Characterising large-scale chromatin looping in mouse meiotic prophase I
Item statusRestricted Access
Embargo end date27/06/2021
MacGregor, Isobel Amy
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.