Pairing Centers mediate meiotic chromosome dynamics in C. elegans

All sexually reproducing animals depend on meiosis, the specialized cell division in which haploid gametes are produced from a diploid germ cell. Meiotic cells undergo one round of replication followed by two rounds of division. A distinctive feature of the first meiotic prophase is the establishment of physical linkages, via crossover recombination, between homologous chromosomes, which are essential for their proper segregation at metaphase I. Prior to the recombination step, however, homologous chromosomes must overcome the problem of finding and recognizing their appropriate partner within the nuclear volume, a process called homolog pairing. Transient interactions between paired homologs are further stabilized by a process called synapsis, in which the synaptonemal complex polymerizes along the entire length of paired chromosome axes. My research aims to address central questions to our understanding of the meiotic process: how do homologous chromosomes find and recognize their correct partner and how is synapsis regulated so that it occurs only between properly paired homologs. In Chapter 1, I present data that Pairing Centers are essential to facilitate proper connections between chromosomes and the nuclear envelope by recruiting a polo-like kinase. In Chapter 2, I identify novel meiotic mutants through a candidate screen and present my preliminary characterization of their meiotic defects. I further describe tools to visualize their chromosome dynamics in vivo.

Proper meiotic chromosome segregation in C. elegans requires homolog pairing, synapsis, and recombination. The mechanisms underlying homologous chromosome pairing remain poorly understood. In C. elegans, as in many other eukaryotes, pairing is accompanied by a global rearrangement of chromosomes. Work from the Dernburg lab and others has found that this rearrangement is driven through the association of special chromosome regions known as Pairing Centers (PCs) with nuclear envelope proteins and cytoskeletal components (Phillips et al. 2005, Sato et al. 2009). Using fluorescent markers for nuclear envelope attachment sites and Pairing Centers, I analyzed prophase chromosome dynamics through real-time imaging and quantitative motion tracking. My results reveal a dramatic increase in chromosome motion at the onset of chromosome pairing that persists after homologous loci are paired. I show that this increased mobility correlates with the formation of NE patches, and that the increase in motion that accompanies meiotic entry is abrogated by knockdown of cytoplasmic dynein. These rapid motions are also sensitive to depolymerization of microtubules by colchicine, but are not affected by treatment with Latrunculin A. In addition, fluorescent labeling of whole chromosomes suggests that meiotic chromosome motion is driven primarily by the PC end of chromosomes and that the chromosome is quite flexible. These data support a model in which meiotic chromosome motion is promoted by a small number of fast, microtubule-dependent, motor-driven movements that augment the smaller, likely diffusive motions seen prior to meiosis. The observation that fast motions persist well after pairing is completed suggests additional roles in chromosome synapsis or recombination, and are consistent with the idea that rapid motions function to destabilize inappropriate, non-homologous interactions.

For most eukaryotes, recombination between homologous chromosomes during meiosis is an essential aspect of sexual reproduction. Meiotic recombination is initiated by programmed double-strand breaks (DSBs) in the DNA, which have the potential to induce mutations if not efficiently repaired. The focus of the work presented here is to better understand the mechanisms that govern the initiation of recombination and regulate the formation of DSBs. The nematode Caenorhabditis elegans was used as a model system for these studies. Here I describe the identification of a novel gene, dsb-1, that is required for DSB formation in C. elegans. Through analysis of DSB-1 I illuminate two important regulatory pathways that control the initiation of meiotic recombination and regulate DSB number.

The first regulatory pathway presented in this work is the crossover assurance checkpoint, which promotes crossover recombination events on all chromosome pairs to ensure successful meiosis. Under the crossover assurance checkpoint, DSB formation is prolonged when one or more homologous chromosome pair fails to form a crossover precursor. This increase in meiotic recombination initiation events gives cells additional opportunities to produce the crossovers necessary for proper chromosome segregation.

I also present evidence for a separate regulatory pathway that functions to limit the number of DSBs. I describe a negative feedback loop that is mediated by DNA-damage response kinases ATM and ATR and acts though DSB-1 to down-regulate DSB formation. This regulatory pathway permits the formation of a limited number of meiotic DSBs, while preventing excess DSB levels. Furthermore, my results demonstrate the resilience of meiotic cells in tolerating excess levels of meiotic DSBs without negative consequences for genomic integrity or crossover regulation.

The work presented here provides important insights into the regulation of meiotic recombination initiation. Although the regulatory pathways described here were identified in C. elegans, recent studies suggest that similar regulatory pathways are likely to be conserved in other organisms. Therefore this work is important not only for understanding DSB regulation in C. elegans, but may also shed light on the regulation of meiotic DSBs in other eukaryotic organisms.

Faithful segregation of homologous chromosomes during meiosis requires pairing, synapsis, and crossing-over. In the nematode Caenorhabditis elegans, homolog pairing and synapsis depend on pairing centers, special regions near one end of each chromosome that interact with the nuclear envelope and cytoplasmic microtubules. Pairing centers are required for nuclear reorganization at the onset of meiosis. Here, I show that pairing centers recruit the Polo-like kinase PLK-2 to induce nuclear envelope remodeling, chromosome pairing, and synapsis. Recruitment of PLK-2 is also required to mediate a cell cycle delay and selective apoptosis of nuclei containing unsynapsed chromosomes, establishing a molecular link between these two quality control mechanisms. I also show that the checkpoint kinase CHK-2 is recruited to the interface of pairing centers and nuclear envelope proteins, consistent with its role in promoting homolog interactions and nuclear reorganization. Interestingly, PLK-2 is later recruited to the axes of paired and synapsed chromosomes where it plays a downstream role in synaptonemal complex dynamics. This work reveals new functions for the conserved family of Polo-like kinases, and advances our understanding of how meiotic processes are properly coordinated to ensure transmission of genetic information from parents to progeny.

Meiosis is the specialized cell division by which haploid egg and sperm cells are generated from mitotically dividing diploid germ cells. During early prophase, the environment of the nucleus is dynamic, as a given chromosome will undergo a series of encounters with homologous and nonhomologous chromosomes. A fundamental problem is how each chromosome is able to discriminate and accurately pair and form stabilized interactions via the synaptonemal complex only with its appropriate partner. It is only within this context of homologous interactions that recombination can take place, which is what allows for genetic diversity among sexually reproducing species.

In Chapters 1 and 2, I will focus on homolog pairing and synapsis and will discuss experiments that point towards the Pairing Center protein HIM-8 functioning as a molecular link that couples these two events. In Chapter 3, I will discuss the role of the chromatin factor GAK-1 in meiosis. I will show that GAK-1 plays a role at multiple steps in meiosis, including recombination and maintenance of the synaptonemal complex. The data suggest that GAK-1 may function as a transcription factor for meiosis-specific genes, which would explain the pleiotropic effects seen in meiosis. These studies will hopefully further our understanding of the molecular basis of aneuploidies.

Sexual reproduction requires meiosis, the unique cell division in which haploid

gametes are generated from a diploid cell. A hallmark of meiotic prophase is the formation of pairwise linkages between homologous chromosomes, which later enable them to segregate away from each other at meiosis I. In most organisms the pairing of homologous chromosomes is reinforced by polymerization of the synaptonemal complex (SC) between paired chromosome axes, or synapsis. The primary questions addressed in this study are: 1) how pairing is accomplished and 2) how synapsis is regulated so that it occurs selectively between homologs. In Chapter I, I present evidence that a bridge of the nuclear envelope proteins comprised of SUN-1 and ZYG-12 connects meiotic chromosomes to the microtubule cytoskeleton, and this connection spanning the nuclear envelope is critical for coordinating homolog pairing and synapsis. I propose a model in which dynein-based forces are used to assess homology between chromosomes and to license subsequent chromosome interactions during meiotic prophase. In Chapter 2, I identify a novel Pairing Center-interacting protein DPY-30 and present my characterization of the germline in a dpy-30 mutant. I explore different possibilities to investigate the functional significance of the interaction between DPY-30 and Pairing Center proteins. I discuss possible roles of DPY-30 in early meiosis based on my preliminary results.