Modeling meiotic chromosomes indicates a size dependent contribution of telomere clustering and chromosome rigidity to homologue juxtaposition.

Meiosis is the cell division that halves the genetic component of diploid cells to form gametes or spores. To achieve this, meiotic cells undergo a radical spatial reorganisation of chromosomes. This reorganisation is a prerequisite for the pairing of parental homologous chromosomes and the reductional division, which halves the number of chromosomes in daughter cells. Of particular note is the change from a centromere clustered layout (Rabl configuration) to a telomere clustered conformation (bouquet stage). The contribution of the bouquet structure to homologous chromosome pairing is uncertain. We have developed a new in silico model to represent the chromosomes of Saccharomyces cerevisiae in space, based on a worm-like chain model constrained by attachment to the nuclear envelope and clustering forces. We have asked how these constraints could influence chromosome layout, with particular regard to the juxtaposition of homologous chromosomes and potential nonallelic, ectopic, interactions. The data support the view that the bouquet may be sufficient to bring short chromosomes together, but the contribution to long chromosomes is less. We also find that persistence length is critical to how much influence the bouquet structure could have, both on pairing of homologues and avoiding contacts with heterologues. This work represents an important development in computer modeling of chromosomes, and suggests new explanations for why elucidating the functional significance of the bouquet by genetics has been so difficult

Aplicación de herramientas citogenéticas en el estudio de las asociaciones cromosómicas durante la meiosis en trigo con fines de mejora genética

Meiosis is one of the most important processes in eukaryotic cells with sexual reproduction, where homologous (equivalent) chromosomes associate, recognise, pair and recombine. Meiosis has been deeply studied because recombination between chromosomes from two species in a hybrid can introduce new genetic variability. In a crop such as wheat, the transfer of genes from related species to introduce new resistance to biotic or abiotic stresses is a target in breeding programs. Thus, going deeper into the knowledge of meiosis and the processes controlling chromosome associations at the initial stages might contribute to wheat breeding. Chromosome associations at the beginning of meiosis initiate at the chromosome ends, where telomeres and subtelomeres are. Although the function of telomeres has been deeply studied, the subtelomere region continues being a meiosis black hole. In addition, the effect of the Ph1 locus on recombination between interspecific chromosomes also hampers the incorporation of genetic variability from relative species wheat. In this work we have shed light into the role of subtelomeres in initial chromosome recognition and pairing between homologous chromosomes in meiosis in the wheat background. Results revealed that the subtelomeric region is crucial in the processes of homologous chromosome recognition and pairing. In addition, we have shown that homoeologous chromosomes from two barley species (Hordeum vulgare and Hordeum chilense) can associate correctly in pairs at the beginning of meiosis but do not recombine in the presence of the Ph1 locus. Recombination between homoeologous chromosomes from these barley species was only allowed in wheat in the ph1b mutant background. The role of subtelomeres and the Ph1 locus in early meiosis events in wheat is discussed.La meiosis es uno de los procesos más importantes en las células eucariotas con reproducción sexual, donde los cromosomas homólogos (equivalentes) se asocian, reconocen, aparean y recombinan. La meiosis ha sido estudiada en profundidad, ya que la recombinación entre cromosomas de dos especies dando lugar a un híbrido puede introducir una nueva variabilidad genética. En un cultivo como el trigo, la transferencia de genes de especies relacionadas para introducir una nueva resistencia al estrés biótico o abiótico es un objetivo principal en los programas de mejoramiento. Por lo tanto, profundizar en el conocimiento de la meiosis y los procesos que controlan las asociaciones de cromosomas en las etapas iniciales podría contribuir al mejoramiento del trigo. Las asociaciones de cromosomas al comienzo de la meiosis se inician en los extremos de los cromosomas, donde se encuentran los telómeros y los subtelómeros. Aunque la función de los telómeros en meiosis se ha estudiado en profundidad, las regiones subteloméricas siguen siendo un agujero negro. Además, el efecto del locus Ph1 en la recombinación entre asociaciones cromosómicas interespecíficas, también obstaculiza la incorporación de la variabilidad genética de las especies relativas de trigo. En este trabajo hemos estudiado el papel de los subtelómeros en el reconocimiento cromosómico inicial y el apareamiento entre cromosomas homólogos en la meiosis en el fondo genético del trigo. Los resultados revelaron que la región subtelomérica es crucial en estos procesos. Además, hemos mostrado que los cromosomas homeólogos de dos especies de cebada (H. vulgare y H. chilense) pueden asociarse correctamente en pares al comienzo de la meiosis, pero no recombinan en presencia del locus Ph1. La recombinación entre cromosomas homeólogos de estas especies de cebada solo se produjo en trigo en el fondo mutante ph1b. Se discute el papel de los subtelómeros y el locus Ph1 en los eventos de meiosis temprana en trigo

Meiotic chromosome dynamics: a structural characterisation

Ph. D. ThesisMeiotic chromosomes are bound in architecturally enforced synapsis during the first meiotic division by a proteinaceous megastructure known as the synaptonemal complex (SC). This molecular scaffold is built between paired homologues, providing a unique three-dimensional environment in which to form genetic crossovers, physical inter-homologue connections critical in ensuring equational segregation at metaphase. The SC structure represents the hallmark of meiotic division, with a striking tripartite appearance, conserved across evolution, in which chromosomally associated lateral elements are connected to a midline central element via transversal filaments. Such that the SC installs between correctly identified maternal-paternal pairs, a genome-wide, sequencebased, homology search is facilitated by rapid chromosomal movements. Cytoskeletal forces power these movements, transmitted through the nuclear envelope (NE) to the chromosome’s telomeric ends via the LINC (Linker of Nucleoskeleton and Cytoskeleton). Importantly, NE recruitment and tethering is mediated by the meiotic telomere complex, consisting of MAJIN, TERB1, and TERB2, without which meiotic progression is stalled. The works herein reveal the structural basis of synapsis in the human SC and chromosome tethering to the nuclear envelope by the meiotic telomere complex. Specifically, I report a complete biophysical characterisation of SYCP1, the transversal filament protein of the human SC, and present crystal structures which represent mechanisms of its assembly within the SC mediated by sequences at both its N-termini (which mediate midline, head-to-head, associations) and C-termini (which undergo pH dependent, back-to-back assembly on the chromosome axis). Further, we solved the crystal structure of the MAJIN-TERB2 complex and characterised its mode of DNA association providing key insights into how NE tethering is achieved. Our findings are discussed within the context of the existing molecular understanding of meiotic chromosome dynamics

Integrating chromatin structure and global chromosome dynamics

DNA associates with proteins to form chromatin which is essential for the compaction of the DNA into the cell nucleus and is highly dynamic in order to allow the different biological processes of the DNA to occur. Chromatin compaction is achieved at different hierarchical levels: the 10nm fibre (DNA associates to nucleosomes formed by different histones), the Higher Order Chromatin fibre and the 300 nm chromosome structures. This study has shown that both H1 and H4 histones play a crucial role in preserving meiotic as well as mitotic chromosome structure and functional genome integrity in Arabidopsis. The role of the different linker histone H1 isoforms as well as the core histone H4 in Arabidopsis thaliana was investigated using T-DNA and RNAi mutant lines which showed different meiotic defects. Chromosomal breaks as well as non-homologous connections in the h4RNAi were linked to 45S/5S rDNA disorganisation, suggesting that H4 preserves chromosome integrity at these rDNA regions. Ath1.1 mutant presented univalents and reduced chiasma frequency at metaphase I, linked to a severe defect in ASY1 localisation on the meiotic chromosome axes. Thus, indicating that histone H1.1 is vital for proper chromatin axis organization that permit normal loading of recombination machinery proteins in Arabidopsis

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