The CHARGE syndrome ortholog CHD-7 regulates TGF-ß pathways in Caenorhabditis elegans.

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant cellular pathways and targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, stress response, and body size determination. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the DAF-7/ tumor growth factor-ß (TGF-ß) pathway. Epistatic analysis places CHD-7 at the level of the DAF-3/DAF-5 complex, but we found that CHD-7 also directly impacts the expression of multiple components of this pathway. Transcriptomic analysis revealed that chd-7 mutants fail to repress daf-9 for execution of the dauer program. In addition, CHD-7 regulates the DBL-1/BMP pathway components and shares roles in male tail development and cuticle synthesis. To explore a potential conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino-mediated knockdown of Chd7 led to a reduction in col2a1 messenger RNA (mRNA) levels, a collagen whose expression depends on TGF-ß signaling. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by overexpression of col2a1 mRNA. We suggest that Chd7 has conserved roles in regulation of the TGF-ß signaling pathway and pathogenic Chd7 could lead to a defective extracellular matrix deposition.

REC-1 and HIM-5 distribute meiotic crossovers and function redundantly in meiotic double-strand break formation in Caenorhabditis elegans.

The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation.

The molecular tug of war between immunity and fertility: Emergence of conserved signaling pathways and regulatory mechanisms.

Reproduction and immunity are energy intensive, intimately linked processes in most organisms. In women, pregnancy is associated with widespread immunological adaptations that alter immunity to many diseases, whereas, immune dysfunction has emerged as a major cause for infertility in both men and women. Deciphering the molecular bases of this dynamic association is inherently challenging in mammals. This relationship has been traditionally studied in fast-living, invertebrate species, often in the context of resource allocation between life history traits. More recently, these studies have advanced our understanding of the mechanistic underpinnings of the immunity-fertility dialogue. Here, we review the molecular connections between reproduction and immunity from the perspective of human pregnancy to mechanistic discoveries in laboratory organisms. We focus particularly on recent invertebrate studies identifying conserved signaling pathways and transcription factors that regulate resource allocation and shape the balance between reproductive status and immune health.

A DNA repair protein and histone methyltransferase interact to promote genome stability in the Caenorhabditis elegans germ line.

Histone modifications regulate gene expression and chromosomal events, yet how histone-modifying enzymes are targeted is poorly understood. Here we report that a conserved DNA repair protein, SMRC-1, associates with MET-2, the C. elegans histone methyltransferase responsible for H3K9me1 and me2 deposition. We used molecular, genetic, and biochemical methods to investigate the biological role of SMRC-1 and to explore its relationship with MET-2. SMRC-1, like its mammalian ortholog SMARCAL1, provides protection from DNA replication stress. SMRC-1 limits accumulation of DNA damage and promotes germline and embryonic viability. MET-2 and SMRC-1 localize to mitotic and meiotic germline nuclei, and SMRC-1 promotes an increase in MET-2 abundance in mitotic germline nuclei upon replication stress. In the absence of SMRC-1, germline H3K9me2 generally decreases after multiple generations at high culture temperature. Genetic data are consistent with MET-2 and SMRC-1 functioning together to limit replication stress in the germ line and in parallel to promote other germline processes. We hypothesize that loss of SMRC-1 activity causes chronic replication stress, in part because of insufficient recruitment of MET-2 to nuclei.

Modeling primary ovarian insufficiency-associated loci in C. elegans identifies novel pathogenic allele of MSH5.

PURPOSE: In women under the age of 40, primary ovarian insufficiency (POI) is a devastating diagnosis with significant prevalence of 1-4% (Rajkovic and Pangas, Semin Reprod Med. 35(3):231-40, 2017). POI is characterized by amenorrhea with elevated levels of follicle stimulating hormone (FSH) and reduced estrogen levels, mimicking the menopausal state. Genetic determinants account for just over 10% of POI cases, yet determining whether particular single nucleotide polymorphisms (SNPs) are pathogenic is challenging. METHODS: We performed exome sequencing on a cohort of women with POI. CRISPR mutagenesis was employed to create a mutation in a conserved amino acid in the nematode protein. Functional relevance was assessed by analysis of bivalents and aberrant DNA morphologies in diakinesis nuclei. RESULTS: We identified a nonsynonymous c.C1051G; p.R351G variant, in a conserved region of the MSH5 protein. Mutation of this conserved amino acid in the C. elegans homolog, msh-5, revealed defective crossover outcomes in the homozygous and hemizygous states. CONCLUSIONS: These studies further implicate MSH5 as a POI gene and c.C1051G; p.R351G variant as likely playing a functional role in mammalian meiosis. This approach also highlights the ability of model organisms, such as C. elegans, to rapidly and inexpensively identify alleles of interest for further studies in mammalian models.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

Cytogenetic signatures of recurrent pregnancy losses.

OBJECTIVES: To investigate the incidence of chromosomal abnormalities in the products of conception (POC) of patients with spontaneous miscarriages (SM) and with recurrent pregnancy losses (RPL) and to determine biological mechanisms contributing to RPL. METHODS: During a 20-year period, 12 096 POC samples underwent classical chromosome analysis. Cytogenetic findings were compared between the SM and RPL cohorts. RESULTS: Analysis of RPL cohort has identified an increased incidence of inherited and de novo structural chromosome abnormalities, recurrent polyploid conceptions, and complex mosaic alterations. These abnormalities are the signature of genomic instability, posing a high risk of genetic abnormalities to offspring independent of maternal age. Predominance of male conceptions in the RPL cohort points toward an X-linked etiology and gender-specific intolerance for certain genetic abnormalities. CONCLUSIONS: Our study showed several possible genetic etiologies of RPL, including parental structural chromosome rearrangements, predisposition to meiotic nondisjunction, and genomic instability. Loss of karyotypically normal fetuses might be attributed to defects in genes essential for fetal development, as well as aberrations affecting the X chromosome. Molecular studies of parental and POC genomes will help to identify inherited defects in genes involved in meiotic divisions and DNA repair to confirm our hypotheses, and to discover novel fetal-essential genes.

Molecular basis of reproductive senescence: insights from model organisms.

PURPOSE: Reproductive decline due to parental age has become a major barrier to fertility as couples have delayed having offspring into their thirties and forties. Advanced parental age is also associated with increased incidence of neurological and cardiovascular disease in offspring. Thus, elucidating the etiology of reproductive decline is of clinical importance. METHODS: Deciphering the underlying processes that drive reproductive decline is particularly challenging in women in whom a discrete oocyte pool is established during embryogenesis and may remain dormant for tens of years. Instead, our understanding of the processes that drive reproductive senescence has emerged from studies in model organisms, both vertebrate and invertebrate, that are the focus of this literature review. CONCLUSIONS: Studies of reproductive aging in model organisms not only have revealed the detrimental cellular changes that occur with age but also are helping identify major regulator proteins controlling them. Here, we discuss what we have learned from model organisms with respect to the molecular mechanisms that maintain both genome integrity and oocyte quality.

DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans.

Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation.

Correction: DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1005788.].

A germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans.

Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation.

xnd-1 regulates the global recombination landscape in Caenorhabditis elegans.

Meiotic crossover (CO) recombination establishes physical linkages between homologous chromosomes that are required for their proper segregation into developing gametes, and promotes genetic diversity by shuffling genetic material between parental chromosomes. COs require the formation of double strand breaks (DSBs) to create the substrate for strand exchange. DSBs occur in small intervals called hotspots and significant variation in hotspot usage exists between and among individuals. This variation is thought to reflect differences in sequence identity and chromatin structure, DNA topology and/ or chromosome domain organization. Chromosomes show different frequencies of nondisjunction (NDJ), reflecting inherent differences in meiotic crossover control, yet the underlying basis of these differences remains elusive. Here we show that a novel chromatin factor, X non-disjunction factor 1 (xnd-1), is responsible for the global distribution of COs in C. elegans. xnd-1 is also required for formation of double-strand breaks (DSBs) on the X, but surprisingly XND-1 protein is autosomally enriched. We show that xnd-1 functions independently of genes required for X chromosome-specific gene silencing, revealing a novel pathway that distinguishes the X from autosomes in the germ line, and further show that xnd-1 exerts its effects on COs, at least in part, by modulating levels of H2A lysine 5 acetylation.

Methodological considerations for mutagen exposure in C. elegans.

Maintenance of the genome requires the continual repair of DNA lesions. Exposure of nematodes to DNA damage-inducing agents is a powerful method to rapidly ascribe a role for specific genes in DNA repair and to define epistatic relationships to other repair genes which allows for the construction of repair pathways. Despite the extensive use of these agents, however, differences in dosing, timing, and handling makes it difficult to compare results across laboratories. We provide herein a consideration of the parameters that influence the results of these exposures and detailed protocols for the exposure to mutagenic inducing agents.

Methodological considerations for heat shock of the nematode Caenorhabditis elegans.

Stress response pathways share commonalities across many species, including humans, making heat shock experiments valuable tools for many biologists. The study of stress response in Caenorhabditis elegans has provided great insight into many complex pathways and diseases. Nevertheless, the heat shock/heat stress field does not have consensus as to the timing, temperature, or duration of the exposure and protocols differ extensively between laboratories. The lack of cohesiveness makes it difficult to compare results between groups or to know where to start when preparing your own protocol. We present a discussion of some of the major hurdles to reproducibility in heat shock experiments as well as detailed protocols for heat shock and hormesis experiments.

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster.

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation.

Genetic and physical interactions reveal overlapping and distinct contributions to meiotic double-strand break formation in C. elegans.

Double-strand breaks (DSBs) are the most deleterious lesions experienced by our genome. Yet, DSBs are intentionally induced during gamete formation to promote the exchange of genetic material between homologous chromosomes. While the conserved topoisomerase-like enzyme Spo11 catalyzes DSBs, additional regulatory proteins-referred to as "Spo11 accessory factors"- regulate the number, timing, and placement of DSBs during early meiotic prophase ensuring that SPO11 does not wreak havoc on the genome. Despite the importance of the accessory factors, they are poorly conserved at the sequence level suggesting that these factors may adopt unique functions in different species. In this work, we present a detailed analysis of the genetic and physical interactions between the DSB factors in the nematode Caenorhabditis elegans providing new insights into conserved and novel functions of these proteins. This work shows that HIM-5 is the determinant of X-chromosome-specific crossovers and that its retention in the nucleus is dependent on DSB-1, the sole accessory factor that interacts with SPO-11. We further provide evidence that HIM-5 coordinates the actions of the different accessory factors sub-groups, providing insights into how components on the DNA loops may interact with the chromosome axis.

An Emerging Animal Model for Querying the Role of Whole Genome Duplication in Development, Evolution, and Disease.

Whole genome duplication (WGD) or polyploidization can occur at the cellular, tissue, and organismal levels. At the cellular level, tetraploidization has been proposed as a driver of aneuploidy and genome instability and correlates strongly with cancer progression, metastasis, and the development of drug resistance. WGD is also a key developmental strategy for regulating cell size, metabolism, and cellular function. In specific tissues, WGD is involved in normal development (e.g., organogenesis), tissue homeostasis, wound healing, and regeneration. At the organismal level, WGD propels evolutionary processes such as adaptation, speciation, and crop domestication. An essential strategy to further our understanding of the mechanisms promoting WGD and its effects is to compare isogenic strains that differ only in their ploidy. Caenorhabditis elegans (C. elegans) is emerging as an animal model for these comparisons, in part because relatively stable and fertile tetraploid strains can be produced rapidly from nearly any diploid strain. Here, we review the use of Caenorhabditis polyploids as tools to understand important developmental processes (e.g., sex determination, dosage compensation, and allometric relationships) and cellular processes (e.g., cell cycle regulation and chromosome dynamics during meiosis). We also discuss how the unique characteristics of the C. elegans WGD model will enable significant advances in our understanding of the mechanisms of polyploidization and its role in development and disease.

idpr: A package for profiling and analyzing Intrinsically Disordered Proteins in R.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are proteins or protein-domains that do not have a single native structure, rather, they are a class of flexible peptides that can rapidly adopt multiple conformations. IDPs are quite abundant, and their dynamic characteristics provide unique advantages for various biological processes. The field of "unstructured biology" has emerged, in part, because of numerous computational studies that had identified the unique characteristics of IDPs and IDRs. The package 'idpr', short for Intrinsically Disordered Proteins in R, implements several R functions that match the established characteristics of IDPs to protein sequences of interest. This includes calculations of residue composition, charge-hydropathy relationships, and predictions of intrinsic disorder. Additionally, idpr integrates several amino acid substitution matrices and calculators to supplement IDP-based workflows. Overall, idpr aims to integrate tools for the computational analysis of IDPs within R, facilitating the analysis of these important, yet under-characterized, proteins. The idpr package can be downloaded from Bioconductor (https://bioconductor.org/packages/idpr/).

Proteasomal subunit depletions differentially affect germline integrity in C. elegans.

The 26S proteasome is a multi-subunit protein complex that is canonically known for its ability to degrade proteins in cells and maintain protein homeostasis. Non-canonical or non-proteolytic roles of proteasomal subunits exist but remain less well studied. We provide characterization of germline-specific functions of different 19S proteasome regulatory particle (RP) subunits in C. elegans using RNAi specifically from the L4 stage and through generation of endogenously tagged 19S RP lid subunit strains. We show functions for the 19S RP in regulation of proliferation and maintenance of integrity of mitotic zone nuclei, in polymerization of the synaptonemal complex (SC) onto meiotic chromosomes and in the timing of SC subunit redistribution to the short arm of the bivalent, and in turnover of XND-1 proteins at late pachytene. Furthermore, we report that certain 19S RP subunits are required for proper germ line localization of WEE-1.3, a major meiotic kinase. Additionally, endogenous fluorescent labeling revealed that the two isoforms of the essential 19S RP proteasome subunit RPN-6.1 are expressed in a tissue-specific manner in the hermaphrodite. Also, we demonstrate that the 19S RP subunits RPN-6.1 and RPN-7 are crucial for the nuclear localization of the lid subunits RPN-8 and RPN-9 in oocytes, further supporting the ability to utilize the C. elegans germ line as a model to study proteasome assembly real-time. Collectively, our data support the premise that certain 19S RP proteasome subunits are playing tissue-specific roles, especially in the germ line. We propose C. elegans as a versatile multicellular model to study the diverse proteolytic and non-proteolytic roles that proteasome subunits play in vivo.

Meiotic dysfunction accelerates somatic aging in Caenorhabditis elegans.

An expanding body of evidence, from studies in model organisms to human clinical data, reveals that reproductive health influences organismal aging. However, the impact of germline integrity on somatic aging is poorly understood. Moreover, assessing the causal relationship of such an impact is challenging to address in human and vertebrate models. Here, we demonstrate that disruption of meiosis, a germline restricted process, shortened lifespan, impaired individual aspects of healthspan, and accelerated somatic aging in Caenorhabditis elegans. Young meiotic mutants exhibited transcriptional profiles that showed remarkable overlap with the transcriptomes of old worms and shared similarities with transcriptomes of aging human tissues as well. We found that meiosis dysfunction caused increased expression of functionally relevant longevity determinants whose inactivation enhanced the lifespan of normal animals. Further, meiotic mutants manifested destabilized protein homeostasis and enhanced proteasomal activity partially rescued the associated lifespan defects. Our study demonstrates a role for meiotic integrity in controlling somatic aging and reveals proteostasis control as a potential mechanism through which germline status impacts overall organismal health.