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.

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.

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 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.

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.

Aging Negatively Impacts DNA Repair and Bivalent Formation in the C. elegans Germ Line.

Defects in crossover (CO) formation during meiosis are a leading cause of birth defects, embryonic lethality, and infertility. In a wide range of species, maternal aging increases aneuploidy and decreases oocyte quality. In C. elegans which produce oocytes throughout the first half of adulthood, aging both decreases oocytes quality and increases meiotic errors. Phenotypes of mutations in genes encoding double-strand break (DSB)-associated proteins get more severe with maternal age suggesting that early meiosis reflects a particularly sensitive node during reproductive aging in the worm. We observed that aging has a direct effect on the integrity of C. elegans meiotic CO formation, as observed by an increase of univalent chromosomes and fusions at diakinesis, with a considerable increase starting at 4 days. We also characterize the possible causes for the age-related changes in CO formation by analyzing both steady-state levels and kinetics of the ssDNA binding proteins RPA-1 and RAD-51. Profound reductions in numbers of both RPA-1 and RAD-51 foci suggests that both DSB formation and early meiotic repair are compromised in aging worms. Using laser microirradiation and γ-irradiation to induce exogenous damage, we show specifically that recruitment of these homologous recombination proteins is altered. Repair defects can be seen in two-and-one-half day-old adults making the loss of germline repair capacity among the earliest aging phenotypes in the worm.

Poly(ADP-ribose) glycohydrolase coordinates meiotic DNA double-strand break induction and repair independent of its catalytic activity.

Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

GCNA Preserves Genome Integrity and Fertility Across Species.

The propagation of species depends on the ability of germ cells to protect their genome from numerous exogenous and endogenous threats. While these cells employ ubiquitous repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. We identified Germ Cell Nuclear Acidic Peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish, and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with the SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit DNA damage, the IDR imparts significant function. This work shows that GCNA protects germ cells from various sources of damage, providing insights into conserved mechanisms that promote genome integrity across generations.

Genome integrity is regulated by the Caenorhabditis elegans Rad51D homolog rfs-1.

Multiple mechanisms ensure genome maintenance through DNA damage repair, suppression of transposition, and telomere length regulation. The mortal germline (Mrt) mutants in Caenorhabditis elegans are defective in maintaining genome integrity, resulting in a progressive loss of fertility over many generations. Here I show that the high incidence of males (him)-15 locus, defined by the deficiency eDf25, is allelic to rfs-1, the sole rad-51 paralog group member in C. elegans. The rfs-1/eDf25 mutant displays a Mrt phenotype and mutant animals manifest features of chromosome fusions prior to the onset of sterility. Unlike other Mrt genes, rfs-1 manifests fluctuations in telomere lengths and functions independently of telomerase. These data suggest that rfs-1 is a novel regulator of genome stability.

Domain-specific regulation of recombination in Caenorhabditis elegans in response to temperature, age and sex.

It is generally considered that meiotic recombination rates increase with temperature, decrease with age, and differ between the sexes. We have reexamined the effects of these factors on meiotic recombination in the nematode Caenorhabditis elegans using physical markers that encompass >96% of chromosome III. The only difference in overall crossover frequency between oocytes and male sperm was observed at 16 degrees . In addition, crossover interference (CI) differs between the germ lines, with oocytes displaying higher CI than male sperm. Unexpectedly, our analyses reveal significant changes in crossover distribution in the hermaphrodite oocyte in response to temperature. This feature appears to be a general feature of C. elegans chromosomes as similar changes in response to temperature are seen for the X chromosome. We also find that the distribution of crossovers changes with age in both hermaphrodites and females. Our observations indicate that it is the oocytes from the youngest mothers-and not the oldest-that showed a different pattern of crossovers. Our data enhance the emerging hypothesis that recombination in C. elegans, as in humans, is regulated in large chromosomal domains.

ATM and ATR Influence Meiotic Crossover Formation Through Antagonistic and Overlapping Functions in Caenorhabditis elegans.

During meiosis, formation of double-strand breaks (DSBs) and repair by homologous recombination between homologs creates crossovers (COs) that facilitate chromosome segregation. CO formation is tightly regulated to ensure the integrity of this process. The DNA damage response kinases, Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) have emerged as key regulators of CO formation in yeast, flies, and mice, influencing DSB formation, repair pathway choice, and cell cycle progression. The molecular networks that ATM and ATR influence during meiosis are still being resolved in other organisms. Here, we show that Caenorhabditis elegans ATM and ATR homologs, ATM-1 and ATL-1 respectively, act at multiple steps in CO formation to ultimately ensure that COs are formed on all chromosomes. We show a role for ATM-1 in regulating the choice of repair template, biasing use of the homologous chromosome instead of the sister chromatid. Our data suggest a model in which ATM-1 and ATL-1 have antagonistic roles in very early repair processing, but are redundantly required for accumulation of the RAD-51 recombinase at DSB sites. We propose that these features of ATM-1 and ATL-1 ensure both CO formation on all chromosomes and accurate repair of additional DSBs.