Dynamics and function of the 3D chromatin structure in the germ line of vertebrates

Abstract

How genomes are organized and regulated within the cell nucleus varies among cell types and taxa. In germ cells of eutherian mammals, chromatin undergoes extensive remodelling to accommodate key meiotic events such as chromosome pairing and recombination. Yet, whether these patterns are conserved in deeper phylogenetic branches of the Vertebrate Tree of Life remains unknown. Exploring the dynamics and function of the three-dimensional organization of the genome during the generation of germ cells across taxa is essential to understand the mechanisms underlying the heritability of chromatin architecture, and its role in fertility and evolution. The main goal of this thesis was to investigate the dynamics of meiosis progression and chromatin folding during spermatogenesis in representative vertebrates (eutherian mammals, marsupials and reptiles), with a special interest in the structural and functional roles of meiotic cohesins. For this aim, three specific objectives were established: (i) characterize meiosis dynamics among vertebrates, (ii) examine the spatial organization of the genome during spermatogenesis, and (iii) identify the structural and functional roles of cohesins during spermatogenesis. This was archived by combining comparative cytological analysis, fluorescence activated cell sorting (FACS) and high-throughput sequencing technologies, including chromosome conformation capture (Hi-C) and single cell RNA sequencing (sc-RNAseq). We generated new Hi-C data from flow-sorted germ cells from representative vertebrates (tammar wallaby and bearded dragon) along with mice lacking the meiotic cohesin subunit RAD21L. This was combined with single cell transcriptomics profiles throughout spermatogenesis. The data generated was compared with available data from other mammals to provide a comprehensive view of the structural and functional roles of chromatin during germ line formation. We observed that the testicular organization of germ cells follows a non-cystic organization in amniotes, with meiotic dynamics being highly conserved among vertebrates, including canonical events such as bouquet formation, double strand break (DSB) formation and repair, homologous chromosomes synapsis, and meiotic sex chromosome inactivation (MSCI). Despite this conservation, lineage-specific dynamics were detected, including the presence of a prominent bouquet in reptiles and a transient configuration in most marsupials, the early pair and clustering of micro-chromosomes in reptiles, and the elongation of telomeres during male prophase I in dasyurids. Additionally, different sex chromosome systems (i.e. XY, XY1Y2 and ZW) revealed distinct strategies of sex chromosome pairing and silencing, with implications for the evolution of sex chromosomes. Our findings also unveiled fundamental principles of chromatin architecture during vertebrate spermatogenesis. At the hierarchical level of DNA loops, we identified a correlation between synaptonemal complex length, DNA loop size and DSB rates that was conserved between species. DNA loop size was likely determined by genome size, with species with large genomes organized into short chromosomal axes with long meiotic DNA loops, but short DNA loops at post-meiotic stages. We also observed a conserved remodelling of chromatin architecture across species, with both compartments and TADs being attenuated during prophase I. Despite these conserved dynamics, our comparative analysis revealed lineage-specific patterns of chromatin organization during meiosis, where chromosome length, chromosome morphology and DNA loop size are key factors. This thesis also uncovered the structural and functional roles of cohesins during mouse spermatogenesis. Loss of the meiotic subunit RAD21L impacts both chromatin architecture and gene expression, resulting in changes at different levels, including longer DNA loops, loss of TADs, compartmentalization strengthening, generation of aberrant interactions, misregulation of key meiotic genes, and partial disruption of the MSCI. Overall, our findings provide new insights into the evolutionary dynamics of meiosis progression and chromatin architecture in vertebrate germ cells, along with the structural and functional roles of meiotic cohesins. This underscores the key role of chromatin remodelling and gene regulation during spermatogenesis, with significant implications for fertility and evolution.

How genomes are organized and regulated within the cell nucleus varies among cell types and taxa. In germ cells of eutherian mammals, chromatin undergoes extensive remodelling to accommodate key meiotic events such as chromosome pairing and recombination. Yet, whether these patterns are conserved in deeper phylogenetic branches of the Vertebrate Tree of Life remains unknown. Exploring the dynamics and function of the three-dimensional organization of the genome during the generation of germ cells across taxa is essential to understand the mechanisms underlying the heritability of chromatin architecture, and its role in fertility and evolution. The main goal of this thesis was to investigate the dynamics of meiosis progression and chromatin folding during spermatogenesis in representative vertebrates (eutherian mammals, marsupials and reptiles), with a special interest in the structural and functional roles of meiotic cohesins. For this aim, three specific objectives were established: (i) characterize meiosis dynamics among vertebrates, (ii) examine the spatial organization of the genome during spermatogenesis, and (iii) identify the structural and functional roles of cohesins during spermatogenesis. This was archived by combining comparative cytological analysis, fluorescence activated cell sorting (FACS) and high-throughput sequencing technologies, including chromosome conformation capture (Hi-C) and single cell RNA sequencing (sc-RNAseq). We generated new Hi-C data from flow-sorted germ cells from representative vertebrates (tammar wallaby and bearded dragon) along with mice lacking the meiotic cohesin subunit RAD21L. This was combined with single cell transcriptomics profiles throughout spermatogenesis. The data generated was compared with available data from other mammals to provide a comprehensive view of the structural and functional roles of chromatin during germ line formation. We observed that the testicular organization of germ cells follows a non-cystic organization in amniotes, with meiotic dynamics being highly conserved among vertebrates, including canonical events such as bouquet formation, double strand break (DSB) formation and repair, homologous chromosomes synapsis, and meiotic sex chromosome inactivation (MSCI). Despite this conservation, lineage-specific dynamics were detected, including the presence of a prominent bouquet in reptiles and a transient configuration in most marsupials, the early pair and clustering of micro-chromosomes in reptiles, and the elongation of telomeres during male prophase I in dasyurids. Additionally, different sex chromosome systems (i.e. XY, XY1Y2 and ZW) revealed distinct strategies of sex chromosome pairing and silencing, with implications for the evolution of sex chromosomes. Our findings also unveiled fundamental principles of chromatin architecture during vertebrate spermatogenesis. At the hierarchical level of DNA loops, we identified a correlation between synaptonemal complex length, DNA loop size and DSB rates that was conserved between species. DNA loop size was likely determined by genome size, with species with large genomes organized into short chromosomal axes with long meiotic DNA loops, but short DNA loops at post-meiotic stages. We also observed a conserved remodelling of chromatin architecture across species, with both compartments and TADs being attenuated during prophase I. Despite these conserved dynamics, our comparative analysis revealed lineage-specific patterns of chromatin organization during meiosis, where chromosome length, chromosome morphology and DNA loop size are key factors. This thesis also uncovered the structural and functional roles of cohesins during mouse spermatogenesis. Loss of the meiotic subunit RAD21L impacts both chromatin architecture and gene expression, resulting in changes at different levels, including longer DNA loops, loss of TADs, compartmentalization strengthening, generation of aberrant interactions, misregulation of key meiotic genes, and partial disruption of the MSCI. Overall, our findings provide new insights into the evolutionary dynamics of meiosis progression and chromatin architecture in vertebrate germ cells, along with the structural and functional roles of meiotic cohesins. This underscores the key role of chromatin remodelling and gene regulation during spermatogenesis, with significant implications for fertility and evolution.

How genomes are organized and regulated within the cell nucleus varies among cell types and taxa. In germ cells of eutherian mammals, chromatin undergoes extensive remodelling to accommodate key meiotic events such as chromosome pairing and recombination. Yet, whether these patterns are conserved in deeper phylogenetic branches of the Vertebrate Tree of Life remains unknown. Exploring the dynamics and function of the three-dimensional organization of the genome during the generation of germ cells across taxa is essential to understand the mechanisms underlying the heritability of chromatin architecture, and its role in fertility and evolution. The main goal of this thesis was to investigate the dynamics of meiosis progression and chromatin folding during spermatogenesis in representative vertebrates (eutherian mammals, marsupials and reptiles), with a special interest in the structural and functional roles of meiotic cohesins. For this aim, three specific objectives were established: (i) characterize meiosis dynamics among vertebrates, (ii) examine the spatial organization of the genome during spermatogenesis, and (iii) identify the structural and functional roles of cohesins during spermatogenesis. This was archived by combining comparative cytological analysis, fluorescence activated cell sorting (FACS) and high-throughput sequencing technologies, including chromosome conformation capture (Hi-C) and single cell RNA sequencing (sc-RNAseq). We generated new Hi-C data from flow-sorted germ cells from representative vertebrates (tammar wallaby and bearded dragon) along with mice lacking the meiotic cohesin subunit RAD21L. This was combined with single cell transcriptomics profiles throughout spermatogenesis. The data generated was compared with available data from other mammals to provide a comprehensive view of the structural and functional roles of chromatin during germ line formation. We observed that the testicular organization of germ cells follows a non-cystic organization in amniotes, with meiotic dynamics being highly conserved among vertebrates, including canonical events such as bouquet formation, double strand break (DSB) formation and repair, homologous chromosomes synapsis, and meiotic sex chromosome inactivation (MSCI). Despite this conservation, lineage-specific dynamics were detected, including the presence of a prominent bouquet in reptiles and a transient configuration in most marsupials, the early pair and clustering of micro-chromosomes in reptiles, and the elongation of telomeres during male prophase I in dasyurids. Additionally, different sex chromosome systems (i.e. XY, XY1Y2 and ZW) revealed distinct strategies of sex chromosome pairing and silencing, with implications for the evolution of sex chromosomes. Our findings also unveiled fundamental principles of chromatin architecture during vertebrate spermatogenesis. At the hierarchical level of DNA loops, we identified a correlation between synaptonemal complex length, DNA loop size and DSB rates that was conserved between species. DNA loop size was likely determined by genome size, with species with large genomes organized into short chromosomal axes with long meiotic DNA loops, but short DNA loops at post-meiotic stages. We also observed a conserved remodelling of chromatin architecture across species, with both compartments and TADs being attenuated during prophase I. Despite these conserved dynamics, our comparative analysis revealed lineage-specific patterns of chromatin organization during meiosis, where chromosome length, chromosome morphology and DNA loop size are key factors. This thesis also uncovered the structural and functional roles of cohesins during mouse spermatogenesis. Loss of the meiotic subunit RAD21L impacts both chromatin architecture and gene expression, resulting in changes at different levels, including longer DNA loops, loss of TADs, compartmentalization strengthening, generation of aberrant interactions, misregulation of key meiotic genes, and partial disruption of the MSCI. Overall, our findings provide new insights into the evolutionary dynamics of meiosis progression and chromatin architecture in vertebrate germ cells, along with the structural and functional roles of meiotic cohesins. This underscores the key role of chromatin remodelling and gene regulation during spermatogenesis, with significant implications for fertility and evolution.