Tarailo-Graovac, Maja et al. (2009) International Worm Meeting "Spatiotemporal analysis of promoter functions implicates spindle assembly checkpoint genes in polyploidization in Caenorhabditis elegans."

Tu, Domena, Wang, Jun, Baillie, David L., Chen, Nansheng, Shih-Chieh Chu, Jeffrey, Tarailo-Graovac, Maja

[International Worm Meeting, 2009]

The importance of spindle assembly checkpoint (SAC) genes for genome stability and healthy cell function has been clearly established. It is known that SAC delays anaphase onset by inhibiting the activity of the APC/C until all of the kinetochores have achieved proper attachment. However, functional roles of the checkpoint components beyond division, as well as transcriptional regulation of the SAC genes during development of a multicellular organism remain largely unexplored. To start investigating these, we studied spatiotemporal expression patterns of the checkpoint genes using the SAC gene promoters driving the expression of GFP. Specifically, we generated transgenic strains carrying extrachromosomal transgenes corresponding to eight SAC genes, five widely conserved core components (mdf-1/MAD1, mdf-2/MAD2, san-1/MAD3, bub-1/BUB1 and bub-3/BUB3) and three SAC components only conserved in higher eukaryotes (hcp-1/CENP-F, hcp-2 and rod-1/ROD). As expected, we found that SAC promoters drive mainly ubiquitous GFP expression during early embryonic stages. However, we also detected SAC gene promoters driven GFP expression in all developmental stages, including tissues and cell types not undergoing division. Importantly, we observed that most of the checkpoint genes are expressed in four or fewer tissues, with a striking overlap in expression in polyploid tissues. Namely, all of the assayed SAC genes show promoter activities in gut, while six of them are also expressed in hypodermal cells. To confirm the observed patterns and to identify expression at the cell level, we have also successfully utilized Mos1-mediated single-copy insertion (MosSCI)1 method to generate single-copy transgenes of SAC gene promoter driven GFP expression at endogeneous levels. The discovery that genes contributing to the surveillance mechanism that prevents aneuploidy in one cell type may be required in promoting polyploidy in a different cell type is an important one. Gut localization of MDF-2/Mad2 checkpoint component was observed previously but its significance has not been explored.2 Taken together, we have identified and analyzed for the first time common SAC gene expression pattern in polyploid tissues, which together with the new strains we have generated will be useful for understanding novel roles of SAC genes. Currently, we are assaying the consequence of the absence of the SAC components on development of gut and hypodermis, polyploid tissues in C. elegans.. 1 Frokjaer-Jensen C., et al. 2008 Nat Genet. 40:1375-1383. 2 Kitagawa, R., and A. M. Rose, 1999 Nat. Cell Biol. 1: 514-521.

Li, Xiao et al. (2019) International Worm Meeting "Cytosolic iron-sulfur protein assembly (CIA) is important for pyrimidine metabolism and immortal germline in C. elegans."

Tarailo-Graovac, Maja, Li, Xiao

[International Worm Meeting, 2019]

Proper integration of iron-sulfur (Fe-S) clusters into corresponding apoproteins is crucial for normal function of many DNA metabolism proteins including FANCJ/dog-1, RTEL1/rtel-1 and ERCC2/xpd-1 [1]. Biogenesis of Fe-S proteins is a multistep process regulated by partnership between mitochondria iron-sulfur cluster (ISC) assembly machinery, the ISC export machinery and the cytoplasm iron-sulfur protein assembly (CIA) system. The CIA targeting complex, consisting of CIAO1, MMS19 and CIAO2B, facilitates the insertion of Fe-S clusters to selected apoproteins [2]. To study the CIA targeting complex, we obtained a knockout allele for the highly conserved orthologous gene mms-19/MMS19, and observed that absence of MMS-19 did not result in an obvious phenotype unless exposed to 5-Fluorouracil (5-FU), a pyrimidine antagonist widely prescribed to treat cancers. Adverse reactions to 5-FU have been well-documented in cancer patients with germline variants in the DPYD gene encoding dihydropyrimidine dehydrogenase (DPD), a cytosolic Fe-S protein known for breaking down pyrimidines [3]. Our analyses of a dpyd-1/DPYD knockout allele revealed similarly strong sensitivity to 5-FU, suggesting that MMS-19 is important for DPD biogenesis in worms. Importantly, we then studied Y18D10A.9/CIAO1 gene and found that it is an essential gene as its knockout results in complete sterility. Further, we also created strains with missense alleles in highly conserved amino acids (invariant from yeast to human), which resulted in either complete sterility similar to the knockout allele or in a heat-triggered mortal germ line (Mrt) phenotype [4]. The Mrt phenotype was described almost two decades ago as a multigenerational phenotype where worms reproduce for several healthy generations but eventually become sterile. The CIAO1 hypomorphic Mrt mutant becomes progressively sterile in a few generations at 25 deg C. As some of the previously described Mrt mutants are in genes important for genome stability (e.g. mrt-2/RAD1) [4], our results suggest that the germline mortality, when CIAO1 is disrupted, may be explained by defective biogenesis of factors important for genome integrity, such as dog-1, rtel-1 and xpd-1. [1] PMID:22678362; [2] PMID: 25583461; [3] PMID: 10071185; [4] PMID: 10646593

Vergara, Ismael A. et al. (2011) International Worm Meeting "Loss-of-Function genomic variations in wild Caenorhabditis elegans."

Wang, Jun, Tarailo-Graovac, Maja, Chen, Nansheng, Vergara, Ismael A.

[International Worm Meeting, 2011]

As revealed by an explosive number of genome sequencing projects, humans carry an enormous amount of genomic variations (GVs). Strikingly, many of these GVs found in apparently healthy individuals are loss-of-function mutations previously associated with genetic diseases, including many types of cancer and mental retardations. How these GVs function in apparently normal and disease humans is not well understood, due to two major challenges. First, the large size of human genes, the large intergenic regions, and the large quantity of GVs residing within each gene together make it challenging to correlate GVs and affected genes. More importantly, it is difficult if not impossible to interrogate the impact of most functionally important GVs on the fitness of their carrier, hence impeding further understanding of their contribution to the pathogenesis of disease conditions in humans. Using Roche/454 (4X coverage) and Illumina sequence reads (72X coverage) in a complementary manner, we have identified various types of GVs with base pair resolution in the wild C. elegans strain CB4856, which was first isolated in an island in Hawaii. Using N2 strain as reference, we have identified 313,219 putative SNPs, 21,332 small indels, 533 large deletions and 208 large insertions, some of which are larger than 10,000 bp. Also, we have detected hundreds of insertions and deletions co-occurring at the same breakpoint. Inspection of the breakpoints has revealed patterns of local duplications as well as events of non-allelic homologous recombination. Among these GVs, we have identified candidate loss-of-function mutations in 141 different essential genes in C. elegans, nine of which are orthologs of human disease genes (OMIM). The biological impact of these loss-of-function GVs on the fitness of CB4856 is being analyzed.

Jean, Francesca et al. (2021) International Worm Meeting "Using WGS to identify intragenic suppressors of zyg-1 in Caenorhabditis elegans reveals the importance of genomic context in phenotypic interpretation"

Stasiuk, Susan, Tarailo-Graovac, Maja, Galbraith, Andrew, Jean, Francesca, Maroilley, Tatiana

[International Worm Meeting, 2021]

Intragenic modifiers can have dramatic effects on phenotypic outcomes, and in disease, can affect attributes such as severity or age of onset. However, the focus of many modifier screens in model systems is the identification and interpretation of extragenic variants rather than understanding the link between intragenic modifiers and phenotype. Accordingly, technological advances in the field, such as the use of whole-genome sequencing (WGS) or CRISPR, tend to be reserved for the discovery and interpretation of extragenic variants, whereas intragenic modifiers still rely on more traditional methods, such as PCR and Sanger sequencing. We demonstrate that intragenic modifier discovery and interpretation following modifier screens benefits greatly from WGS and CRISPR technologies by subjecting strains generated from a chemical mutagenesis suppressor screen using a temperature-sensitive zyg-1(it25) allele to WGS, filtering for intragenic variants using an in-house pipeline, and validating with CRISPR and homology-directed repair. Following this method, we rapidly identify that approximately 10% of our strains contain intragenic zyg-1 variants. By re-creating the intragenic variants in the parental strain, we show that most intragenic variants suppress the original phenotype, but this is not universal. Based on this, we encourage extending variant validation using CRISPR to intragenic variants to determine whether they are truly capable of modifying the original primary variant and to what extent. For those intragenic variants that are capable of suppressing, the position of the variant in comparison to the original primary variant matters; closer modifying variants are more likely to be true suppressors and have a higher suppression ability, which has important implications in understanding complex alleles in disease. Finally, genomic context, as provided by WGS, plays an important role in our interpretation of the contribution of the variant to suppression dynamics. For instance, several of the strains contain extragenic variants that, in addition to the intragenic variant, are proposed to enhance suppression. In contrast, we identified a strain with substantial genetic burden, dampening the suppression ability of the intragenic variant. Altogether, this work describes a methodology for the high-throughput identification of intragenic modifiers and highlights the importance of thoroughly understanding the phenotypic contribution of intragenic modifiers.

Maroilley, Tatiana et al. (2021) International Worm Meeting "Characterizing complex genomic rearrangements in C. elegans using short-read Whole Genome Sequencing"

Maroilley, Tatiana, Tarailo-Graovac, Maja, Li, Xiao, Oldach, Matthew, Jean, Francesca, Stasiuk, Susan

[International Worm Meeting, 2021]

Structural variants (SVs) and complex rearrangements are a significant, but understudied, part of genomic natural diversity. They can also result in various phenotypes in Caenorhabditis elegans. Additionally, certain strains, known as balancers, carry various types of spontaneous or induced (X-rays, gamma irradiation, chemical mutagens, or more recently CRISPR-Cas9) chromosomal rearrangements. Because those rearrangements abolish or reduce crossover events between large portions of the C. elegans genome, balancers have been used for decades to maintain lethal mutations, facilitate strain construction, or capture mutations. However, despite the advent of sequencing technologies, detecting SVs and complex rearrangements is challenging and thus, in most mutagen-induced balancers, the chromosomal rearrangements have not been characterized at the molecular level. High-throughput WGS sequencing, based on short-reads (srWGS), is the most economical, accurate, and widely supported technology at this time. Yet, the length of short-reads is often reported as limiting the detection of complex genetic events. Long-read technologies are then recommended as an alternative despite their increased costs. Here, we applied srWGS alongside tailored bioinformatics approaches to unravel complex genomic rearrangements in several C. elegans balancer genomes. Our method successfully identified complex balancer rearrangements such as reciprocal translocation (eT1), large deletion (eDf43), chromosome fusion (sC4), and free duplication (sDp3). We were also able to identify and experimentally confirm several chromoanagenesis events. Therefore, our results show that we can maximize the possibilities of srWGS by implementing meticulous analytical approaches. Higher confidence in complex variant calls from srWGS will help to molecularly characterize C. elegans balancers and in the future, to decipher the full spectrum of natural genetic variation in C. elegans.

Vergara, Ismael A. et al. (2009) International Worm Meeting "Polymorphic segmental duplications in the nematode Caenorhabditis elegans."

Tarailo-Graovac, Maja, Baillie, David L., Mah, Allan K., Huang, Jim C., Chen, Nansheng, Vergara, Ismael A., Johnsen, Robert C.

[International Worm Meeting, 2009]

The nematode Caenorhabditis elegans was the first multicellular organism to have its genome fully sequenced. Over the last 10 years since the original publication in 1998, the C. elegans genome has been scrutinized and the last gaps were filled in November 2002, which present a unique opportunity for examining genome-wide segmental duplications. Here, we performed analysis of the C. elegans genome in search for segmental duplications using a new tool-OrthoCluster-we have recently developed. We detected 3,484 duplicated segments-duplicons-ranging in size from 234 bp to 108 kb. The largest pair of duplicons, 108 kb in length each located in the left arm of Chromosome V, was further characterized. They are nearly identical at the DNA level (99.7% identity) and each duplicon contains 26 putative protein coding genes, most of them chemosensory genes. These large duplicons are flanked by nearly-identical Cemar1 transposable elements, suggesting that this segmental duplication occurred by unequal crossing over enhanced by the presence of these transposons. Furthermore, the junction is located in the intronic region of a gene confirmed by EST data, suggesting that this segmental duplication gave birth to a new gene in C. elegans. Genotyping of 76 wild-type N2 strains obtained from different labs in the C. elegans community revealed that not all strains contain this duplication. In fact, only 29 strains carry this large segmental duplication, suggesting a very recent duplication event in C. elegans genome. This report represents the first demonstration that the C. elegans laboratory wild-type N2 strains can acquire large-scale differences.

Chu, Jeffrey S C et al. (2011) International Worm Meeting "Comparative genomics reveals novel regulatory mechanism for the transcription factor RFX/DAF-19 in C. elegans."

Baillie, David, Uyar, Bora, Trinh, Joanne, Chen, Nansheng, Johnsen, Bob, Chu, Jeffrey S C, Wang, Jun, Tu, Domena, Tarailo-Graovac, Maja

[International Worm Meeting, 2011]

RFX transcription factors play important roles in cilia biogenesis and maintenance by transcriptionally regulate ciliary genes. Their target genes have been associated with a number of disease conditions collectively called ciliopathies. daf-19 is the only known member of the RFX family in C. elegans. RFX regulation of ciliary genes is conserved between humans and C. elegans. The DNA binding site, known as the X-box motif, is also conserved. Thus, C. elegans has been effectively used as a model organism to identify RFX target genes. Past studies have identified target genes by searching for X-box motifs in C. elegans using bioinformatics, functional genomics, and comparative genomics methods. All of the DAF-19 target genes studied to date possess a single consensus X-box motif. Some genes (bbs-2 and osm-5) were found to possess two X-box motifs. We hypothesize that tandem X-box motifs could have cooperative roles. To test this hypothesis, we compared the gene sets between 4 Caenorhabditis species to find C. elegans genes with multiple X-box motifs within 500-bp promoter region. The C. elegans gene set is an extensively curated set, but this is not the case for the remaining Caenorhabditis species. In order to employ comparative genomics for X-box motif searches, we improved the gene sets for 3 Caenorhabditis species using genBlastG, a homology based gene predictor that we recently developed. Using comparative genomics with the improved gene set, we identified 15 genes that have conserved X-box motifs in all species and have multiple X-box motifs in C. elegans. We examined one gene, F25B4.2, in detail. Using singly integrated reporter constructs, we have shown that F25B4.2 is expressed in ciliated neurons. This expression is dependent on the two 15-bp X-box motifs as well as DAF-19 indicating that F25B4.2 is a DAF-19 target gene. When the proximal motif is removed, expression in ciliated neurons is ablated. When the distal motif is removed, we observed an elevated expression suggesting the distal motif has a repressive role. This is the first to report a putative repressive X-box motif in C. elegans. Our data suggest that two X-box motifs cooperate together to regulate specific expression level of F25B4.2. We model that having multiple X-box motifs in the promoter could achieve specific expression level. Our identifications of X-box motifs will improve our understanding on RFX mediated regulation in C. elegans and in other organisms including humans.

Maja Tarailo et al. (2007) International Worm Meeting "such mutants bypass MDF-1/Mad1 requirement without delaying mitosis."

Ann Rose, Maja Tarailo

[International Worm Meeting, 2007]

The interplay between the kinetochores and the spindle is monitored by the conserved surveillance mechanism, spindle assembly checkpoint (SAC), which delays sister chromatid separation until stable bipolar attachment is achieved. If this delay is bypassed by reducing SAC function, anaphase may start prematurely, resulting in missegregation of chromosomes and loss of genetic fidelity. In humans, such genomic instability may cause miscarriage, birth defects or promote neoplasia by amplifying oncogenes or by reducing tumor suppressor gene dosage. In Caenorhabditis elegans spindle assembly checkpoint gene mdf-1/MAD1 is essential for long-term survival and fertility.1 We described 11 suppressors of the mdf-1(gk2) lethality, ten identified in an EMS mutagenesis screen and one isolated using the dog-1(gk10) (deletions of guanine-rich DNA) strain.2, 3 We hypothesized that, if anaphase onset is delayed in mdf-1(gk2) strain, then there would be more time for proper attachment in the absence of the spindle checkpoint. Using time-lapse imaging of early embryonic cells and germline mitotic division, we demonstrate that there are two classes of suppressors.3 The major class of suppressors compensates for the loss of the checkpoint by delaying mitotic progression. This class includes two known suppressors and anaphase promoting complex/cyclosome (APC/C) components, emb-30/APC44 and fzy-1/CDC202, and four new such (suppressors of spindle checkpoint defect) genes.3 One of the new such genes was found to be an APC5-like gene not previously identified as a component of the APC/C in C. elegans.3 This analysis revealed that apc-5 and apc-10 components have paralogs in the C. elegans genome. The second, newly identified class of suppressors has normal anaphase onset suggesting an alternate mechanism of rescuing mdf-1(gk2)/MAD1 lethality. These are of particular interest, since they represent unexplored functions that render SAC nonessential for long-term survival and fertility in C. elegans. We are focusing our efforts on the mechanism by which this class of suppressors rescues the checkpoint lethality. Funded by CIHR, Canada. References: 1 Kitagawa, R. and Rose, A. M. (1999). Nat Cell Biol. 1: 514-521. 2 Kitagawa, R, et al. (2000). Curr. Biol. 12: 2118-2123. 3 Tarailo, M, et al. (2007). doi: 10.1534/genetics.106.067918. 4 Furuta, T, et al.(2000). Mol Biol Cell. 11: 1401 - 1419.