Chu, Diana S. et al. (2013) International Worm Meeting "Spectrum: Building Pathways to Biomedical Research Careers for Girls and Women of Color."

Tanner, Kimberly D., Chu, Diana S., Parangan-Smith, Audrey G.

[International Worm Meeting, 2013]

Women of color are largely absent from the biomedical research community. Despite some progress made in encouraging girls to pursue career paths in science, few materials exist that specifically attract girls of color to these careers. To address this need, the Science Education Partnership and Assessment Laboratory (SEPAL) at San Francisco State University (SFSU) developed the Spectrum program. This program partners SFSU biomedical scientists who are women of color (including Biology faculty, undergraduate students, Masters students, and alumni in doctoral and biotechnology positions) with middle and high school girls and teachers. These teams collaborate to: 1) co-sponsor after-school science clubs targeting girls of color in high needs public schools, 2) develop a mentoring community of women of color trainees in biomedical research, 3) create video biographies highlighting the research programs and experiences of women of color biologists, and 4) partner with the local and national Expanding Your Horizons organizations to disseminate Spectrum activities. During its initial four years, Spectrum engaged 456 middle and high school girls (45% Latina, 13% African American, 22% Asian, 11% Unknown, 7% White) across nine clubs providing ~20 hours of academic enrichment for each girl, including two field trips to the laboratories of SFSU women of color biologists. Evaluation data shows increases in the percentage of participating girls who agree with the following statements: 1) I have heard a woman scientist talk about why she likes science (pre: 72%, post: 96%), and 2) I have met a woman scientist who is like me (pre: 36%, post: 65%);. Spectrum has also developed two video resources highlighting women of color from SFSU. 1) Women of Color Doing Biomedical Science: Inspiring Stories from Women of Color Biomedical Researchers and 2) From Us to Us: Advice on Careers in Biomedical Sciences for Girls & Women of Color. These are available on the SEPAL website and have been distributed through partnerships with national organizations supporting girls in science. Spectrum is supported by the National Institutes of Health through award #R25RR024307.

Diana S Chu et al. (2002) West Coast Worm Meeting "Identification of proteins involved in C. elegans sperm chromosome condensation."

Diana S Chu, Barbara J Meyer, John R Yates III, Hongbin Liu

[West Coast Worm Meeting, 2002]

The transfer of genetic material to subsequent generations requires the efficient packaging of DNA into chromatin during gamete formation. Spermatogenesis in C. elegans provides an ideal system to study the mechanisms of chromatin condensation during the processes of mitosis, meiosis, and spermiogenesis. Sperm chromatin is tightly compacted after meiosis and remains transcriptionally inactive in the spermatid until after fertilization, when it is decondensed. The proteins and mechanisms involved in this efficient packaging and subsequent disassembly of specialized chromatin are unknown. We are interested in characterizing proteins that are required specifically for sperm chromatin condensation. A proteomic approach is being utilized in which gamete chromatin is purified and associated proteins identified using a multidimensional protein identification technique (MudPIT). MudPIT utilizes two tandem liquid chromatography steps to provide resolution of complicated peptide mixtures before peptide identification through tandem mass spectrometry. Computer comparison against a predicted peptide database from the C. elegans genome is used to identify corresponding proteins. Progress in identification of candidate proteins and assessment of function in sperm formation will be presented.

Diana S Chu et al. (2003) International Worm Meeting "A Subtractive Proteomic Approach to Identify Sperm Chromatin-Associated Factors required for Male Fertility"

Barbara J Meyer, John R Yates, Hongbin Liu, Diana S Chu

[International Worm Meeting, 2003]

During spermatogenesis, chromosomes are partitioned and specifically packaged to ensure transmission of the intact paternal genome to the next generation. This packaging not only protects the sperm DNA from damage, it also aids in transcriptional silencing and efficient sperm motility. Important paternal factors required for development, like SPE-11, are also tightly associated with sperm chromatin. Proteins that assemble or associate with sperm chromatin are vital for fertility, as specific cases of human male infertility result directly from defective sperm chromatin packaging. Sperm-specific chromatin packaging is a conserved event from worms to humans; however, little is known about the basic cellular components necessary to achieve it. We are conducting a large-scale identification and analysis of sperm chromatin-associated proteins that are important for sperm chromatin packaging and fertility in C. elegans. A subtractive proteomic approach comparing sperm and oocyte chromatin has initially identified over 200 sperm-specific chromatin-associated proteins. Interestingly, 21% (44 out of 202) of the proteins identified show homology to mouse or human proteins that are linked to roles in spermatogenesis, gametogenesis, or meiosis. Initial candidates for further characterization were selected through homology with proteins linked to either fertility in other organisms or chromatin organization. Interesting candidates have emerged from this analysis including C. elegans protamine candidates, specific histone variants, and chromatin-associated phosphatases and kinases. Temporal and spatial requirements for initial candidate proteins are being assessed by immunofluorescence to help define how proteins participate in sperm-specific functions. As an example, E03A3.4 is a histone H3 variant that localizes specifically between homologs in metaphase I and sisters in metaphase II of meiosis and around mature sperm DNA, a pattern distinct from the centromeric localization of another histone H3 variant, CENP-A. E03A3.4 also colocalizes with the Aurora-like kinase, AIR-2, that is involved in chromosome segregation, condensation, cohesion, and cytokinesis. This finding represents the first description of a histone H3 variant localizing specifically between homologs and sisters during sperm meiosis. Functional roles in sperm chromatin assembly, fertilization, and embryogenesis of E03A3.4 and other candidate proteins are currently being assessed through RNAi or mutant analysis.

Diana S Chu et al. (2005) International Worm Meeting "Abundance Correlated Subtractive Proteomic Analysis of Sperm Chromatin Identifies Conserved Fertility Factors"

Hongbin Liu, John R Yates, Barbara J Meyer, Diana S Chu

[International Worm Meeting, 2005]

Though male infertility is a major contributor to reproductive failure, few molecular causes have been identified. To find proteins important for male gamete formation, we have used C. elegans to conduct a large-scale functional proteomic analysis of sperm chromatin, a fundamental cellular component whose integrity is required for fertility from worms to humans. To focus our analysis, selective approaches provided enrichment, prioritization, and subtraction of proteins to narrow 1000 proteins identified by proteomic analysis to 132 abundant sperm-enriched chromatin associated proteins. From this narrowed set of candidates, we found several sperm-specific proteins required meiotic progression and chromosome segregation during spermatogenesis. In addition, we also identified key proteins involved in kinetochore function, splicing regulation, and chromosome compaction with unexpected preferential enrichment during spermatogenesis. For example, HTAS-1, a histone H2A variant was identified as the first known C. elegans sperm nuclear basic protein. Importantly, though male reproductive genes evolve rapidly, 15% of abundant sperm chromatin enriched proteins are homologous to factors linked to fertility in mammals. Other C. elegans sperm chromatin enriched proteins identified that are conserved in mammals, but not previously implicated in fertility, may reveal universal features of male fertility and previously unknown etiologies of reproductive failure.

Beyene, Joseph et al. (2013) International Worm Meeting "The Role of Sperm Specific PP1 Phosphatase GSP-3/4 in Kinetochore Localization and Function During Spermatogenesis."

Beyene, Joseph, Chu, Diana

[International Worm Meeting, 2013]

Male infertility affects millions of couples within the US. Male fertility and sperm function depend upon proper chromosome segregation during meiosis. However, little is known about the molecular components required for chromosome segregation during sperm meiosis. Our lab examines the role of the PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) in C. elegans sperm meiosis. GSP-3/4 are 98% identical, sperm-specific, and localize to sperm chromosomes. Deletion of the gsp-3/4 genes causes failure of sperm chromosome segregation during meiosis. Though we have found that GSP-3/4 colocalize with components of the kinetochore, which attach microtubules to chromosomes, the specific role of GSP-3/4 in chromosome segregation remains unknown. Thus, we hypothesize that GSP-3/4 are required for the correct localization and function of kinetochore components during spermatogenesis. Consistent with this, we have found that kinetochore components HCP-2 and KNL-1 mislocalize in male gsp-3/4 mutants, suggesting a dependency upon GSP-3/4 for kinetochore localization.To visualize kinetochore localization and microtubule dynamics during the male meiotic divisions, we used immunolocalization of wild-type and gsp-3/4 mutants. In wild-type animals, HCP-2 surrounds dividing chromosomes then concentrates in the spindle midzone at the end of anaphase 2. In contrast, in gsp-3/4 mutants HCP-2 improperly concentrates in the spindle midzone at anaphase 1. Subsequent division failure in meiosis II results in the formation of incompletely separated chromosome "rods". Furthermore, KNL-1, which surrounds dividing chromosomes and localizes around the "lagging x" during anaphase 1, is also mislocalized in gsp-3/4 mutants. This also results in chromosome rod formation due to meiosis II division failure. Taken together, this data suggests a kinetochore component dependency upon GSP-3/4 for proper localization and function.Studies examining the role of GSP-3/4 in regulating additional kinetochore components are underway. Overall, this work indicates that kinetochore components likely have sperm-specific localization and dynamics that depend upon GSP-3/4.

Vlachos IS et al. (2016) Nucleic Acids Res "DIANA-mirExTra v2.0: Uncovering microRNAs and transcription factors with crucial roles in NGS expression data."

Vlachos IS, Hatzigeorgiou AG, Chatzopoulos S, Lykokanellos F, Paraskevopoulou MD, Georgiou P, Karagkouni D, Vergoulis T, Georgakilas G, Christodoulou F, Dalamagas T

[Nucleic Acids Res, 2016]

Differential expression analysis (DEA) is one of the main instruments utilized for revealing molecular mechanisms in pathological and physiological conditions. DIANA-mirExTra v2.0 (http://www.microrna.gr/mirextrav2) performs a combined DEA of mRNAs and microRNAs (miRNAs) to uncover miRNAs and transcription factors (TFs) playing important regulatory roles between two investigated states. The web server uses as input miRNA/RNA-Seq read count data sets that can be uploaded for analysis. Users can combine their data with 350 small-RNA-Seq and 65 RNA-Seq in-house analyzed libraries which are provided by DIANA-mirExTra v2.0.The web server utilizes miRNA:mRNA, TF:mRNA and TF:miRNA interactions derived from extensive experimental data sets. More than 450 000 miRNA interactions and 2 000 000 TF binding sites from specific or high-throughput techniques have been incorporated, while accurate miRNA TSS annotation is obtained from microTSS experimental/in silico framework. These comprehensive data sets enable users to perform analyses based solely on experimentally supported information and to uncover central regulators within sequencing data: miRNAs controlling mRNAs and TFs regulating mRNA or miRNA expression. The server also supports predicted miRNA:gene interactions from DIANA-microT-CDS for 4 species (human, mouse, nematode and fruit fly). DIANA-mirExTra v2.0 has an intuitive user interface and is freely available to all users without any login requirement.

Curran, Tyler S et al. (2013) International Worm Meeting "Giving Light to Sperm-Specific Phosphatases."

Chu, Diana, Mateo, Leslie, Curran, Tyler S

[International Worm Meeting, 2013]

Despite affecting millions in the U.S. alone, male infertility at the molecular level is poorly understood. Phenotypes of male infertility include improperly segregated sperm chromosomes and sperm immotility. For example, in mice these same phenotypes have been linked to defects in a sperm-specific isoform of protein phosphatase one (PP1) isoform, PP1g2. Our research in Caenorhabditis elegans has identified two sperm-specific PP1s GSP-3 and GSP-4, which are 98%; identical. We have shown that GSP-3/4 are functional homologues of PP1g2; targeted deletion of gsp-3/4 genes causes aberrant chromosome segregation during meiosis and immotility in mature sperm. PP1g2 and GSP-3/4 likely do not act alone, as PP1s rely on interacting proteins for catalytic activity and substrate specificity. Thus, many studies focus on individual PP1-protein complexes. While some sperm-specific PP1-protein complexes are starting to be identified, many of the interacting proteins and their functions remain unknown. To that end, we aim to identify the interacting proteins of GSP-3/4 in C. elegans. Previous work has given us candidate interacting proteins of GSP-3/4. Targeted deletions of gsp-3/4 disrupt major sperm protein (MSP) localization, a key protein in sperm motility. In addition, we have shown that GSP-3/4 localization is similar to that of kinetochores during sperm meiotic divisions; thus we expect GSP-3/4 may interact with kinetochore proteins. Our study is generating worms expressing green fluorescent protein (GFP) tagged GSP-3/4 (GSP-3/4::GFP). GFP will allow for live imaging of the enzymes and will also be a target for co-immunoprecipitation (co-IP) of GSP-3/4::GFP and their interacting proteins. Thus far, we have used Gateway Cloning (Invitrogen) to create GSP-3/4::GFP constructs in Mos1 single copy insert (MosSCI) compatible vectors. Using microinjection into MosSCI compatible EG6699 worms we have integrated single copies gsp-4::gfp into genomic DNA. We are currently working to integrate gsp-3::gfp constructs and validate fusion protein function. Our future studies will utilize these strains for the identification of interacting proteins by co-IP, as well as live imaging of GSP-3/4::GFP during spermatogenesis.

Nera, Bernadette et al. (2009) International Worm Meeting "Characterizing the roles of enhanced RNAi genes eri-1 and eri-3 in sperm development and function."

Ratner, Dmitry, Cintra, Thais, Nera, Bernadette, Chu, Diana

[International Worm Meeting, 2009]

Generation of functional sperm depends on timely control of the gene expression at specific stages of spermatogenesis. RNA interference (RNAi) pathways are involved in regulating gene expression and are required for male fertility in other organsims. In C. elegans, enhanced RNAi mutants, eri-1 and eri-3, are male sterile. Our lab is interested in investigating the role of these genes in proper sperm formation. Previous studies have found eri mutants are sterile. However, it is unknown if the sterility is caused by a lack of germ cell production or if germ cells are produced but not functional. We have found that eri mutant hermaphrodites produce less progeny than wild type at both permissive and restrictive temperatures. At 25 deg C eri mutants did not produce any viable progeny but were still able to produce oocytes and sperm. This indicates that though eri mutant sperm cannot fertilize, they are capable of generating a signal for oocyte maturation. In order to analyze how and where sperm formation is defective, temperature shifted eri mutant and wild type males were fixed and stained with the DNA dye DAPI. Overall the progression of germ cell nuclei appears to be normal; however, we observed chromosomes during late pachytene appear more diffusely compacted. In addition we observed defects in sperm meiotic chromosome segregation, with the presence of abnormally sized and shaped sperm nuclei, suggesting defects in chromosome segregation or integrity. We are currently analyzing the localization of components of the core chromosome segregation machinery to investigate how eri-1 and eri-3 mutants are defect in chromosome segregation. RNAi is important for genomic integrity in other organisms such as zebrafish, mice, and flies where small RNAs are important for transposon silencing. To investigate genomic integrity in C. elegans, we analyzed double strand break formation and germ cell apoptosis in eri mutants. Our preliminary results show that at 25C eri mutants do not generate more double stranded breaks than wild-type.Thus, unlike RNAi components in other organisms, eri mutants may not play a role in transposon silencing in C. elegans. The link between the enhanced RNAi phenotype of the eri mutants and chromosome defects resulting in abnormal sperm suggests that sperm-specific microRNAs may play an important role in sperm development.

Quintanilla, Luis et al. (2015) International Worm Meeting "Phosphoregulation balance allows successful segregation of chromosomes during male meiosis."

Cintra, Thais, Quintanilla, Luis, Cota, Vanessa, Chu, Diana

[International Worm Meeting, 2015]

Sperm meiosis, a highly regulated process, relies on proper microtubule attachments and timely removal of cohesion for chromosome segregation to take place. Phosphoregualtion has been demonstrated to play a role in the stabilizing and destabilizing of microtubule attachments at the kinetochore, a multi-layered structure connected to chromosomes. One example can be seen in human mitotic studies, where AIR-2 promotes microtubule destabilization while PLK-1 promotes microtubule stabilization. In another case, kinase AIR-2's destabilizing activity is opposed by PP1 Phosphatase GSP-2 in C. elegans oocyte meiosis. Although phosphoregulation plays a role in meiosis, little is understood about its regulatory mechanisms specifically in sperm meiosis. Our lab has discovered two 98% identical sperm-specific PP1 Phosphatases called GSP-3 and GSP-4 (GSP-3/4). They colocalize with the kinetochore and are essential for sperm meiotic chromosome segregation. Deletion of these phosphatases leads to delayed resolution of the single X chromosome, failure of chromosome segregation at meiosis II, and aneuploidy. We thus hypothesize that GSP-3/4 are dually responsible for regulating kinetochore microtubule interactions and directly or indirectly mediating the removal of cohesion proteins. Preliminary results show mislocalization of outer kinetochore kinase, BUB-1 during anaphase I in GSP-3/4 mutants. BUB-1 is prematurely released in the mid-zone, possibly explaining the lack of sister chromatic separation during the second part of meiosis. Indeed, we also see mislocalization of other outer kinetochore proteins like HCP-2, which supports role of GSP-3/4 regulating kinetochore microtubule interactions. Furthermore, we find that localization of AIR-2, the cohesion and microtubule destabilizing kinase, behaves normally in GSP3/4 mutant males. These results suggest that cohesion proteins are not impaired. In fact, our preliminary results indicate that REC-8, COH-3, and COH-4 are not mislocalized. Surprisingly, we see that LAB-1, a cohesion protector protein, is absent on the lagging X chromosome during anaphase 1, but remains intact between autosomes. Though LAB-1 absence suggests that cohesion would be cleaved early, we see a delay in X chromosome resolution. Thus, GSP-3/4 regulation of cohesion requires further investigation. Lastly, our preliminary results investigating the role of PLK-1, which mediates centriole duplication and kinetochore attachments, show that centrioles duplicate and separate normally during male meiosis in gsp-3/4 mutants. Overall the data presented begins to shed light on the relationship between phosphatases and kinases at the kinetochore in sperm meiosis.

Cohen, Robert D. et al. (2013) International Worm Meeting "A Functional Genomic Screen for the Telomerase RNA in C. elegans."

Smith, Christopher, Cohen, Robert D., Chu, Diana

[International Worm Meeting, 2013]

Telomerase maintains telomere length in eukaryotes by synthesizing telomere repeat patterns on the chromosome ends. It is composed of a protein component (TERT) with reverse transcriptase activity, and an RNA component (TERC) that acts as a template for the reverse transcriptase domain. As dysfunction in telomere length maintenance has been implicated in cancer and aging, the study of telomerase has emerged as an attractive research area for therapeutic intervention. Although identification of TERT genes in newly sequenced genomes has been achieved by homologous search with known TERT genes, the same cannot be said for TERC genes, as primary sequence is not well conserved. TERC identification has relied on co-purification experiments; however, these methods are costly and labor intensive, and as a result, TERC discovery has been limited. To identify candidate TERC genes in a set of genomes from closely related species, we have developed a novel bioinformatic strategy. C. elegans is an ideal organism for the technique, because its TERC gene is unknown, a rich body of bioinformatic resources are available, and RNAi assays are available to test candidate gene function by reverse genetics. Although primary sequence is not well conserved in TERCs, one feature is dependably reproduced - the 1.5x template repeat pattern (eg, 9 bases of the telomere repeat: TTAGGCTTA), which serves as a template for TERT. Searching the C. elegans genome for all instances of this sequence yields 20503 loci. We used the Mauve genome aligner to identify syntenic regions between the Caenorhabditis genomes. 1.5x template repeats conserved at the same syntenic locus in each species are high probability TERC gene candidates. As a positive control, this bioinformatic approach analyzed five vertebrate genomes to identify the human TERC gene, which has been previously published. We have verified expression and RNAi knockdown of two TERC gene candidates, and are currently developing a C. elegans telomere length assay to confirm a reduced telomere length phenotype. Identification of the C. elegans TERC would provide an important tool to elucidate telomerase function in C. elegans.