Hormad1 mutation disrupts synaptonemal complex formation, recombination, and chromosome segregation in mammalian meiosis

Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell-specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1-/-) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1-/- testes and ovaries as shown by the drastic decrease in the γH2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with cH2AX to the sex body during pachytene. BRCA1, ATR, and γH2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes γH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1-/- ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1-/- oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing. © 2010 Shin et al

The DSIF Subunits Spt4 and Spt5 Have Distinct Roles at Various Phases of Immunoglobulin Class Switch Recombination

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription of S regions and AID, expression array analysis suggests that Spt4 and Spt5 regulate a distinct subset of transcripts in CH12F3-2A cells. Curiously, Spt4 is critically important in suppressing cryptic transcription initiating from the intronic Sμ region. Depletion of Spt5 reduced the H3K4me3 level and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status and S region cleavage. H3K4me3 modification level thus correlated well with the DNA breakage efficiency. Therefore we conclude that Spt5 plays a role similar to the histone chaperone FACT complex that regulates H3K4me3 modification and DNA cleavage in CSR. Since Spt4 is not involved in the DNA cleavage step, we suspected that Spt4 might be required for DNA repair in CSR. We examined whether Spt4 or Spt5 is essential in non-homologous end joining (NHEJ) and homologous recombination (HR) as CSR utilizes general repair pathways. Both Spt4 and Spt5 are required for NHEJ and HR as determined by assay systems using synthetic repair substrates that are actively transcribed even in the absence of Spt4 and Spt5. Taken together, Spt4 and Spt5 can function independently in multiple transcription-coupled steps of CSR

Characterising large-scale chromatin looping in mouse meiotic prophase I

Meiotic prophase I pachytene meiocytes exhibit a highly characteristic chromatin architecture. Running the length of every chromosome, the chromatin is packaged into sequential loop arrays emanating from a proteinaceous core, known as the synaptonemal complex (SC). The configuration of these chromatin loop arrays, including loop density and positioning, has been proposed to significantly impact on the distribution of meiotic recombination, a key process in the promotion of the faithful segregation of homologous chromosomes at the first meiotic division. This relationship is primarily based on observations made in lower-order organisms, therefore this thesis sought to characterise the fundamental principles of meiotic chromatin organisation at the level of individual chromatin loops in mice. To investigate chromosome organisation in mouse meiosis, fluorescence in situ hybridisation (FISH) was conducted to map a single autosomal loop at the HoxA locus on chromosome 6 in pachytene spermatocytes. This approach defined a consistent ~1.3 Mb chromatin loop emanating from the SC. Higher resolution FISH analysis demonstrated that chromatids are tightly clustered when in proximity to the SC but become more separate as the chromatin extends into the loop. Furthermore, the topology of the HoxA loop was shown to be altered in cohesin mutant mice (Smc1β-/- and Smc1β-/-,1α), in which whole chromosome morphology is known to be disrupted, thus supporting the validity of the chromatin loop map and its cohesindependent regulation. To understand the role of transcription in the maintenance of meiotic chromatin loop architecture in pachytene spermatocytes, FISH analyses were performed following acute transcriptional inhibition. Inhibition led to no significant change in SC length. However, the total nuclear area was substantially reduced as autosomal chromatin was drawn closer to the SC, with no significant change in chromatin compaction. A relatively subtle response was seen on the grossly transcriptionally silent sex chromosomes. On RNase treatment, a similar, yet less substantial, change in chromosome morphology was observed. Collectively, these findings demonstrate that chromatin loop organisation is dependent on a transcriptional component. In mice, the frequency of meiotic crossovers (COs; a product of meiotic recombination) is sexually dimorphic, with an approximately two-fold reduction in male CO frequency relative to females. FISH-based analyses revealed that chromatin loop extensions are significantly longer and chromatid separation substantially greater in spermatocytes, relative to oocytes. Chromatid separation was also found to be significantly greater at two CO hotspots in juvenile males, which experience a reduction in inter-homolog interactions compared to their adult counterparts. Cumulatively, these data indicate that differences in the frequency of inter-homolog interactions and COs correspond with differences in the relative spatial positioning of chromatids. Together, these findings advance present understanding of the fundamental features of meiotic chromatin architecture at the level of individual chromatin loops in murine meiocytes. Furthermore, this research provides insight into the nuclear environment in which meiotic recombination occurs, which ultimately has wide-reaching clinical implications relating to infertility, specific developmental disorders and spontaneous miscarriage, in which the legitimate segregation of meiotic chromosomes is perturbed

Characterization of INO80 Chromatin-Remodeling Activity During Germ Cell Development

The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale chromatin modifications within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for post-meiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well-described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in sterility of the animal due to the failure to repair DNA damage during meiotic recombination. Specifically, we observed a disruption in the Fanconi Anemia repair pathway, where early elements of the pathway were present on the chromosomal axes while BRCA1 remained absent. From these observations, we propose a model where INO80 activity is required to prepare the chromatin landscape local to the break site, creating the physical space necessary for the localization of downstream DNA repair proteins. In conclusion, this work provides deeper insight in to the critical nature of chromatin-remodeling activity for spermatogenesis, particularly during meiotic recombination, and a foundation for future studies into the genomic functions of the INO80 complex.Doctor of Philosoph

Understanding crossover control using A. thaliana and S. cerevisiae

During meiosis, homologous chromosomes pair, synapse, and recombine to facilitate accurate chromosome segregation in meiosis I. Meiotic recombination is facilitated by programmed double-strand breaks that can be repaired either as crossovers or non-crossovers. In most organisms, crossover distribution along chromosomes is non-random in that crossovers are more evenly spaced than null expectations. The inhibition of closely spaced events is known as interference. Despite the fact that interference was originally observed almost a century ago, fundamental questions regarding its underlying mechanisms still exist. I discuss key unanswered questions regarding interference as well as the most commonly referenced models that have been proposed to explain the interference mechanism. We have developed a visual assay (the FTL system) for the detection of crossovers, gene conversions and interference in A. thaliana. This assay involves monitoring the segregation of fluorescent proteins in the pollen grains of qrt1 mutants. qrt1 mutants exhibit pollen tetrads i.e. the fusion of the four meiotic products, which allows for advanced statistical analyses previously only available in yeasts. The development and applications of this system are discussed. Humans, S. cerevisiae and A. thaliana have at least two pathways for producing crossovers, which include a primary pathway that is subject to interference and a secondary pathway that is interference-insensitive. Using the FTL system, we demonstrate that AtMUS81 is a mediator of the interference-insensitive pathway in A. thaliana. Atmus81 mutants are sensitive to a wide range of DNA damaging agents and exhibit decreased pollen viability and crossover frequency. The remaining crossovers in the Atmus81 mutant are subject to interference. Meiotic recombination occurs in the context of chromatin and chromatin context is often invoked to explain why recombination occurs preferentially in some genomic regions. Using a technique called FAIRE, we demonstrate that double-strand break hotspots and regions of open chromatin have a positive but complex association in S. cerevisiae. We also show that subtelomeric border regions and regions surrounding tRNA genes are enriched for meiosis-specific open chromatin. Centromeres exhibit constitutive enrichment of open chromatin

Investigating meiotic DNA double-strand break interference in S. cerevisiae

Meiotic recombination is initiated by the programmed formation of DNA double-strand breaks (DSBs), however the presence of these types of lesions can easily compromise the integrity of the cell. As such, cells have developed multiple regulatory pathways to control when, how many and where DSBs are formed in order to ensure a proper timing (temporal regulation), number (quantitative regulation) and distribution (spatial regulation) of DSBs across the genome. Among the pathways involved in shaping the distribution of DSBs, Tel1-dependent DSB interference has emerged as one of the core negative feedbacks that pattern the DSB landscape. However, how Tel1-dependent DSB interference operates in space and what features are involved is poorly understood. The work presented in this thesis uses: (i) specific techniques to study Tel1-dependent DSB interference (Chapter 3) and the influence of the chromosome structure on Tel1-dependent DSB interference using meiotic sister chromatid cohesin rec8Δ mutants (Chapter 4) at specific genomic locations; (ii) a novel method to map Spo11-DSBs genome wide (CC-seq) to study the effect that Tel1 exerts on a genome-wide scale (Chapter 5 and 6); (iii) a simulation platform that mimics the effect that DSB interference would impose over the distribution of DSBs in silico (MATLAB: DSBSim) to investigate the process of Tel1–dependent DSB interference (Chapter 6) and to explore the features—such as Tel1 kinase activity, chromosome structure and chromosome compaction—that influence such a phenomenon (Chapter 7). Collectively, this thesis further elucidates the mechanisms underpinning the process of Tel1-dependent DSB interference and therefore contribute to our understanding of how DSBs are spatially regulated in S. cerevisiae during meiosis

Investigating the spatial regulation of meiotic recombination in S. cerevisiae

In order for a species to engage in and reap the evolutionary benefits of sexual reproduction, a subset of cells in each individual must undergo a complex ordeal known as meiosis—a specialised cell division. By halving the genome content and “shuffling the deck”, meiosis generates genetically diverse haploid gametes (eggs, sperm) or spores from diploid cells. Such a monumental task is by no means easy or risk free: during the meiotic programme, cells intentionally damage their own genomes through widespread induction of DNA double-strand breaks (DSBs) in order to initiate homologous recombination—a DNA-repair process—and subsequent crossover (CO) formation. The success of meiosis is, however, not left up to chance. Rather, a complicated web of regulation acts at multiple stages to ensure this dangerous tradeoff pays dividends. Notably, the spatial pattern of meiotic recombination across the genome is complex and non-random. Whilst ultimately stochastic in nature, recombination events within any given meiotic cell display relatively even distributions along each chromosome—a phenomenon mediate by processes of “interference” acting at two key stages in meiosis: DSB and CO formation. Despite wide ranging historical observation, relatively little is known about how either form of interference is accomplished. Genome-wide mapping of recombination within S. cerevisiae has, however, provided a unique opportunity to investigate the underlying mechanisms. By computationally and mathematically analysing genome-wide data, work presented throughout this thesis seeks to: (i) investigate CO distribution and CO interference within various DNA damage response and DNA repair mutants (Tel1ATM, Mec1ATR, Rad24, Msh2) (Chapter 2) (ii) develop novel approaches to DSB mapping (Chapter 3) (iii) characterise the hyperlocal regulation of DSB formation (Chapter 3) and (iv) examine the mechanics of DSB interference (Chapter 4). Moreover, widely applicable simulation platforms for investigating DSB and CO formation have been developed (Chapter 2, 4). Collectively, this thesis further elucidates the mechanisms that underpin the spatial regulation of meiotic recombination in S. cerevisiae

Regulation and function of the Synaptonemal Complex during meiosis in Saccharomyces cerevisiae

The Synaptonemal Complex (SC) is a proteinaceous structure that connects homologous chromosomes lengthwise during meiotic prophase. In budding yeast, the SC consists of two parallel axes that become connected by the central element protein, Zip1 that extends along the chromosome axes (Sym, Engebrecht et al. 1993). Extension of the SC is coordinated to crossover formation by a group of proteins known as the ‘ZMM’s (Zip1, Zip2, Zip3, Zip4, Msh4, Msh5 and Mer3) (Borner, Kleckner et al. 2004). Work outlined here demonstrates a role for the mismatch repair paralogue, Msh4 in preventing SC extension from being de-coupled from crossover formation. Furthermore, increased temperature serves as a positive effector for this decoupling. These findings suggest that SC extension is highly regulated to ensure that it is coupled with crossing over. As well as its role in crossover formation (Storlazzi, Xu et al. 1996), the work outlined here demonstrates an independent role for Zip1 in promoting the segregation of non-exchange chromosome pairs (NECs). Zip1 pairs the centromeres of NECs in pachytene through to metaphase I, where it aids their segregation at the first meiotic division. The localisation and function of Zip1 at the centromeres of non-exchange chromosomes depends on Zip3 and Zip2, respectively. Zip1 is observed at the centromeres of all chromosomes following SC disassembly through to the first meiotic division, where it promotes the segregation of exchange pairs also. A model is suggested whereby Zip1 promotes the segregation of both non-exchange and exchange chromosome pairs by tethering homologous centromeres throughout meiotic prophase. Finally, a parallel pathway for NEC segregation is also described that depends upon the spindle checkpoint component, Mad3. When both ZIP1 and MAD3 are deleted, NECs segregate at random