Periodic Reporting for period 4 - 3D-loop (Mechanism of homology search and the logic of homologous chromosome pairing in meiosis)
Reporting period: 2024-07-01 to 2024-12-31
Homologous recombination (HR) is a universal DNA double-strand breaks repair pathway that promotes cell viability in genotoxic conditions and suppresses tumor formation in humans. HR also performs the genetic and mechanical work underlying the recognition, pairing and HR of parental homologous chromosomes during canonical meiosis, and is thus at the basis of sexual reproduction in most eukaryotes. The both depend on HR’s ability to identify and use an intact homologous DNA molecule as a repair template. How does the break site achieve accurate and efficient identification of a single homologous donor in the genome and nucleus? How is this mechanism regulated to achieve homologs attachment in meiosis? The goal of “3D-loop” is to tackle these two long lasting conundrums, in the model eukaryote S. cerevisiae.
The first goal of 3D-loop is to characterize the multi-step and multi-scale process of homology search in cells, which we presume involves two rounds of homology assessment. A first round efficiently selects for semi-homologous DNA molecules. A second round increases the stringency of the donor selection process. The two are separated by a DNA strand invasion reaction. The identification and effective competition between homologous regions is dictated by the probability of their spatial encounter. Therefore, we aim at defining both the molecular and spatial determinants of these two rounds of homology assessment. This primarily experimental work will feed into a computational framework of homology search, whose purpose is to integrate different types of molecular and contact genomic data in order to define in quantitative terms the respective contributions of the various factors involved in homology search.
The second goal of 3D-loop is to define the nature of the meiosis-specific regulation of the homology search process that results in three unexplained, yet central phenomena for sexual reproduction:
• the homolog bias: i.e. the preferred usage of the homolog rather than the “by default” sister chromatid.
• the obligatory crossover, which ensures at least one attachment and HR point per pair of homologs.
• the crossover interference; the inhibition of crossover designation in the vicinity of an already designated crossover.
To this end, we aim at simultaneously tracking chromatin folding, pairing, as well as the individual HR reaction in a population of S. cerevisiae cells undergoing synchronous meiosis. We developed an ambitious experimental system to achieve such multi-scale study, so as to investigate the role of chromatin folding in regulating the HR reaction and homolog pairing in meiosis.
The goal of 3D-loop is thus to define both the basic mechanism and the regulations of homology search in cells, and their interplay with spatial genome organization.
The first goal of 3D-loop is to characterize the multi-step and multi-scale process of homology search in cells, which we presume involves two rounds of homology assessment. A first round efficiently selects for semi-homologous DNA molecules. A second round increases the stringency of the donor selection process. The two are separated by a DNA strand invasion reaction. The identification and effective competition between homologous regions is dictated by the probability of their spatial encounter. Therefore, we aim at defining both the molecular and spatial determinants of these two rounds of homology assessment. This primarily experimental work will feed into a computational framework of homology search, whose purpose is to integrate different types of molecular and contact genomic data in order to define in quantitative terms the respective contributions of the various factors involved in homology search.
The second goal of 3D-loop is to define the nature of the meiosis-specific regulation of the homology search process that results in three unexplained, yet central phenomena for sexual reproduction:
• the homolog bias: i.e. the preferred usage of the homolog rather than the “by default” sister chromatid.
• the obligatory crossover, which ensures at least one attachment and HR point per pair of homologs.
• the crossover interference; the inhibition of crossover designation in the vicinity of an already designated crossover.
To this end, we aim at simultaneously tracking chromatin folding, pairing, as well as the individual HR reaction in a population of S. cerevisiae cells undergoing synchronous meiosis. We developed an ambitious experimental system to achieve such multi-scale study, so as to investigate the role of chromatin folding in regulating the HR reaction and homolog pairing in meiosis.
The goal of 3D-loop is thus to define both the basic mechanism and the regulations of homology search in cells, and their interplay with spatial genome organization.
The project led to a 3 high-profile primary research publications, 2 preprints currently in revision, a methodological publication, a patent, and a software.
We investigated how the break and the donor site can get together in the nuclear space, and the role of various factors in regulating this access. We showed we Hi-C and functional assays that the loop organization of chromatin by a conserved protein complex called Cohesin initially impinged on homology search by regulating the “visibility” of the genome by the broken region. We could delineate various factors spatially organizing the broken region and localizing cohesin around the break site. This work thus uncovered, and identified functions for, the local and genome-wide spatial chromatin reorganization occurring in response to a DNA break. These advances led to a publication (Piazza, Bordelet et al. 2021) and a conceptual review (Savocco & Piazza, 2021). They were also expanded to study the role of loop formation by condensin in the repair of programmed DNA break integral to mating-type switching in yeast (Piveteau et al. BioRxiv 2024). Realizing the technical limitations of Hi-C, we developed a method to directly detect contacts made by single-stranded DNA (the active entity during homology search). The resulting ssHi-C methodology caps our effort to chart all HR steps in cells, and can be straightforwardly applied to study various DNA metabolic processes in any organism (Patent submitted in 2023). This methodology enabled revealing the mechanism that underlies the expansion of homology search genome-wide, and that resides in the assembly of long and rigid Rad51-ssDNA filaments (Dumont et al. Mol. Cell 2024). Coupling of these Hi-C methodologies and functional HR assays is proving extremely informative to define the molecular functions of several HR proteins in homology search within this new framework.
In parallel we addressed the functional role of various molecular actors involved in the early stages of HR and participating of the fidelity of the pathway. It entailed methodological improvements (Rietz et al., JoVE 2022). We developed an exquisitely sensitive assay for detection of rare chromosomal translocations, which enables studying the kinetics of their formation and the role of essential factors in HR-mediated genomic instability (Reitz et al. Genes Dev 2023). We also revealed the control exerted by transcription by RNA Polymerase II on early DNA joint molecules formed during HR, the mechanism of this interference, and its role in promoting genome stability (Djeghmoum & Piazza, BioRxiv 2025). Finally, we developed a stochastic model of HR, which we use to infer the quantitative contributions of the various chromatin and HR factors partaking in the search process.
The conjoined study of HR and chromatin organization in meiosis required the assembly of a 150 kb synthetic chromosomal region. We obtained a fully debugged synthetic region. This genetic engineering feat allowed us for the first time to map at high resolution chromatin folding along large regions of individual homologs, and determine their trans interactions profile in wild-type cells. This new-generation experimental system opens the way to the rationale study of the role of chromatin structure in regulating HR and homolog pairing in meiosis, through controlled cis and trans perturbations. It will give rise to a high-impact publication.
We investigated how the break and the donor site can get together in the nuclear space, and the role of various factors in regulating this access. We showed we Hi-C and functional assays that the loop organization of chromatin by a conserved protein complex called Cohesin initially impinged on homology search by regulating the “visibility” of the genome by the broken region. We could delineate various factors spatially organizing the broken region and localizing cohesin around the break site. This work thus uncovered, and identified functions for, the local and genome-wide spatial chromatin reorganization occurring in response to a DNA break. These advances led to a publication (Piazza, Bordelet et al. 2021) and a conceptual review (Savocco & Piazza, 2021). They were also expanded to study the role of loop formation by condensin in the repair of programmed DNA break integral to mating-type switching in yeast (Piveteau et al. BioRxiv 2024). Realizing the technical limitations of Hi-C, we developed a method to directly detect contacts made by single-stranded DNA (the active entity during homology search). The resulting ssHi-C methodology caps our effort to chart all HR steps in cells, and can be straightforwardly applied to study various DNA metabolic processes in any organism (Patent submitted in 2023). This methodology enabled revealing the mechanism that underlies the expansion of homology search genome-wide, and that resides in the assembly of long and rigid Rad51-ssDNA filaments (Dumont et al. Mol. Cell 2024). Coupling of these Hi-C methodologies and functional HR assays is proving extremely informative to define the molecular functions of several HR proteins in homology search within this new framework.
In parallel we addressed the functional role of various molecular actors involved in the early stages of HR and participating of the fidelity of the pathway. It entailed methodological improvements (Rietz et al., JoVE 2022). We developed an exquisitely sensitive assay for detection of rare chromosomal translocations, which enables studying the kinetics of their formation and the role of essential factors in HR-mediated genomic instability (Reitz et al. Genes Dev 2023). We also revealed the control exerted by transcription by RNA Polymerase II on early DNA joint molecules formed during HR, the mechanism of this interference, and its role in promoting genome stability (Djeghmoum & Piazza, BioRxiv 2025). Finally, we developed a stochastic model of HR, which we use to infer the quantitative contributions of the various chromatin and HR factors partaking in the search process.
The conjoined study of HR and chromatin organization in meiosis required the assembly of a 150 kb synthetic chromosomal region. We obtained a fully debugged synthetic region. This genetic engineering feat allowed us for the first time to map at high resolution chromatin folding along large regions of individual homologs, and determine their trans interactions profile in wild-type cells. This new-generation experimental system opens the way to the rationale study of the role of chromatin structure in regulating HR and homolog pairing in meiosis, through controlled cis and trans perturbations. It will give rise to a high-impact publication.
The synthetic biology approaches part of both work packages represents new gold standard experimental systems to investigate the interplay between HR and chromatin folding, and as such are of major interest for the DNA repair and meiosis research fields. Likewise, the various methodological developments to chart HR in cells overcome decades of vexation in the field, and already revealed new roles for various proteins in homology search. Furthermore, the development of a general computational frameworks of HR integrating molecular and chromosomal scale information has never been achieved. This framework will enable to quantitatively interpret the experimental data, and in turn generate predictions for future investigations. Coupling of these experimental innovations and computational approach form the bedrock for several future and ongoing projects, supported by competitive national grants. We expect it to provide a general, integrated and quantitative understanding of the respective and compounded roles of various conserved nucleoprotein factors acting across scales in homology search, in both mitotic and meiotic cells.