Abby F Dernburg et al. (2001) International C. elegans Meeting "Chromosome remodeling during meiosis"

Abby F Dernburg, Bobby Hogg, Carol Anderson

[International C. elegans Meeting, 2001]

This abstract is being submitted as a placeholder for a revised abstract to be entered during the week of April 20. This delay is necessary because we would like to include data from different lab personnel, and my rotation students (currently comprising the entire lab) will be making their final lab decisions that week. We are working on several questions regarding chromosome and chromatin remodeling. In particular, we are interested in how chromatin is remodeled during meiosis to accomplish chromosome pairing, crossing-over, and segregation.

Liu, Chenshu et al. (2021) International Worm Meeting "Rewiring quality control in C.elegans meiosis using a new chemically-induced proximity system"

Liu, Chenshu, Dernburg, Abby F.

[International Worm Meeting, 2021]

During the extended meiotic prophase, homologous chromosomes must pair, culminating with the assembly of synaptonemal complex (SC) along their lengths (synapsis). Synapsis is essential for crossover formation between homologs, and thus for their accurate segregation. In C.elegans, defects in synapsis are monitored by a quality control program called the synapsis checkpoint, as one or more pairs of unsynapsed chromosomes lead to a cell cycle delay and can eventually trigger apoptosis. Previous work from our lab revealed that the synapsis checkpoint requires the presence of unsynapsed pairing centers (PCs), special regions on each chromosome that promote homolog pairing and synapsis. However, how cells detect defects in SC assembly remains unknown. In C.elegans, the Polo-like kinase PLK-2 shows dynamic subnuclear localization during meiotic prophase: it is first recruited to PCs during early prophase, and following synapsis it relocalizes to the SC. This suggests that the localization of PLK-2 might be part of the signal that triggers the synapsis checkpoint. To test this idea, we deployed a new chemically-induced proximity (CIP) system that we engineered by modifying a core component of the auxin-inducible degradation system. We engineered mutations into the F-box protein TIR1 to prevent it from interacting with other ubiquitin ligase components. By fusing one protein to this TIR1 sequence and another to a "degron" peptide, we can induce proximity between the two tagged proteins using the small molecule indole acetic acid (auxin). With this system, we successfully targeted PLK-2 to specific chromosomal/nuclear structures. We found that ectopic targeting of PLK-2 to X-chromosomal PCs following synapsis was sufficient to induce apoptosis. Importantly, such induced apoptosis did not require HUS-1, an essential component of the DNA damage checkpoint, but was abrogated by mutation of PCH-2, an essential component of the synapsis checkpoint. By combining this CIP system with various meiotic mutants, I will also discuss about how PLK-2 coordinates with other meiotic kinases and the nucleoskeleton during meiotic quality control and cell cycle progression. Together, we have developed a simple, versatile CIP system and leveraged it to better understand the mechanisms underlying meiotic progression and quality control. This CIP system will enable a wide variety of new experiments in C. elegans and other model organisms.

Rog, Ofer et al. (2013) International Worm Meeting "In vivo visualization of chromosome synapsis in C. elegans."

Rog, Ofer, Dernburg, Abby F.

[International Worm Meeting, 2013]

Meiosis is the special cell division process that enables the production of haploid gametes. During meiotic prophase, chromosomes form linkages with their homologous partners to enable reductional segregation during the first meiotic division. Essential to this process is the pairwise alignment of homologous chromosomes along their entire lengths. In most eukaryotes this alignment is reinforced through synapsis, the assembly of a structurally conserved polymer called the synaptonemal complex (SC), which links homologous chromosomes. The SC promotes genetic exchanges (crossovers) between homologs, and likely regulates their number and location. Synapsis is a dynamic process, the details of which are difficult to infer from images of fixed cells or tissues. Our goal is to illuminate this process through analysis in living nematodes, using fluorescently tagged SC components and high-resolution time-lapse microscopy. Our observations have revealed that initiation of synapsis is a relatively infrequent event that is rate-limiting for completion of synapsis, consistent with evidence that initiation is subject to strict regulation. Initiation occurs at the "Pairing Centers" - regions near one end of each chromosome where initial pairing of homologous chromosomes is achieved. Once initiated, synapsis extends along the chromosome at a rate of ~160nm per minute. Individual chromosomes thus complete synapsis within 20-30 minutes of initiation. In C. elegans, synapsis is accompanied by rapid chromosome motions driven by dynein, which is coupled through the nuclear envelope to the Pairing Centers. Under conditions where this motion is severely abrogated, synapsis remains processive but is ~5-fold slower. This suggests that chromosome motion promotes homolog alignment, enabling synapsis to proceed more rapidly. Moreover, it reveals a novel function for the rapid chromosome motions that have been observed in diverse organisms during meiotic prophase.

Glazier, Christina M et al. (2011) International Worm Meeting "Asymmetric chromosome behavior during oocyte meiosis."

Glazier, Christina M, Dernburg, Abby F

[International Worm Meeting, 2011]

Meiosis is the specialized cell division that gives rise to haploid gametes. In order to reduce the chromosome complement by half, meiosis involves one DNA replication event followed by two chromosome segregation events. For proper segregation to occur, homologous chromosomes must pair, synapse, and undergo crossover recombination to form physically linked bivalents. A microtubule-based spindle then separates the bivalents so homologous pairs are pulled to opposite spindle poles. Sister chromatids then realign at the metaphase plate and segregate to opposite poles during meiosis II. In animals, oocyte meiosis gives rise to a single functional gamete, rather than four, and the other meiotic products are typically extruded into a polar body that does not contribute to the developing zygote. In cases where the two homologous copies of a chromosome missegregate, or nondisjoin, at the first meiotic division, the expectation is that each gamete would have an equal probability of receiving either zero or two copies of that chromosome. However, in C. elegans, it has long been recognized that XO progeny (males) greatly exceed the number of triplo-X (Dumpy) hermaphrodites, both in wild-type broods and in mutants with nonrecombinant (achiasmate) X-chromosomes, such as him-5 or him-8. I am exploring the basis for this unusual inheritance pattern to learn more about the mechanisms underlying meiotic chromosome segregation. One hypothesis that I am testing is that the excess of males over triplo-X hermaphrodites could result from a preferential missegregation of achiasmate X chromosomes into the polar body. I am developing genetic and cytological assays to determine whether this asymmetric chromosome behavior is specific to the X chromosome, or instead reflects an intrinsic asymmetry of the spindle. Eventually I also plan to identify genetic factors that alter the segregation behavior, perhaps resulting in more random behavior.

Whittle, Christina M. et al. (2011) International Worm Meeting "Purification of intact germline nuclei: Towards a high-resolution spatiotemporal map of meiotic chromatin in C. elegans."

Dernburg, Abby F., Whittle, Christina M.

[International Worm Meeting, 2011]

C. elegans as a model has traditionally lent itself well to genetic and cytological studies with strengths being the well-characterized, invariant cell lineage leading to relatively few tissue types. However, biochemical studies thus far have been largely limited to whole animal extracts leaving tissue-specific studies consigned to the resolution afforded by cytology. C. elegans simplicity as a metazoan model gives it enormous untapped potential as a system in which to do tissue-specific biochemistry in primary cells. Methods for isolating specific tissues to analyze protein-protein or protein-DNA interactions would bridge the gap between cytological analysis, which provides low-resolution but tissue-specific information, and the molecular interaction data obtained from whole animal extracts, such as ChIP-Seq. Germline processes, such as meiosis and the specialized chromatin inherent to its execution, provide an ideal test case and intriguing subject for examination. We employed a nuclear envelope tagging strategy to tissue-specifically label nuclei with GFP and both employing and simplifying an approach recently reported in plants (1), in C. elegans. We obtained GFP::ZYG-12 driven specifically in the germline under the pie-1 promoter (2) and isolated intact nuclei. To circumvent the use of antibodies for purification, which could impair vital downstream applications such as ChIP, we utilized an unusual GFP binding protein (GBP) derived from camelid antibodies (3) to magnetically label only the GFP tagged nuclei, followed by bulk isolation. We are now optimizing the purification protocol, as well as generating meiosis-specific tagged nuclei. We will present preliminary results that demonstrate the feasibility and purity of germ nuclei isolated for biochemical applications such as ChIP-seq. References: 1. Deal RB and Henikoff S. (2010) Dev Cell. 18(6):1030-40. 2. Malone et al. (2003) Cell. Vol. 115 (7) pp. 825-36. 3. Rothbauer et al. (2008). Mol Cell Proteomics. 7, 282-9.

Kim, Yumi et al. (2015) International Worm Meeting "The meiotic chromosome axis mediates feedback control of CHK-2 activity during meiosis."

Dernburg, Abby F., Kostow, Nora, Kim, Yumi

[International Worm Meeting, 2015]

A series of events during the meiotic cell cycle enables the segregation of homologous chromosomes to form haploid gametes. Early in meiosis, chromosomes are reorganized around a central axis, and then pair, synapse, and recombine with their homologs. In C. elegans, a meiosis-specific paralog of the DNA damage checkpoint kinase CHK-2 is required for pairing, synapsis, and recombination. We have found that CHK-2 promotes pairing and synapsis by phosphorylating a family of zinc finger proteins (HIM-8, ZIM-1, ZIM-2 and ZIM-3) that bind to pairing centers, specialized regions of each chromosome that play essential roles in meiotic chromosome dynamics. Phosphorylation of these proteins by CHK-2 primes their recruitment of the Polo-like kinase PLK-2 to promote pairing and synapsis. A phospho-specific antibody has revealed that CHK-2 activity normally declines once all chromosomes have accomplished synapsis and crossing-over, but is prolonged in mutants that disrupt crossover formation, suggesting that CHK-2 activity is regulated by a feedback circuit that monitors crossover formation. Interestingly, CHK-2 activity is not extended in mutants that disrupt the meiotic HORMA domain proteins HIM-3, HTP-1, or HTP-3, which are components of meiotic chromosome axes, despite their inability to form crossovers. Deleting HIM-3 or HTP-1/2 suppresses the extension of CHK-2 activity in other meiotic mutants, indicating that each of these proteins plays an essential role in sensing and/or signaling meiotic defects for CHK-2 regulation. Feedback control is also abrogated by mutations in HTP-3 that prevent recruitment of HTP-1/2 or HIM-3 to the chromosome axis, indicating that interactions among these axis-associated proteins are essential for meiotic feedback regulation. Our findings establish the molecular basis for checkpoint mechanisms targeting CHK-2 that coordinate meiotic chromosome dynamics with cell cycle progression. .

Liu, Chenshu et al. (2021) International Worm Meeting "Nuclear lamina cooperates with inner nuclear membrane proteins to counteract LINC-mediated forces during oogenesis in C. elegans"

Wang, John, Dernburg, Abby F., Liu, Chenshu, Wu, Fan, Lung, Zoe

[International Worm Meeting, 2021]

The nuclear lamina is essential to protect genome integrity from mechanical stress. This requirement is more stringent in some tissues and developmental events. During oogenesis, meiotic nuclei are situated in a stressful environment filled with cytoskeletons. The nuclear lamina, consisting of the lamin proteins, is a conserved component of the nuclear envelope that can confer mechanical rigidity to the meiotic nuclei. C.elegans expresses a single lamin protein, LMN-1, which is similar to mammalian B-type lamin. Loss of LMN-1 results in near-complete sterility, with hypercondensed chromatin observed in many germline nuclei. The exact functions of LMN-1 in meiotic nuclei during oogenesis, however, remains unclear. Using the auxin-inducible degradation system, we found that acute depletion of LMN-1 in C.elegans germline recapitulated nuclear collapse seen in lmn-1 homozygotes during late stages of meiotic prophase. LMN-1 depletion also led to persistent DNA double strand breaks and elevated apoptosis, but germline apoptosis is neither sufficient nor required for nuclear collapse. We further observed prolonged and excessive clustering of the LINC complex proteins SUN-1 and ZYG-12 at the nuclear envelope (NE) upon LMN-1 acute depletion. Importantly, co-depletion of SUN-1 or ZYG-12, or inhibition of dynein-mediated forces, rescued the nuclear collapse triggered by acute LMN-1 depletion. By contrast, co-depletion of the inner nuclear membrane proteins EMR-1/LEM-2 or SAMP-1 rendered nuclei susceptible to collapse even earlier, at the time of meiotic entry. Live imaging demonstrated that shrinkage of the NE preceded chromosome hypercondensation during nuclear collapse, and that before NE shrinkage happened, LINC complex asymmetrically redistribute to one side of the NE in a dynein-dependent manner. Finally, the connection between the pairing center regions of the chromosomes and the NE, albeit being important for LINC complex function during homolog pairing, is dispensable for nuclear collapse caused by LMN-1 depletion. Together our results suggest that lamin cooperates with additional inner nuclear membrane proteins to protect meiotic nuclei from collapse by antagonizing forces exerted by dynein and transmitted through the LINC complex during oogenesis. Our work has also established an inducible system for modeling laminopathy.

Harper, Nicola C. et al. (2011) International Worm Meeting "A Polo-like kinase coordinates meiotic chromosome dynamics."

Harper, Nicola C., Rillo, Regina, Bhalla, Needhi, Jover-Gil, Sara, Dernburg, Abby F.

[International Worm Meeting, 2011]

Accurate segregation of homologous chromosomes in meiosis relies on their pairing, synapsis and recombination. Previous work from our lab has characterized special regions of C. elegans chromosomes termed pairing centers (PCs) which are specifically bound by a family of zinc finger (ZnF) proteins and facilitate homolog pairing and synapis. Homolog interactions are first established in the "transition zone" region of the gonad, which is marked by nuclei with clustered chromosomes. In the transition zone, PCs colocalize with patches of the nuclear envelope (NE) proteins SUN-1 and ZYG-12 to form a bridge between chromosomes and the cytoplasmic microtubule network. This connection facilitates homolog pairing and restricts synapsis to occur specifically between properly paired homologs. PC-mediated chromosome connections to the cytoskeleton appear to be a variation of the widely conserved meiotic bouquet, which is typically mediated by telomeres. Major unanswered questions have been how PCs exert their functions and how components of the NE bridging patches are established at meiotic entry and maintained until homolog pairing and synapsis are complete. We have defined essential roles for the Polo-like kinase PLK-2 in coordinating these meiotic processes. We find that PC function is imparted by the recruitment of PLK-2 to PCs through an interaction with the ZnF proteins. This recruitment promotes SUN-1 phosphorylation at a key serine residue (Ser12), NE patch aggregation, and homolog pairing and synapsis. Loss of PLK-2 results in partial defects in homolog pairing and synapsis because the closely related PLK-1 can partially substitute for its role at PCs. Deletion of all four PC ZnF proteins, or the absence of both PLK-1 and PLK-2, completely abolishes chromosome clustering, phosphorylation of SUN-1 at Ser12, NE patch formation, and homolog interactions. We also find that PLK-2 is required for two critical responses to unsynapsed chromosomes: a cell cycle delay that maintains chromosome connections with the cytoskeleton to facilitate continued homology search, and the preferential apoptosis of nuclei containing unsynapsed chromosomes. This work reveals novel roles for Polo-like kinases and expands our understanding of meiotic regulatory mechanisms that ensure accurate transmission of genetic information from parents to progeny.

Kim, Yumi et al. (2015) International Worm Meeting "Dynamic phosphoregulation of axis proteins underlies chromosome remodeling during meiosis."

Kostow, Nora, Rog, Ofer, Kim, Yumi, Corbett, Kevin D., Dernburg, Abby F., Rosenberg, Scott C.

[International Worm Meeting, 2015]

Segregation of chromosomes in meiosis requires their dramatic, stepwise reorganization during meiotic prophase. Each chromosome must pair, synapse, and recombine with its homolog to achieve the stable bivalent structures that biorient and then divide during Meiosis I. A fundamental mystery is how these events are coordinated during the meiotic cell cycle. Pairing, synapsis, and recombination depend on the formation of linear "axes" along each chromosome at meiotic entry, followed by assembly of the synaptomemal complex (SC) between paired axes. Chromosome axes in C. elegans are comprised of cohesins and four related HORMA domain proteins: HIM-3, HTP-1, HTP-2, and HTP-3. We recently reported that the largest of these proteins, HTP-3, recruits the other three paralogs through short peptide sequences (closure motifs) in its C-terminal tail, which are bound by the HORMA domains of HTP-1, HTP-2, and HIM-3 (Kim, Rosenberg, et al., 2014). Here we show that these interactions are dynamically regulated by phosphorylation of the closure motifs in HTP-3 by two meiotic kinases, CHK-2 and PLK-2. Phosphorylation of the four central motifs reduces their binding affinity for HIM-3, and occurs in two temporally distinct waves. In early meiosis, phosphorylation along the entire axis by CHK-2 is required for efficient synapsis. A second wave of phosophorylation by PLK-2 occurs after crossover formation, and specifies the "short arm" of the bivalent where cohesion will be released during the first meiotic division. This second wave of HTP-3 phosphorylation requires both crossover formation and recruitment of PLK-2 to the chromosomes through a binding site in a SC component, SYP-1. Thus, phosphorylation-dependent regulation of HORMA domain protein assembly promotes dynamic remodeling of chromosome axes during meiotic progression, and is essential for proper segregation of holocentric chromosomes in C. elegans meiosis.

Wei-meng Woo et al. (2004) West Coast Worm Meeting "The extracellular matrix protein F-spondin is essential for muscle attachment and epidermal elongation"

Chris Suh, Yishi Jin, Andrew D Chisholm, Mei Ding, Christie Emigh, Wei-Meng Woo, Jian Cao

[West Coast Worm Meeting, 2004]

In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to vab-13 and ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.