Tag: Diana Libuda

Investigating sexual dimorphic P-granule structures during germ cell development in C. elegans

Presenter: Madison Studer – Neuroscience

Faculty Mentor(s): Acadia DiNardo, Diana Libuda

Session: (In-Person) Poster Presentation

Proper egg and sperm development is crucial for the faithful passage of the genome from one generation to the next. To prevent infertility and genomic instabilities linked to congenital disabilities, the process of sperm and egg development is tightly regulated by small RNA pathways. These pathways silence genes that disrupt the genome and maintain silencing across generations independent of DNA sequence, termed transgenerational epigenetic inheritance. In Caenorhabditis elegans, the components of small RNA pathways localize to P-granules, liquid-like condensates that form around the nuclei of developing sperm and eggs. ZNFX-1, a recently discovered structural P-granule component, is required for genome maintenance and fertility. Although ZNFX-1 is known to be involved in transgenerational epigenetic inheritance during egg development, the role of ZNFX-1 during sperm development remains unknown. Preliminary data from the Libuda Lab suggests that ZNFX-1 has sex-specific localization, indicating distinct sex-specific mechanisms for genome maintenance in egg and sperm development. To determine the sexually dimorphic localization patterns of ZNFX-1 during sperm and egg development, I am examining GFP-tagged ZNFX-1 localization in wild type and mutant P-granule strains. This work will reveal the sex-specific role of ZNFX-1 in small RNA pathways and provide insights into the molecular mechanisms that maintain genomic integrity and fertility.

Investigating the role of H3K9 methyl transferases in heat-induced DNA damage

Presenter: Philip Nosler – Biology

Faculty Mentor(s): Nicole Kurhanewicz, Diana Libuda

Session: (In-Person) Oral Panel—Bio-Zebrafish and DNA

Exposure to elevated temperature is a major cause of male infertility observed across both animals and plants. A primary consequence of heat stress is the accumulation of unusually high levels of DNA damage in developing sperm. Previous work from the Libuda Lab demonstrated that, similarly to humans, a single acute heat exposure is sufficient to produce high levels of DNA damage in developing sperm, but not in developing eggs in the model organism Caenorhabditis elegans. Further, mobilization of transposons, segments of DNA that can move autonomously throughout the genome, was associated with heat-induced DNA damage specifically in sperm. Normally, transposon movement is strictly repressed in the germline via chromatin modifications, which affect chromosome structure and regulate gene expression. Specifically, transposon genes are silenced in the germline via a particular chromatin modification: methylation of histone H3 lysine 9 by the methyltransferases SET-25, SET-32, and MET-2. Using an existing mutant strain for set-25 and a double mutant for met- 2;set-25, I found that DNA damage is elevated following heat stress, suggesting set-25 and met-2 repress heat-induced DNA damage. Currently, I am further assessing the roles of set-25, set-32, and met-2 in heat-induced DNA damage using single and double mutant strains. Overall, this work will further our understanding of the mechanisms underlying heat-induced male infertility.

Meiotic recombination is regulated by dosage of synaptonemal complex proteins

Presenter: Amelia Dayton — Biology

Faculty Mentor(s): Cori Cahoon, Diana Libuda

Session: (In-Person) Oral Panel—Daily Dose of Proteins

Meiosis is a specialized cell division that produces haploid gametes, such as sperm and eggs. To ensure each parental genome is inherited properly, cells must pair homologous chromosomes, induce DNA double-strand breaks, repair these breaks as crossovers, and segregate the chromosomes. The synaptonemal complex (SC), a large protein structure, assembles between homologs and facilitates crossing over, which ensures accurate chromosome segregation. Using Caenorhabditis elegans, previous work in the Libuda lab showed that two SC proteins, SYP-2 and SYP-3, have dosage-dependent functions in regulating crossing over. SYP-2 dosage is critical for regulating early crossover steps, while SYP-3 dosage influences the timing of crossover establishment. Crossovers are nonrandomly positioned on chromosomes, and whether SYP dosage influences crossover position remains unclear. To this end, I am using single nucleotide polymorphisms to characterize the positions and rates of crossovers in cells with altered SYP-2 and SYP-3 dosages. Preliminary data on Chromosome X shows that SYP-dosage is crucial for proper crossover positioning. Since the sex chromosomes often behave differently from autosomes, I am also determining the effect of SYP- dosage on crossovers across Chromosome II to establish whether autosomes show similar changes in crossover positioning. Overall, these experiments will define the dosage-dependent manner that SYP-2 and SYP-3 regulate recombination to promote fertility.

Investigating the Role of Transposons in Temperature-Induced DNA Damage During Spermatogenesis

Presenter(s):  Colin Maxwell − Biology

Faculty Mentor(s): Diana Libuda, Nicole Kurhanewicz Codd

Oral Session 3M

Research Area: Biological Science

Meiosis is a specialized form of cell division that sexually reproducing organisms use to generate haploid sex cells. Developing sperm are particularly sensitive to temperature fluctuations, with some studies indicating that exposure to elevated temperature increases DNA damage in spermatocytes, but not oocytes. Although temperature-induced DNA damage has been observed, the underlying molecular mechanisms remain unknown. DNA transposons are mobile genetic elements that produce double-strand DNA breaks (DSBs) when excised from the genome. Additionally, transposons can excise from the genome under heat stress.

I hypothesize that heat stress causes transposon excision which may be observed as a linear relationship between transposon copy number and the quantity of DSBs in developing spermatocytes exposed to elevated temperature. To test this hypothesis, I conducted an immunofluorescence screen of wild type Caenorhabditis elegans strains with varying transposon copy numbers. Using deconvolution microscopy, DSBs were visualized via the recombinase RAD-51, a protein involved in the early stages of meiotic DSB repair. Quantification of RAD-51 foci was performed to determine the frequency of temperature-induced DSB formation. Preliminary results demonstrate that the CB4856 strain with ~15 copies of Tc1, a class of transposons active in C. elegans, exhibited half the amount of DSBs as the Bristol N2 strain with ~30 copies of Tc1 displayed upon heat shock. In contrast, comparisons of DSB quantities between additional strains with varying Tc1 copy numbers show no clear relationships. Taken together, these results indicate temperature-induced DNA damage in spermatocytes has multiple mechanisms, with excision of Tc1 transposons as one possible mechanism.

Investigating the Role Of Genomic Positioning in Directing Meiotic Double-Strand DNA Break Repair

Presenter(s): Rachel David − Biochemistry

Faculty Mentor(s): Diana Libuda, Erik Toraason

Oral Session 3M

Research Area: Biological Sciences

Funding: UO Alden Scholar Research Award, NICHD Grant

During meiosis, the specialized form of cell division that produces gametes, cells utilize recombination to maintain genomic integrity and promote proper chromosome segregation, ensuring fertility. Double-strand DNA breaks (DSBs), which serve as substrates for homologous recombination, are intentionally induced during meiosis. A fraction of DSBs must be repaired
as crossover recombination events with the homologous chromosome to forge a physical connection required to facilitate proper chromosome segregation. Although DSBs are induced across the genome, crossovers in C. elegans are preferentially formed along chromosome arms and not the center of chromosomes. What determines this crossover preference along chromosome arms is not well known. Intriguingly, previous studies in C. elegans have indicated that the crossover landscape is not determined by chromatin marks or specific sequence motifs. However, proximity of a DSB to the synaptonemal complex (SC), a meiosis-specific proteinaceous structure that connects homologous chromosomes together, has been suggested to influence DSB repair outcomes. To determine how genomic positioning of a DSB affects its repair outcome, I am exploiting genetic assays developed by the Libuda lab that enable controlled induction of a single DSB at a known genomic location and assess how that induced DSB was repaired. Utilizing CRISPR/Cas9 genome editing, I have targeted these assays to four unique loci that differ in position both along the chromosome length and in proximity to the SC. Together, our studies will elucidate how the position of a DSB within the genome influences how it is repaired to maintain genome integrity.

SMC-5/6 Facilitates Efficient DSB Repair During Meiosis in C. elegans

Presenter(s): Cordell Clark − Biology

Faculty Mentor(s): Diana Libuda, Erik Toraason

Poster 73

Oral Session 3M

Research Area: Biological Science

Funding: OURS Program Summer 2017, Dr. Diana Libuda’s laboratory is funded in part by an NICHD grant

Sexually reproducing organisms depend upon meiosis to form haploid sex cells necessary for reproduction. Despite the inherent risks of DNA damage to genome integrity, meiotic cells intentionally induce double strand DNA breaks (DSBs) throughout the genome. A specific and limited number of DSBs must be repaired as crossovers with the homologous chromosome to promote proper chromosome segregation. DSBs are induced in excess of the permitted number of crossovers. DSBs not repaired as crossovers must be repaired to maintain genomic integrity. In the Libuda lab, we have designed an assay to determine the repair outcome of a single induced DSB. We have demonstrated that in addition to the homologous chromosome, the sister chromatid is used as a repair template during DSB repair in C. elegans meiosis. My research aims to uncover the mechanisms that facilitate intersister repair, which are currently unknown. Experiments in multiple species have demonstrated that SUMOylation is required for crossover formation. Our preliminary immunofluorescence experiments in SUMO deficient mutants reveal SUMOylation is required for DSB repair in C. elegans oocytes. Prior research has hypothesized that the SMC5/6 complex, which contains a SUMO ligase subunit (NSE-2), facilitates intersister recombination. Using our intersister repair assay, I have demonstrated that the SMC5/6 complex is required for efficient intersister repair and intersister crossovers. My ongoing experiments are directly testing whether the SMC5/6 complex promotes intersister recombination via the NSE-2 SUMO ligase subunit. Overall, our studies are defining the mechanisms that facilitate intersister recombination to ensure genome integrity during sperm and egg development.

Characterizing Early DNA Break Repair in C. Elegans

Presenter(s): Nicole Szczepanski

Faculty Mentor(s): Diana Libuda & Austin Harvey

Poster 19

Session: Sciences

Accurate chromosome segregation is critical for the formation of viable gametes by the specialized cell division of meiosis. During meiosis, programmed double strand DNA breaks (DSBs) are formed and repaired by recombination mechanisms to maintain genomic integrity and to promote proper chromosome segregation. In order to better understand early repair dynamics of DSBs, we intended to devise a strain via CRISPR with an early repair phenotype closer to wildtype phenotype for future live imaging experiments. In past experiments, endogenously tagged GFP::RAD-51 mutants were utilized, but strayed from the usual wildtype phenotype. RAD-51 is a conserved recombinase that indicates an early repair stage of DSBs and is required for all meiotic recombination events. Using immunofluorescence, DSBs display distinct early repair dynamics through differential RAD-51 foci, leading to the hypothesis that these distinct dynamics indicate different DSB repair outcomes. Using the C. elegans model, we found that endogenously tagged GFP::RAD-51 mutants did not show a more wildtype RAD-51 foci phenotype after inheriting two copies of wildtype RAD-51 compared to worms that did not inherit the duplication. We also found that there is a significant difference between RAD-51 foci in early pachytene and late pachytene, the former having larger volumes and stronger intensities, representing interhomolog repair outcomes. In addition, interhomolog crossover repair outcomes show smaller, dimmer foci than do noncrossover outcomes. This indicates differential DSB end-resectioning between different stages within meiosis and between different repair outcomes.

SMC-5/6 E3 SUMO ligase subunit NSE-2 is required for robust repair of meiotic DNA double-strand breaks

Presenter(s): Alina Salagean

Faculty Mentor(s): Diana Libuda & Erik Toraason

Oral Session 3 M

Most organisms utilize meiosis, a specialized form of cell division, to produce haploid gametes such as sperm and eggs. Failure to maintain genomic integrity during meiosis can cause infertility and cancer. Using the model organism Caenorhabditis elegans, previous work has demonstrated that the conserved Structural Maintenance of Chromosomes 5/6 complex (SMC-5/6) is required for robust repair of double-strand DNA breaks (DSBs) in late meiotic prophase I. The specific mechanisms by which SMC-5/6 promotes DSB repair remain unknown. One subunit of the SMC- 5/6 complex, the E3 SUMO ligase NSE-2, has been implicated in DNA repair in multiple organisms. To identify the specific contributions of NSE-2 to meiotic DSB repair and fertility, we generated four nse-2 null mutants using CRISPR/Cas9 genome editing and assessed their phenotypes associated with genome integrity across generations. Utilizing these nse-2 mutants, we find that similarly to SMC-5, NSE-2 is required for a germ line-response to exogenous DNA damage. In contrast, unlike SMC-5, NSE-2 is not required for maintenance of fertility over generations. These data suggest NSE-2 is required for either a specific subset of functions of the SMC-5/6 complex or the efficient function of SMC-5/6. Our future experiments will utilize both genetic assays and immunofluorescence imaging techniques to distinguish between these hypotheses. Taken together, our research defines mechanisms preserving genomic integrity and fertility across generations.

Mechanisms of sister chromatid repair during meiotic double-strand DNA break repair

Presenter(s): Anna Horacek

Faculty Mentor(s): Diana Libuda & Erik Toraason

Oral Session 3 M 

Poster 68

Session: Sciences

Most sexually reproducing organisms utilize meiosis, a specialized form of cell division, to generate haploid gametes such as eggs and sperm. Meiotic cells utilize recombination to repair double-strand DNA breaks (DSBs) with either the sister chromatid or homologous chromosome as a repair template. Although recombination with the homologous chromosome has been extensively studied, little is known about engagement of the sister chromatid during meiotic DSB repair. To characterize sister chromatid recombination, we have developed a sister chromatid repair (SCR) assay in Caenorhabditis elegans. The SCR assay contains engineered nucleotide polymorphisms enabling the detection of gene conversions between sister chromatids, which arise from the nonreciprocal exchange of sequences between chromosome templates and indicate recombination intermediates. Analysis of these conversions tracts indicate that throughout meiotic prophase I: 1) sister chromatid repair intermediates remain central to the site of DSB induction; and, 2) sister chromatid repair is highly processive, as template switching is not observed. Interestingly, the length of conversion tracts, indicating the extent of DSB resectioning, changes in the presence of a homolog repair template. In the absence of a homolog repair template, the conversion tract size is uniform throughout prophase I. When a homolog repair template is present, large conversion tracts (>210bp) are observed only in late prophase I, suggesting the presence of the homolog repair template may affect the extent of DSB resectioning in late prophase I. Taken together, our work presents a comprehensive analysis of meiotic sister chromatid recombination and defines mechanisms fundamental to the preservation of genomic integrity.

Piwi-piRNA pathway protein PRG-1 represses in temperature-induced DNA damage in spermatocytes

Presenter(s): Fountane Chan

Faculty Mentor(s): Diana Libuda & Nicole Kurhanewicz

Oral Session 3 M

Poster 47

Session: Sciences

Half of infertility cases worldwide involve male-factor subfertility. As awareness and frequency of male infertility has grown, it is increasingly important to understand the underlying mechanisms of these major human health concerns. Developing sperm are particularly sensitive to fluctuations in temperature, requiring a narrow isotherm of 2-7°C below core body temperature. Although both oocytes and spermatocytes undergo meiosis, the specialized form of cell division that produces haploid sex cells, elevated gonadal temperatures have been shown both to impair only male fertility and produce excess DNA damage specifically in spermatocytes. Preliminary work using the powerful roundworm model Caenorhabditis elegans suggests the Piwi-piRNA pathway, a highly conserved genome maintenance pathway, is involved in temperature-induced DNA damage. Absence of worm-specific Argonaute proteins (WAGO), primary effector proteins of the Piwi pathway, results in considerably elevated DNA damage upon heat-shock. Interestingly, a panel of mutants deficient in the C. elegans Piwi protein, PRG-1, which functions upstream of WAGOs, demonstrate highly variable degrees of heat-induced DNA damage. This variability is likely due to acquired mutations stemming from inadequate germline surveillance over multiple generations. To circumvent this issue by controlling the number of generations a strain is without PRG-1, we generated a conditional knockdown mutant of PRG-1. Utilizing this mutant, we found that in the absence of PRG-1 after one generation, spermatocytes demonstrate exacerbated levels of heat-induced DNA damage, similar to WAGO null mutants. Taken together, my data suggest a key role for PRG-1 and male-specific components of the Piwi pathway in heat-induced DNA damage in spermatocytes.