nor arm cohesion during metaphase I. We propose that Rec8 then provides cohesion. Furthermore, we found evidence for interaction of Rec8 with proteins involved in meiotic HR: Rad50 and Rad51 and/or Dmc1 co-immunoprecipitate with Rec8. Possibly, Rec8 provides also a basis for assembly of meiotic recombination complexes. We hypothesize that the replacement of mitotic cohesin Scc1 by meiotic cohesin Rec8 was necessary to allow the assemly of AEs. The AEs in turn were required both for the altered regulation of cohesion and for blocking the sister chromatid as template for repair of meiotic DSBs by HR.From chapters 4-6 it has become clear that cohesins form the basis of AEs in meiosis. AEs are single axial structures that are shared by the two sister chromatids. At the beginning of diakinesis, this shared chromatid axis disappears, and at the end of diakinesis, two axes appear, which each support one individual chromatid (I call these "single-chromatid axes"). I wandered if cohesin plays a role in the establishment of the single-chromatid axis at the end of diakinesis. I therefore wrote an overview of chromosomal axial structures in mitosis and meiosis ( chapter 7 ). I included in my overview the literature from the pre-immunocytochemistry era, when axes were visualized by silver staining. Silver staining reveals axial structures in mitosis and meiosis. In mitosis, a shared sister chromatid axis, (one axis supporting two sister chromatids) is not normally seen, whereas many publications have appeared about the mitotic single-chromatid axis. Some protein components which make part of the mitotic single-chromatid axis have been identified, as well as the DNA sequences (the scaffold attachment regions or SARs) by which chromatin is attached to this axis. with respect to meiosis, in contrast, many publications have appeared about the silver-stainable shared chromatid axis (which should correspond to the AE), while little is known about the meiotic single-chromatid axis. Although no hard conclusions could be drawn about the role of cohesin in the establishment of the single-chromatid axis in mitosis or meiosis, many ideas for useful and new experiments came up, which can fill the gap in today's knowledge of chromosomal axial structures.</p

Silencing markers are retained on pericentric heterochromatin during murine primordial germ cell development

Background: In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins l

corona Is Required for Higher-Order Assembly of Transverse Filaments into Full-Length Synaptonemal Complex in Drosophila Oocytes

The synaptonemal complex (SC) is an intricate structure that forms between homologous chromosomes early during the meiotic prophase, where it mediates homolog pairing interactions and promotes the formation of genetic exchanges. In Drosophila melanogaster, C(3)G protein forms the transverse filaments (TFs) of the SC. The N termini of C(3)G homodimers localize to the Central Element (CE) of the SC, while the C-termini of C(3)G connect the TFs to the chromosomes via associations with the axial elements/lateral elements (AEs/LEs) of the SC. Here, we show that the Drosophila protein Corona (CONA) co-localizes with C(3)G in a mutually dependent fashion and is required for the polymerization of C(3)G into mature thread-like structures, in the context both of paired homologous chromosomes and of C(3)G polycomplexes that lack AEs/LEs. Although AEs assemble in cona oocytes, they exhibit defects that are characteristic of c(3)G mutant oocytes, including failure of AE alignment and synapsis. These results demonstrate that CONA, which does not contain a coiled coil domain, is required for the stable ‘zippering’ of TFs to form the central region of the Drosophila SC. We speculate that CONA's role in SC formation may be similar to that of the mammalian CE proteins SYCE2 and TEX12. However, the observation that AE alignment and pairing occurs in Tex12 and Syce2 mutant meiocytes but not in cona oocytes suggests that the SC plays a more critical role in the stable association of homologs in Drosophila than it does in mammalian cells

SPO11-Independent DNA Repair Foci and Their Role in Meiotic Silencing

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11YF/YF), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11YF/YFand Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number

An ES-Like Pluripotent State in FGF-Dependent Murine iPS cells

Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions

Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin) machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation

A Novel Mouse Synaptonemal Complex Protein Is Essential for Loading of Central Element Proteins, Recombination, and Fertility

The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes

The Radially Swollen 4 Separase Mutation of Arabidopsis thaliana Blocks Chromosome Disjunction and Disrupts the Radial Microtubule System in Meiocytes

The caspase-family protease, separase, is required at the onset of anaphase to cleave the cohesin complex that joins replicated sister chromatids. However, in various eukaryotes, separase has acquired additional and distinct functions. A single amino-acid substitution in separase is responsible for phenotypes of the Arabidopsis thaliana mutant, radially swollen 4 (rsw4). This is a conditional mutant, resembling the wild type at the permissive temperature (∼20°C) and expressing mutant phenotypes at the restrictive temperature (∼30°C). Root cells in rsw4 at the restrictive temperature undergo non-disjunction and other indications of the loss of separase function. To determine to what extent separase activity remains at 30°C, we examined the effect of the mutation on meiosis, where the effects of loss of separase activity through RNA interference are known; and in addition, we examined female gametophyte development. Here, we report that, at the restrictive temperature, replicated chromosomes in rsw4 meiocytes typically fail to disjoin and the cohesin complex remains at centromeres after metaphase. Meiotic spindles appear normal in rsw4 male meiocytes; however the mutation disrupts the radial microtubule system, which is replaced by asymmetric arrays. Surprisingly, female gametophyte development was relatively insensitive to loss of separase activity, through either rsw4 or RNAi. These effects confirm that phenotypes in rsw4 result from loss of separase activity and establish a role for separase in regulating cell polarization following male meiosis

Mouse HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins, are depleted from synapsed chromosome axes with the help of TRIP13 AAA-ATPase

Meiotic crossovers are produced when programmed double-strand breaks (DSBs) are repaired by recombination from homologous chromosomes (homologues). In a wide variety of organisms, meiotic HORMA-domain proteins are required to direct DSB repair towards homologues. This inter-homologue bias is required for efficient homology search, homologue alignment, and crossover formation. HORMA-domain proteins are also implicated in other processes related to crossover formation, including DSB formation, inhibition of promiscuous formation of the synaptonemal complex (SC), and the meiotic prophase checkpoint that monitors both DSB processing and SCs. We examined the behavior of two previously uncharacterized meiosis-specific mouse HORMA-domain proteins-HORMAD1 and HORMAD2-in wild-type mice and in mutants defective in DSB processing or SC formation. HORMADs are preferentially associated with unsynapsed chromosome axes throughout meiotic prophase. We observe a strong negative correlation between SC formation and presence of HORMADs on axes, and a positive correlation between the presumptive sites of high checkpoint-kinase ATR activity and hyper-accumulation of HORMADs on axes. HORMADs are not depleted from chromosomes in mutants that lack SCs. In contrast, DSB formation and DSB repair are not absolutely required for depletion of HORMADs from synapsed axes. A simple interpretation of these findings is that SC formation directly or indirectly promotes depletion of HORMADs from chromosome axes. We also find that TRIP13 protein is required for reciprocal distribution of HORMADs and the SYCP1/SC-component along chromosome axes. Similarities in mouse and budding yeast meiosis suggest that TRIP13/Pch2 proteins have a conserved role in establishing mutually exclusive HORMAD-rich and synapsed chromatin domains in both mouse and yeast. Taken together, our observations raise the possibility that involvement of meiotic HORMA-domain proteins in the regulation of homologue interactions is conserved in mammals

Interplay between Synaptonemal Complex, Homologous Recombination, and Centromeres during Mammalian Meiosis

The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC–dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC–dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post–SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1−/− implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes centromere and chiasma function