Periodic Reporting for period 2 - CrossOver (Meiotic crossing-over: from spatial distribution to in situ chromosomal architecture)
Reporting period: 2023-07-01 to 2024-12-31
To haploidize their genome, sexually reproducing organisms use a specialized cell division program called meiosis. This process includes one round of DNA replication followed by two consecutive rounds of chromosome segregation: meiosis I and II. While meiosis II is similar to mitosis, meiosis I involves unique events. In most organisms, the physical linkage and separation of maternal and paternal chromosomes require homologous recombination and crossing-over. Crossovers, which occur at different chromosomal positions in different cells, tend to be widely and evenly spaced along chromosomes due to a phenomenon called crossover interference.
In the past 30 years, significant progress has been made in understanding the sequence of molecular events leading to crossing-over. However, it is still unclear how cells spatially regulate these events to achieve precise crossover patterning.
This research aims to address this fundamental question by developing two novel approaches to explore key aspects of meiotic recombination with unprecedented detail:
1. We are developinmg new methodology (HJmap) to create a genome-wide map of Holliday junctions, the central recombination intermediates that mark future crossover sites. This will help us understand how chromosomal context influences crossing-over globally.
2. Using electron cryotomography, we are visualizing the architecture of crossover-designated recombination intermediates in situ and in 3D. This is expecedt to reveal how structural features of chromosomes affect the crossing-over process locally.
By understanding how cells implement genetic exchange through crossing-over, this research will shed light on the molecular basis of heredity—the passing of traits from parents to offspring. This knowledge is crucial for society as it can enhance our understanding of genetic diseases and contribute to advancements in biotechnology.
In the past 30 years, significant progress has been made in understanding the sequence of molecular events leading to crossing-over. However, it is still unclear how cells spatially regulate these events to achieve precise crossover patterning.
This research aims to address this fundamental question by developing two novel approaches to explore key aspects of meiotic recombination with unprecedented detail:
1. We are developinmg new methodology (HJmap) to create a genome-wide map of Holliday junctions, the central recombination intermediates that mark future crossover sites. This will help us understand how chromosomal context influences crossing-over globally.
2. Using electron cryotomography, we are visualizing the architecture of crossover-designated recombination intermediates in situ and in 3D. This is expecedt to reveal how structural features of chromosomes affect the crossing-over process locally.
By understanding how cells implement genetic exchange through crossing-over, this research will shed light on the molecular basis of heredity—the passing of traits from parents to offspring. This knowledge is crucial for society as it can enhance our understanding of genetic diseases and contribute to advancements in biotechnology.
First Work Package (Aim 1): Developing New Methods for Cleaving and Mapping Double Holliday Junctions (dHJs)
Our first goal focuses on creating new techniques to specifically cut and map double Holliday junctions (dHJs), which are crucial intermediates in meiotic recombination. We have made significant progress in understanding how the cleavage of dHJs affects cells during meiosis. We have already submitted a manuscript showing that dHJs are essential for maintaining the stability of the synaptonemal complex during the first phase of meiosis (Henggeler et al., submitted).
Additionally, we have made substantial advancements in mapping dHJs across the genome, especially using a technique called GLOE-seq after inducing the resolution of dHJs. GLOE-seq has shown that DNA nicks introduced during the resolution of dHJs can be mapped near DNA double-strand break hotspots. We are now combining this method with mutations in certain factors to see how they affect the number and location of dHJs. The upcoming months are promising, as we expect to finalize our initial results for publication. We have also developed an alternative method for mapping dHJs using a peptide that binds to Holliday junctions. This work is nearly complete, and we are preparing the figures for our first publication describing this new approach.
Second Work Package (Aim 2): Visualizing Recombining Chromosomes with CryoET
Our second goal involves using cryo-electron tomography (cryoET) to visualize chromosomes during recombination. Since our initial proposal, we have made significant progress. We have developed a workflow to identify filament-forming proteins in cell spreads. This has revealed that the filaments seen in cryoET images, which are specific to meiosis, are actually polymers of metabolic enzymes, not related to the synaptonemal complex as initially thought. These findings have been published in two studies (Hugener et al., Cell, 2024; Wettstein et al., Dev Cell, 2024). This unexpected discovery has opened up a new area of research.
We have learned a lot about the capabilities and limitations of cryoET in studying meiotic cells. We found that imaging the synaptonemal complex in cryoFIB milled sections is very challenging. However, we successfully established cryoET imaging of cell spreads and combined it with correlative light and electron microscopy (CLEM). This has allowed us to capture detailed snapshots of the synaptonemal complex at different stages of its assembly. We have also created several new strains of cells with fluorescently tagged key recombination factors, which will help us locate recombination nodules using CLEM.
Our first goal focuses on creating new techniques to specifically cut and map double Holliday junctions (dHJs), which are crucial intermediates in meiotic recombination. We have made significant progress in understanding how the cleavage of dHJs affects cells during meiosis. We have already submitted a manuscript showing that dHJs are essential for maintaining the stability of the synaptonemal complex during the first phase of meiosis (Henggeler et al., submitted).
Additionally, we have made substantial advancements in mapping dHJs across the genome, especially using a technique called GLOE-seq after inducing the resolution of dHJs. GLOE-seq has shown that DNA nicks introduced during the resolution of dHJs can be mapped near DNA double-strand break hotspots. We are now combining this method with mutations in certain factors to see how they affect the number and location of dHJs. The upcoming months are promising, as we expect to finalize our initial results for publication. We have also developed an alternative method for mapping dHJs using a peptide that binds to Holliday junctions. This work is nearly complete, and we are preparing the figures for our first publication describing this new approach.
Second Work Package (Aim 2): Visualizing Recombining Chromosomes with CryoET
Our second goal involves using cryo-electron tomography (cryoET) to visualize chromosomes during recombination. Since our initial proposal, we have made significant progress. We have developed a workflow to identify filament-forming proteins in cell spreads. This has revealed that the filaments seen in cryoET images, which are specific to meiosis, are actually polymers of metabolic enzymes, not related to the synaptonemal complex as initially thought. These findings have been published in two studies (Hugener et al., Cell, 2024; Wettstein et al., Dev Cell, 2024). This unexpected discovery has opened up a new area of research.
We have learned a lot about the capabilities and limitations of cryoET in studying meiotic cells. We found that imaging the synaptonemal complex in cryoFIB milled sections is very challenging. However, we successfully established cryoET imaging of cell spreads and combined it with correlative light and electron microscopy (CLEM). This has allowed us to capture detailed snapshots of the synaptonemal complex at different stages of its assembly. We have also created several new strains of cells with fluorescently tagged key recombination factors, which will help us locate recombination nodules using CLEM.
We have already made significant breakthroughs in our project, including the development of FilamentID (Hugener et al., 2024). By the end of the project, we expect to have developed at least one new method for monitoring Holliday junctions across the entire genome and to have gained insights into the mechanisms that control their positioning. Overall, we anticipate making key discoveries that will enhance our understanding of how maternal and paternal chromosomes recombine during meiosis.