Golden, Andy et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "EMB-1, SUCH-1, and GFI-3, three Anaphase-Promoting Complex subunits, are required for the meiotic divisions of fertilized embryos"

Stein, Kathryn, Nesmith, Jessica, Golden, Andy, Shakes, Diane

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Chromosome segregation during meiosis and mitotis requires the activity of the Anaphase-Promoting Complex (APC), a multi-subunit E3 ubiquitin ligase that ubiquitylates target proteins, which are then degraded by the 26S proteasome. RNAi and mutational analysis has revealed that most APC subunits in C. elegans are required for the meiotic divisions in fertilized embryos. Depletion of any of these subunits results in embryos arrested in metaphase of meiosis I. Temperature-sensitive alleles of emb-1 share this arrest phenotype at non-permissive temperatures. Alleles of emb-1 also enhance other APC mutants at permissive temperatures and are suppressed by mutants that suppress other APC mutan ts. We mapped and cloned emb-1; it encodes an 81 amino acid protein with no known function. The Oegema lab has demonstrated that EMB-1 co-purifies with APC subunits. Together, these data suggest that EMB-1 is a novel regulator or subunit of the APC.

Andy Golden (2004) East Coast Worm Meeting "The Myt1 ortholog in C. elegans is essential for oocyte maturation."

Andy Golden

[East Coast Worm Meeting, 2004]

Myt1, a member of the WEE1 kinase family, is an essential negative regulator of the CDK/cyclin complex that acts during the G2/M transition of the meiotic cell cycle in Xenopus oocytes. Using RNA interference (RNAi), we have shown C. elegans oocytes depleted of the Myt1 homolog, WEE-1.3, fail to develop and mature normally within the oviduct of the hermaphrodite. At the earliest stages of WEE-1.3 depletion, the oocytes appear to mature precociously. Despite this precocious maturation, oocytes are ovulated normally. However, such WEE-1.3-depleted oocytes are fertilization-incompetent in the presence of wild-type sperm and appear to remain in an extended M-phase state within the uterus. With longer exposure to wee-1.3 RNAi, the ability of the hermaphrodite germline to produce normal oocytes becomes greatly compromised. Our explanation is that CDK-1, the presumed WEE-1.3 target, is an abundant maternal protein that must to be kept inactive during critical phases of oocyte development. Presumably, in the absence of WEE-1.3, CDK-1 is precociously activated in immature oocytes. This model is strengthened by our observation that CDK-1 depletion suppresses the wee-1.3 RNAi maturation/fertilization defects. Our experimental results confirm that WEE-1.3 acts through CDK-1 in the proper regulation of the G2/M transition during C. elegans oocyte development and maturation.

Kropp, Peter et al. (2019) International Worm Meeting "Understanding Multiple Mitochondrial Dysfunctions Syndrome with C. elegansUnderstanding Multiple Mitochondrial Dysfunctions Syndrome with C. elegans."

Golden, Andy, Kropp, Peter

[International Worm Meeting, 2019]

Understanding Multiple Mitochondrial Dysfunctions Syndrome with C. elegans Peter Kropp1, Andy Golden1 1: National Institute of Diabetes, Digestive, and Kidney Disorders, NIH, Bethesda,MD, USA While individual rare diseases are by definition uncommon (affecting less than 200,000 people in the US), one in ten individuals in the United States is affiliated with a rare disease. Of the roughly 7,000 rare diseases, ~80% are monogenic. We can model rare diseases in the nematode Caenorhabditis elegans (C. elegans) thanks to conservation of >40% of genes between humans and C. elegans including most monogenic disease genes. Utilizing powerful CRISPR/Cas9 genome editing tools I recreate specific patient alleles in the orthologous C. elegans genes. This approach allows me to understand the molecular, cellular, and systemic implications of mutations in individual patient disease genes. Multiple mitochondrial dysfunctions syndrome (MMDS) is a collection of perinatal-fatal diseases. Each type of MMDS (1-6) has known monogenic origins in genes of the Iron-Sulphur cluster (ISC) synthesis pathway. ISCs are essential cofactors of many proteins with a broad range of cellular functions. MMDS is specifically associated with mutations in genes responsible for the maturation and trafficking of Fe-S clusters, but the precise impact of each mutation remains unknown. I am focused on the factor NFU1 (lpd-8 in C. elegans) that is responsible for trafficking ISCs to the appropriate mitochondrial target proteins and the monogenic cause of MMDS1. I hypothesize that tissue-level analyses of patient-specific mutations in lpd-8 will reveal previously unknown roles for this factor in ISC delivery and further explain the symptoms observed in MMDS1. Analysis of patient alleles reveals defects in germline development, muscle function, and lipid storage in homozygous mutant animals. Preliminary evidence indicates that these findings are due to increased oxidative stress in the affected tissues. Ongoing work will determine the mechanisms for these dysfunctions and to what extent each is cell-autonomous. I will also conduct a genetic suppressor screen to identify extragenic mutations that restore cellular functions to these mutants. These findings will not only elucidate the role of disease genes in ISC maturation and trafficking, but also provide broader understanding of how mitochondrial dysfunction impacts human disease. Further, suppressor screens may identify potential therapeutic targets that modulate expression or function of the ISC pathway.

Graham, Carina et al. (2019) International Worm Meeting "Modeling NGLY1 Deficiency in C. elegans."

Graham, Carina, Golden, Andy

[International Worm Meeting, 2019]

NGLY1 Deficiency is an autosomal recessive disorder characterized by global developmental delays, neurological abnormalities, alacrima, hypotonia, auditory neuropathies, liver fibrosis, and hyperkinetic movement disorders. Since its discovery in 2012, fewer than 80 patients have been diagnosed worldwide. The nematode C. elegans represents an opportunity to model such ultra-rare diseases due to its easy husbandry, high orthology with the human genome, and receptivity to the CRISPR-Cas9 genome editing system. To model NGLY1 Deficiency, the orthologous nematode gene png-1 was altered via the CRISPR-Cas9 system. In addition to a full gene deletion, patient-specific single-nucleotide mutations were replicated in png-1. Phenotypes were characterized via physiological, chemical, and genetic screens. It was found that png-1 mutants display a hypersensitivity to proteasomal stress. When proteasome activity is inhibited by drug treatment or RNAi, png-1 mutant worms display larval arrest or lethality not seen in wild-type worms. Additionally, png-1 mutant worms display abnormalities in neuronal patterning. After establishment of phenotypic assays, mutant worms were subjected to random chemical mutagenesis, and proteasomal inhibition assays were repeated on survivors. Lines of worms were established from individuals who showed suppression of the larval arrest phenotype. These worms theoretically carry mutations in genes that represent potential therapeutic targets for human patients.

Fabritius, Amy et al. (2013) International Worm Meeting "Defining genetic pathways of disease through genetic suppression screening in C. elegans."

Fabritius, Amy, Golden, Andy

[International Worm Meeting, 2013]

Orthologs of genes responsible for many human monogenic diseases exist in C. elegans. We are using mutants in these orthologs in C. elegans to perform EMS mutagenesis suppressor screens to find genes that are involved in various disease pathways. I am using mutants in the C. elegans type IV collagens and the ubiquitin-activating enzyme in suppressor screens to identify potential novel therapeutic targets. Humans have six type IV collagens (alpha1-alpha6) and C. elegans has two type IV collagens (emb-9 and let-2). The type IV collagens in both species are a major constituent of basement membranes and play an important role in signaling and cell adhesions. Type IV collagens form a heterotrimer along their long helical central domain and higher order net-like structures through interactions of the amino and carboxyl termini. Mutations in the human type IV collagens can cause Alport syndrome or brain small vessel disease. Mutations in either gene in C. elegans lead to embryonic lethality or larval arrest (Kramer, 2005). The ubiquitin-activating enzyme in human (UBA1or UBE1) and in C. elegans (uba-1) is required for activation of ubiquitin for downstream ligation of ubiquitin by the E2 and E3 ubiquitin ligases and subsequent protein degradation by the proteasome. Mutations in UBA1 cause early-onset spinal muscular atrophy and some cancers. UBA-1 is the only known ubiquitin-activating enzyme in C. elegans and uba-1(it129ts) allele causes embryonic lethality, larval lethality, sperm-specific lethality and change in size at restrictive temperature (Kulkarni and Smith, 2008). We have obtained and are characterizing potential suppressors of an allele of emb-9. We are also continuing mutagenesis screens to find additional interactions and suppressors of uba-1 and additional disease genes. These results will help further define genetic pathways and potentially identify genes that could serve as therapeutic targets to suppress disease symptoms.

Fabritius, Amy et al. (2015) International Worm Meeting "Defining genetic pathways of disease through genetic suppression screens in Caenorhabditis elegans."

Fabritius, Amy, Golden, Andy

[International Worm Meeting, 2015]

There are approximately 7,000 rare diseases known in humans, many of which are mongenic (caused by a mutation in a single gene). C. elegans has orthologs of approximately 50 percent of the genes responsible for human diseases. We are using mutations in these orthologous genes in C. elegans to perform EMS mutagenesis suppressor screens to find genes that are involved in various disease pathways. This will provide insight into the genetic and molecular mechanisms of these diseases, offering possible therapeutic targets. We are using mutants in the C. elegans type IV collagens and the ubiquitin-activating enzyme in suppressor screens to identify potential novel therapeutic targets.Humans have six type IV collagens (alpha1-alpha6) and C. elegans has two type IV collagens (emb-9 and let-2). The type IV collagens in both species are a major constituent of basement membranes and play an important role in cell signaling and cell adhesions. Mutations in the human type IV collagens can cause Alport syndrome or brain small vessel disease. Mutations in either gene in C. elegans lead to embryonic lethality or larval arrest.The ubiquitin-activating enzyme in human (UBA1or UBE1) and in C. elegans (uba-1) is required for activation of ubiquitin for downstream ligation of ubiquitin by the ubiquitin ligases and subsequent substrate degradation by the proteasome. Mutations in human UBA1 cause early-onset spinal muscular atrophy and are involved in some cancers. UBA-1 is the only known ubiquitin-activating enzyme in C. elegans and the temperature sensitive (ts) uba-1(it129) allele causes embryonic lethality, larval lethality and sperm-specific sterility at restrictive temperatures.We have obtained suppressors of the larval arrest and embryonic lethality phenotypes in ts alleles at restrictive temperatures of emb-9, let-2 and uba-1. Through whole-genome sequencing, RNAi candidate testing and recreation of the suppressor mutation in the mutant background, we have identified several genes responsible for suppression. One suppressor of the emb-9(hc70ts) allele is dig-1, a giant member of the immunoglobulin superfamily. We are currently testing this and other candidates to confirm their identities as suppressors and to characterize the suppressor mutations as well as the genetic and molecular nature of the suppressor activity that allows the animals with the mutation to survive. These results will help further define genetic pathways and potentially identify genes that could serve as therapeutic targets to suppress disease symptoms.

Shakes, Diane et al. (2009) International Worm Meeting "EMB-1 is a novel protein involved in the metaphase-to-anaphase transition."

Golden, Andy, Shakes, Diane

[International Worm Meeting, 2009]

The Anaphase Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that promotes the metaphase-to-anaphase transition during meiotic and mitotic divisions. Temperature-sensitive (ts) mutants in mat-1, mat-2, mat-3, emb-27, and emb-30 arrest as 1-cell embryos, stuck in metaphase of meiosis I. These five genes code for five subunits of the APC. The ts alleles of emb-1 have grabbed our attention because their arrest phenotype is indistinguishable from the APC mutants. Furthermore, genetic doubles constructed between emb-1(hc62) and the APC mutants cannot be maintained at the permissive temperature, a common feature of any APC double mutant. Additionally, suppressors that suppress the APC mutants (Stein et al., 2007) also suppress emb-1. What is EMB-1 you may ask? We mapped emb-1 to a tiny interval on LG III and used RNAi to phenocopy the 1-cell arrest phenotype. Rescue and sequencing confirmed that emb-1 codes for a novel protein with no known homologies outside of Caenorhabditis species. Localization studies are underway. We propose that EMB-1 is a novel subunit or regulator of the APC in C. elegans.

Zafra Martinez, Isabella et al. (2019) International Worm Meeting "Creation of a Database of C. elegans Orthologs for Human Rare Disease Genes."

Zafra Martinez, Isabella, Golden, Andy

[International Worm Meeting, 2019]

There are approximately 7,000 known rare diseases in humans, with about 80% of these being monogenic. When combined, nearly 10% of the United States population has a rare disease. Unfortunately, treatments have been successfully developed for less than 5% of these rare diseases. Therefore, there is a great need for more researchers to employ model organisms as tools for studying the molecular mechanisms underlying these rare diseases in order to ultimately develop effective treatments. Fortunately, whole-genome and whole-exome sequencing has allowed identification of disease-associated alleles for many of these rare diseases. The nematode Caenorhabditis elegans has 20,000 protein-coding genes, with nearly 40% of these being estimated to have human orthologs. This, along with its physiological simplicity and short life cycle, makes C. elegans a great model organism to study genes associated with human disease. Using modern genome-editing tools, such as the CRISPR-Cas9 system, we can create mutations in the worm that mimic the mutations in the patients that are afflicted by these monogenic rare diseases. In doing this, we can study the phenotypes observed in the worm that are a result of the mutation created in the gene of interest, which are considered phenologs of the human disease. Here we present the creation of a database in collaboration with the NIH's Undiagnosed Disease Program (UDP) and WormBase with the goal of indicating which monogenic disease genes identified by the UDP have C. elegans orthologs. In this database, we indicate which of these genes in the worm have the corresponding amino acid that is mutated in the disease condition. The ultimate goal of this project is to merge our database with WormBase to highlight C. elegans genes that researchers can consider generating the missense allele associated with a human disease to study its biology and the underlying genetic mechanisms. Because of the high degree of gene conservation across species, such databases in other model organisms should stimulate similar research projects to better understand the function of these genes.

Jaramillo-Lambert, Aimee et al. (2017) International Worm Meeting "top-2 is required for proper chromosome segregation during male meiosis in C. elegans."

Golden, Andy, Jaramillo-Lambert, Aimee

[International Worm Meeting, 2017]

During sexual reproduction, haploid gametes (i.e. eggs and sperm) are generated from diploid precursors through the specialized cell division of meiosis. Meiosis reduces ploidy by following one round of DNA replication with two rounds of chromosome segregation (MI and MII respectively). In MII sister chromatids segregate from each other similar to mitosis; but in MI, it is the homologs that segregate, which requires pairing, synapsis, and recombination. Type II DNA topoisomerases are enzymes that play a crucial role in chromosome fidelity by disentangling topological problems that arise in double stranded DNA. Topo II is a large ATP-dependent, homodimeric enzyme. Each subunit breaks one DNA strand, passes a second unbroken strand through the break, and then reseals the break. Thus, during mitotic divisions, Topo II enzymes solve topological problems that arise during replication, transcription, sister chromatid segregation, and recombination. However, the exact role Topo II plays during meiosis has not been fully elucidated. Previously, we have shown that a novel allele of Topo II, top-2(it7ts), uniquely disrupts the segregation of homologous chromosomes during the meiotic divisions of spermatogenesis but not oogenesis. TOP-2 is expressed throughout the germ lines of both spermatogenic and oogenic germ lines, localizing along the lengths of chromosomes during meiotic prophase. In top-2(it7ts) mutants, localization of TOP-2 is disrupted in both spermatogenesis and oogenesis leading us to question why only meiotic chromosome segregation in spermatogenesis is affected. A major difference between spermatogenesis and oogenesis is that chromosome morphology differs after pachynema. We hypothesize that TOP-2 plays a role in chromosome remodeling/architecture during late meiotic prophase thus facilitating homologous chromosome segregation during spermatogenesis. Preliminary evidence evaluating the role of top-2 in chromosome architecture has found that meiotic chromosome axis components, the meiotic cohesins, COH-3 and COH-4, localize normally during spermatogenesis, however, the other meiotic cohesin, REC-8, may be prematurely removed from chromosomes. We are continuing to investigate the role of top-2 in chromosome structure through the examination of additional architectural components.

Rogers, Pippa et al. (2021) International Worm Meeting "Modeling Rare Genetic Diseases in C. elegans: Neuromuscular Junction Involvement in Multiple Mitochondrial Dysfunctions Syndrome 1"

Kropp, Peter, Rogers, Pippa, Golden, Andy

[International Worm Meeting, 2021]

A disease is defined as rare if there are fewer than 200,000 affected people in the United States. There are approximately 7,000 rare human diseases, the majority of which are thought to be genetic. Model organisms give hope for improved ability to diagnose, understand, and treat these diseases. The nematode Caenorhabditis elegans has about 20,000 protein-coding genes, an estimated 40% of which have human homologs. C. elegans as a research tool has many benefits, including being a complex, multicellular system and having a short lifecycle. This, along with the power of more accessible CRISPR-Cas9 editing strategies, allow researchers to make pathogenic mutation lines, as well as full length deletions of disease-causing genes, and more easily observe phenotypic outcomes. Multiple Mitochondrial Dysfunctions Syndrome (MMDS) is a class of six autosomal recessive diseases caused by mutations in various nuclear-encoded mitochondrial genes, but with similar disease outcomes. Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1), one of these syndromes, is caused by pathogenic mutations in NFU1. NFU1 is an iron-sulfur cluster transfer protein that primarily delivers 4Fe-4S clusters to target apoproteins. The C. elegans nfu-1 gene produces the protein NFU-1, which is orthologous to human NFU1, so five CRISPR/Cas9 alleles were made with pathogenic mutations, as well as a full-length deletion of nfu-1. Presently, the phenotypes of the mutant animals are being characterized in order to better understand the function of the protein and identify the mechanisms and molecular pathways by which the protein is regulated. It was previously shown that three of the MMDS1 patient alleles made in C. elegans (G148R, G166C, and C168F) showed reduced thrashing when placed in liquid. This leads to the question: was the reduced thrashing caused by a problem with neurons, muscles, or both? To determine whether neuronal and/or muscle cells have impaired function, a series of drug assays were used to pinpoint neuromuscular junction issues. It is now hypothesized that mutations in the disease-causing gene of MMDS1 affect the availability and/or binding of neurotransmitters. These results create strong ties between the known genetic mutations and disease symptoms.