Work spearheaded by post-doctoral fellow Tadasu Nozaki, in the Kleckner laboratory, and recently published in Nature (PDF), uses a powerful new approach to examine pairing of homologous chromosomes during meiosis, in real time, at individual loci.
Meiosis is the modified cellular program by which a diploid progenitor cell produces haploid gametes for sexual reproduction (e.g. sperm and egg). The key unique feature of this program is a complex series of interactions between homologous chromosomes (“Mom” and “Dad”). As one central feature, organized maternal and paternal homologs recognize one another and pair intimately along their lengths. Pairing occurs at stage when chromosomes are long and thin. The Kleckner lab wishes to understand how pairing occurs, and in such a way that it does not produce a tangled mess. The challenges involved are illustrated dramatically for an organism with many very long chromosomes (Figure 1).
At the time of pairing, chromosomes are organized into arrays of chromatin loops that emanate from a DNA/protein structural axis meshwork. Both homolog recognition and spatial juxtaposition of homolog axes mediated by recombination complexes that are physically and functionally linked to those structural axes. As a result, local recombination-related events can spread along the chromosomes to promote the whole chromosome (axis-mediated) pairing.
Nozaki et al, studied this process as it occurs in real time, in living cells of budding yeast. Local fluorescent tags, inserted at corresponding positions on homologous chromosomes, were tracked in three-dimensions (Figure 2), at high spatio-temporal resolution over long time periods (~40 nm spatial resolution at 10 sec intervals for the entire pairing period of ~2-3 hours or at 1 min intervals for more than 10 hours, thus encompassing all of meiosis.
Figure 2
Nozaki’s key discovery is that homologous loci move from quite far apart (~2 µm) to very close together (wih their axes separated by only ~100 nm) extremely rapidly, in only 6 min (Figure 3). This phenomenon was named Rapid Homolog Juxtaposition (RHJ). Nozaki’s work further showed that RHJ occurs nearly synchronously over significant distances along a single chromosome pair.
Figure 3
Together these findings led Nozaki and colleagues to a new conceptual framework for pairing that could be general to many organisms. In brief, pairing on a particular chromosome (or chromosomal region) is nucleated by a first RHJ event. This first event will automatically draw adjacent regions closer together in space, thereby allowing efficient homology searching and RHJ nearby. As this process propagates along the chromosome, it will progressively draw the entire homolog pair together, out of the “pairing pool” of other chromosomes, in a topologically regular outcome. One major challenge for the future is to understand how the earliest stages of pairing regulated so as to ensure that searching occurs only while pairing progresses, rather than among all possible regions, everywhere in the genome, at all times.
Nozaki’s analysis of RHJ provided an additional surprise. A second major feature of the meiotic interhomolog interaction program (and another favorite topic of Kleckner laboratory studies) is “crossover interference”. Discovered over 100 years ago, interference is a one-dimensional spatial patterning process: DNA crossover recombination products occur at different positions in different nuclei but nonetheless tend to be evenly spaced, as if occurrence of an event at one position “interferes” with occurrence of another event nearby. This process requires communication along the chromosomes, the basis for which is not understood. RHJ analyses reveals that implementation of interference is directly (temporally and functionally) linked to homolog pairing. Detailed analysis shows that RHJ involves a prominent relatively long-lived intermediate stage (“coalignment”), with very rapid movement into and then beyond that stage (Figure 3), and that implementation of crossover patterning triggers exit from the intermediate stage. One implication is that the many recombination complexes that mediate pairing are “poised” at the intermediate stage, awaiting instructions as to which will become crossovers. Another implication is that crossover patterning chases coalignment pairing through the genome. This effect is likely to be critical. At the intermediate stage, homolog axes are linked by many “undifferentiated” recombination interactions which have the potential, in a crowded nucleus, to entrap unrelated chromosomes into entanglements. Crossover interference removes most of these interactions, keeping only the few that will become crossovers. Via this effect, topological simplification will be achieved in direct linkage to progression of pairing. Future work can determine the basis for rapid movements into and out of the intermediate stage and can apply RHJ analysis to further elucidate the mechanism of crossover patterning.
Dr. Nozaki’s collaborator in this work was Staff Scientist Beth Weiner. The spot detection/denoising algorithm used for this work was created by Frederick Chang for his MCB PhD.
by Nancy Kleckner
