Genetics最新文献

The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation, and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis. However, a detailed understanding of this process is lacking. Here, we leverage CUT&RUN to define the emerging H3K9me3 landscape of the zebrafish embryo with high sensitivity and temporal resolution. Despite the prevalence of DNA transposons in the zebrafish genome, we found that LTR transposons are preferentially targeted for embryonic H3K9me3 deposition, with different families exhibiting distinct establishment timelines. High signal-to-noise ratios afforded by CUT&RUN revealed new, emerging sites of low-amplitude H3K9me3 that initiated before the major wave of zygotic genome activation (ZGA). Early sites of establishment predominated at specific subsets of transposons and were particularly enriched for transposon sequences with maternal piRNAs and pericentromeric localization. Notably, the number of H3K9me3 enriched sites increased linearly across blastula development, while quantitative comparison revealed a >10-fold genome-wide increase in H3K9me3 signal at established sites over just 30 min at the onset of major ZGA. Continued maturation of the H3K9me3 landscape was observed beyond the initial wave of bulk establishment.

Pentatricopeptide repeat (PPR) proteins bind RNA and are present in mitochondria and chloroplasts of Eukaryota. In fungi, they are responsible for controlling mitochondrial genome expression, mainly on the posttranscriptional level. Candida albicans is a human opportunistic pathogen with a facultative anaerobic metabolism which, unlike the model yeast Saccharomyces cerevisiae, possesses mitochondrially encoded respiratory Complex I (CI) subunits and does not tolerate loss of mtDNA. We characterized the function of 4 PPR proteins of C. albicans that lack orthologs in S. cerevisiae and found that they are required for the expression of mitochondrially encoded CI subunits. We demonstrated that these proteins localize to mitochondria and are essential to maintain the respiratory capacity of cells. Deletion of genes encoding these PPR proteins results in changes in steady-state levels of mitochondrial RNAs and proteins. We demonstrated that C. albicans cells lacking CaPpr4, CaPpr11, and CaPpr13 proteins show no CI assembly, whereas the lack of CaPpr7p results in a decreased CI activity. CaPpr13p is required to maintain the bicistronic NAD4L-NAD5 mRNA, whereas the other 3 PPR proteins are likely involved in translation-related assembly of mitochondrially encoded CI subunits. In addition, we show that CaAep3p, which is an ortholog of ScAep3p, performs the evolutionary conserved function of controlling expression of the ATP8-ATP6 mRNA. We also show that C. albicans cells lacking PPR proteins express a higher level of the inducible alternative oxidase (AOX2) which likely rescues respiratory defects and compensates for oxidative stress.

PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related "modifier' genes (i.e. genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.

Scanning electron microscopy is the method of choice to visualize the surface structures of animals, fungi, plants, or inorganic objects at the highest resolution and often with impressive appeal. Numerous scanning electron microscope (SEM) images exist of Drosophila melanogaster, one of the most important model organisms in genetics and developmental biology, which have been taken partly for esthetics and often to solve scientific questions. Our work presents a collection of images comprising many prominent anatomical details of D. melanogaster in excellent quality to create a research and teaching resource for all Drosophilists.

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

Shc (Src homologous and collagen) proteins function in many different signaling pathways where they mediate phosphorylation-dependent protein-protein interactions. These proteins are characterized by the presence of two phosphotyrosine-binding domains, an N-terminal PTB and a C-terminal SH2. We describe a previously unrecognized Caenorhabditis elegans Shc gene, shc-3 and characterize its role in stress response. Both shc-3 and shc-1 are required for long-term survival in L1 arrest and survival in heat stress, however, they do not act redundantly but rather play distinct roles in these processes. Loss of shc-3 did not further decrease survival of daf-16 mutants in L1 arrest, suggesting that like SHC-1, SHC-3 functions in the insulin-like signaling pathway. In the absence of SHC-3, DAF-16 nuclear entry and exit are slowed, suggesting that SHC-3 is required for rapid changes in DAF-16 signaling.

Patterns of lineal descent play a critical role in the development of metazoan embryos. In eutelic organisms that generate a fixed number of somatic cells, invariance in the topology of their cell lineage provides a powerful opportunity to interrogate developmental events with empirical repeatability across individuals. Studies of embryonic development using the nematode Caenorhabditis elegans have been drivers of discovery. These studies have depended heavily on high-throughput lineage tracing enabled by 4D fluorescence microscopy and robust computer vision pipelines. For a range of applications, computer-aided yet manual lineage tracing using 4D label-free microscopy remains an essential tool. Deep learning approaches to cell detection and tracking in fluorescence microscopy have advanced significantly in recent years, yet solutions for automating cell detection and tracking in 3D label-free imaging of dense tissues and embryos remain inaccessible. Here, we describe embGAN, a deep learning pipeline that addresses the challenge of automated cell detection and tracking in label-free 3D time-lapse imaging. embGAN requires no manual data annotation for training, learns robust detections that exhibits a high degree of scale invariance, and generalizes well to images acquired in multiple labs on multiple instruments. We characterize embGAN's performance using lineage tracing in the C. elegans embryo as a benchmark. embGAN achieves near-state-of-the-art performance in cell detection and tracking, enabling high-throughput studies of cell lineage without the need for fluorescent reporters or transgenics.

RNA-binding proteins (RBPs) play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the adenosine deaminase acting on RNA family of RBPs and the sole adenosine-to-inosine RNA-editing enzyme in Caenorhabditis elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RBPs that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein family of RBPs. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and RNA editing-independent mechanisms in regulating embryogenesis.

Neurons are highly polarized cells with dendrites and axons. Dendrites, which receive sensory information or input from other neurons, often display elaborately branched morphologies. While mechanisms that promote dendrite branching have been widely studied, less is known about the mechanisms that restrict branching. Using the nematode Caenorhabditis elegans, we identify rabr-1 (for Rab-related gene 1) as a factor that restricts branching of the elaborately branched dendritic trees of PVD and FLP somatosensory neurons. Animals mutant for rabr-1 show excessively branched dendrites throughout development and into adulthood in areas where the dendrites overlay epidermal tissues. Phylogenetic analyses show that RABR-1 displays similarity to small GTPases of the Rab-type, although based on sequence alone, no clear vertebrate ortholog of RABR-1 can be identified. We find that rabr-1 is expressed and can function in epidermal tissues, suggesting that rabr-1 restricts dendritic branching cell-nonautonomously. Genetic experiments further indicate that for the formation of ectopic branches rabr-1 mutants require the genes of the Menorin pathway, which have been previously shown to mediate dendrite morphogenesis of somatosensory neurons. A translational reporter for RABR-1 reveals a subcellular localization to punctate, perinuclear structures, which correlates with endosomal and autophagosomal markers, but anticorrelates with lysosomal markers suggesting an amphisomal character. Point mutations in rabr-1 analogous to key residues of small GTPases suggest that rabr-1 functions in a GTP-bound form independently of GTPase activity. Taken together, rabr-1 encodes for an atypical small GTPase of the Rab-type that cell-nonautonomously restricts dendritic branching of somatosensory neurons, likely independently of GTPase activity.