CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat

Abstract

SUMMARYDuring meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synapotonemal complex (SC) construction in hexaploid wheat (2n=42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarisation (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganisation of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarisation were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues.

Observation of Extensive Chromosome Axis Remodeling during the “Diffuse-Phase” of Meiosis in Large Genome Cereals

“…The presence of ASY1 found here also mirrors its recently reported cytological persistence in wheat (Sepsi et al, 2016), indicating that our observations of ASY1 in tinsel structure and desynapsis may form part of an extended functional role late in meiosis I.…”

ImmunoFISH: Simultaneous Visualisation of Proteins and DNA Sequences Gives Insight Into Meiotic Processes in Nuclei of Grasses

“…The bulk chromatin was labelled by DAPI in both cases. This enabled us to correlate centromere–telomere dynamics and that of the bulk chromatin to the development of the synaptonemal complex ( Sepsi et al, 2017 ; Figures 3 , 4 ).…”

“…A parafilm coverslip was applied and slides were replaced in the plastic box for 30–60 min at RT. Rabbit anti-CENH3 primary antibody was prepared as described by Sepsi et al (2017) . Guinea pig anti-ASY1 antibody was developed by an immunisation program using a recombinant protein including the Horma-domain of A. thaliana ASY1 protein while the ZYP1 antibody was produced against a recombinant protein matching the C-terminal region of the A. thaliana ZYP1B protein ( Osman et al, 2018 ).…”

“…Chromosome–chromosome interactions leading to homologous pairing at meiosis are accompanied by programmed changes in chromatin organisation and structure. Distinct stages of chromatin remodelling and movement with association of landmark chromosomal regions imprint the period of early meiosis and result in a distinctive nuclear polarisation ( Colas et al, 2008 ; Heslop-Harrison and Schwarzacher, 2011 ; Sepsi et al, 2017 ). These chromatin dynamics are facilitated by meiosis-specific functional protein complexes, whose presence and localisation at particular chromosomal regions reflect the progression through meiosis ( Schwarzacher, 2003 ; Cahoon and Hawley, 2016 ).…”

“…ImmunoFISH combines FISH to label chromosomes and chromosome regions with immunohistochemistry and high-resolution microscopy, to study chromatin dynamics and specific meiotic proteins simultaneously, providing a precise timing of prophase I ( Martín et al, 2017 ; Sepsi et al, 2017 ). The protocol presented here was developed for the analysis of meiotic cells of Triticeae and focused on the three-dimensional (3D) preservation of nuclei and long-term storage without the need for time-consuming embedding and tissue sectioning.…”