Biology subjects

Crichton, J. H.

Publications and source records attributed to Crichton, J. H..

6 recordsLinked to original sources

bioRxiv2023

Computational Tools for the Analysis of Meiotic Prophase I Images

Prophase I is a remarkable stage of meiotic division during which homologous chromosomes pair together and exchange DNA by meiotic recombination. Fluorescence microscopy of meiotic chromosome spreads is a central tool in the study of this process, with chromosome axis proteins being visualised as extended filaments upon which recombination proteins localise in focal patterns. Chromosome pairing and recombination are dynamic processes, and hundreds of recombination foci can be present in some meiotic nuclei. As meiotic nuclei can exhibit significant variations in staining patterns within and between nuclei, particularly in mutants, manual analysis of images presents challenges for consistency, documentation, and reproducibility. Here we share a combination of complementary computational tools which can be used to partially automate the quantitative analysis of meiotic images. 1) The segmentation of axial and focal staining patterns, to automatically measure chromosome axis length and count axis-associated (and non-axis associated) recombination foci; 2) Quantification of focus position along chromosome axes to investigate spatial regulation; 3) Simulation of random distributions of foci within the nucleus or along the chromosome axes to statistically investigate observed foci-axis associations and foci-foci associations; 4) Quantification of chromosome axis proximity to investigate relationships with chromosome synapsis/asynapsis; 5) Quantification of and orientation of focus-axis distances. Together these tools provide a framework to perform routine documentation and analysis of meiotic images, as well as opening up routes to build on this initial output and perform more detailed analyses.

cell biology↗

bioRxiv2022

Parallel recruitment pathways contribute to synaptonemal complex assembly during mammalian meiosis

During meiosis, the synaptonemal complex (SC) assembles between paired chromosomes, binding them together in close apposition, and facilitating recombination. SC assembly is thought to occur through the hierarchical zipper-like recruitment of axial elements, followed by transverse filaments and then central elements. However, the rapidity of SC formation in mammals has hitherto hindered investigation of its assembly mechanisms and their relationship with recombination. Using super-resolution imaging of separation-of-function mouse mutants, we show that, contrary to the hierarchical assembly model, central element protein SYCE2 is recruited to recombination sites early in SC assembly, and independently of SYCP1-containing transverse filaments. Further, SYCE2-TEX12 binds DNA in vitro, and SYCE2-containing bridges physically link paired chromosomes at recombination sites prior to transverse filament recruitment and chromosome synapsis. These data suggest that mammals integrate parallel recruitment pathways to assemble a mature SC: one recruiting central element proteins to recombination sites, and another recruiting transverse filaments to chromosomes.

cell biology↗

bioRxiv2022

Structural maturation of SYCP1-mediated meiotic chromosome synapsis through conformational remodelling by molecular adapter SYCE3

In meiosis, a supramolecular protein structure, the synaptonemal complex (SC), assembles between homologous chromosomes to facilitate their recombination. Mammalian SC formation is thought to involve hierarchical zipper-like assembly of an SYCP1 protein lattice that recruits stabilising central element (CE) proteins as it extends. Here, we combine biochemical approaches with separation-of-function mutagenesis in mice to uncover that, rather than stabilising the SYCP1 lattice, the CE protein SYCE3 actively remodels this structure during synapsis. We find that SYCP1 tetramers undergo conformational change into 2:1 heterotrimers upon SYCE3-binding, removing their assembly interfaces and disrupting the SYCP1 lattice. SYCE3 then establishes a new lattice by its self-assembly mimicking the role of the disrupted interface in tethering together SYCP1 dimers. SYCE3 also interacts with CE complexes SYCE1-SIX6OS1 and SYCE2-TEX12, providing a mechanism for their recruitment. Thus, SYCE3 remodels the SYCP1 lattice into a CE-binding integrated SYCP1-SYCE3 lattice to achieve long-range synapsis by a mature SC.

biochemistry↗

bioRxiv2017

Tex19.1 Regulates Acetylated SMC3 Cohesin and Prevents Aneuploidy in Mouse Oocytes

Age-dependent oocyte aneuploidy, a major cause of Down syndrome, is associated with declining sister chromatid cohesion in postnatal oocytes. Here we show that cohesion in postnatal mouse oocytes is regulated by Tex19.1. We show that Tex19.1-/- oocytes have defects in the maintenance of chiasmata, mis-segregate their chromosomes during meiosis, and transmit aneuploidies to the next generation. By reconstituting aspects of this pathway in mitotic somatic cells, we show that Tex19.1 regulates an acetylated SMC3-marked subpopulation of cohesin by inhibiting the activity of the E3 ubiquitin ligase UBR2 towards specific substrates, and that UBR2 itself has a previously undescribed role in negatively regulating acetylated SMC3. Lastly, we show that acetylated SMC3 is associated with meiotic chromosome axes in oocytes, but that this is reduced in the absence of Tex19.1. These findings indicate that Tex19.1 maintains acetylated SMC3 and sister chromatid cohesion in postnatal oocytes, and prevents aneuploidy in the female germline.

genetics↗

bioRxiv2017

Tex19.1 Regulates Meiotic DNA Double Strand Break Frequency in Mouse Spermatocytes

Meiosis relies on the SPO11 endonuclease to generate the recombinogenic DNA double strand breaks (DSBs) required for homologous chromosome synapsis and segregation. The number of meiotic DSBs needs to be sufficient to allow chromosomes to search for and find their homologs, but not excessive to the point of causing genome instability. Here we report that meiotic DSB frequency in mouse spermatocytes is regulated by the mammal-specific gene Tex19.1. We show that the chromosome asynapsis previously reported in Tex19.1-/- spermatocytes is preceded by reduced numbers of recombination foci in leptotene and zygotene. Tex19.1 is required for the generation of normal levels of Spo11-dependent DNA damage during leptotene, but not for upstream events such as MEI4 foci formation or accumulation of H3K4me3 at recombination hotspots. Furthermore, we show that mice carrying mutations in the E3 ubiquitin ligase UBR2, a TEX19.1-interacting partner, phenocopy the Tex19.1-/- recombination defects. These data show that Tex19.1 and Ubr2 are required for mouse spermatocytes to generate sufficient meiotic DSBs to ensure that homology search is consistently successful, and reveal a hitherto unknown genetic pathway regulating meiotic DSB frequency in mammals.\n\nAuthor SummaryMeiosis is a specialised type of cell division that occurs during sperm and egg development to reduce chromosome number prior to fertilisation. Recombination is a key step in meiosis as it facilitates the pairing of homologous chromosomes prior to their reductional division, and generates new combinations of genetic alleles for transmission in the next generation. Regulating the amount of recombination is key for successful meiosis: too much will likely cause mutations, chromosomal re-arrangements and genetic instability, whereas too little causes defects in homologous chromosome pairing prior to the meiotic divisions. This study identifies a genetic pathway requiredto generate robust meiotic recombination in mouse spermatocytes. We show that male mice with mutations in Tex19.1 or Ubr2, which encodes an E3 ubiquitin ligase that interacts with TEX19.1, have defects in generating normal levels of meiotic recombination. We show that the defects in these mutants impact on the recombination process at the stage when programmed DNA double strand breaks are being made. This defect likely contributes to the chromosome synapsis and meiotic progression phenotypes previously described in these mutant mice. This study has implications for our understanding of how this fundamental aspect of genetics and inheritance is controlled.

genetics↗

bioRxiv2017

Defects in Meiotic Recombination Delay Progression Through Pachytene in Mouse Spermatocytes

During meiosis, recombination, synapsis, chromosome segregation and gene expression are coordinately regulated to ensure successful execution of this specialised cell division. In many model organisms, checkpoint controls can delay meiotic progression to allow defects or errors in these processes to be repaired or corrected. Mouse spermatocytes possess quality control checkpoints that eliminate cells with persistent irreparable defects in chromosome synapsis or recombination, and here we show that a spermatocyte checkpoint regulates progression through pachytene to accommodate delays in meiotic recombination. We have previously show that the appearance of early recombination foci is delayed in Tex19.1-/- spermatocytes during leptotene/zygotene, but some Tex19.1-/- spermatocytes still successfully synapse their chromosomes. Therefore, we have used autosomally synapsed Tex19.1-/- mouse spermatocytes to assess the consequences of delayed recombination on progression through pachytene. We show that these pachytene spermatocytes are enriched for early recombination foci. This skew is not accompanied by cell death and likely reflects delays in the generation and/or maturation of recombination foci. Moreover, patterns of axis elongation, chromatin modifications, and histone H1t expression are also all skewed towards earlier substages of pachytene suggesting these events are co-ordinately regulated. Importantly, the delay in histone H1t expression in response to loss of Tex19.1 does not occur in a Spo11 mutant background, suggesting that histone H1t expression is being delayed by a recombination-dependent checkpoint. These data indicate that a recombination-dependent checkpoint operates in mouse spermatocytes that can alter progression through pachytene to accommodate spermatocytes with some types of recombination defect.

genetics↗