PiRNA-4447944 promotes castration-resistant growth and metastasis of prostate cancer by inhibiting NEFH expression through forming the piRNA-4447944-PIWIL2-NEFH complex.

Castration-resistant prostate cancer (CRPC) is the leading cause of prostate cancer (PCa)-related death in males, which occurs after the failure of androgen deprivation therapy (ADT). PIWI-interacting RNAs (piRNAs) are crucial regulators in many human cancers, but their expression patterns and roles in CRPC remain unknown. In this study, we performed small RNA sequencing to explore CRPC-associated piRNAs using 10 benign prostate tissues, and 9 paired hormone-sensitive PCa (HSPCa) and CRPC tissues from the same patients. PiRNA-4447944 (piR-4447944) was discovered to be highly expressed in CRPC group compared with HSPCa and benign groups. Functional analyses revealed that piR-4447944 overexpression endowed PCa cells with castration resistance ability in vitro and in vivo, whereas knockdown of piR-4447944 using anti-sense RNA suppressed the proliferation, migration and invasion of CRPC cells. Additionally, enforced piR-4447944 expression promoted in vitro migration and invasion of PCa cells, and reduced cell apoptosis. Mechanistically, piR-4447944 bound to PIWIL2 to form a piR-4447944/PIWIL2 complex and inhibited tumor suppressor NEFH through direct interaction at the post-transcriptional level. Collectively, our study indicates that piR-4447944 is essential for prostate tumor-propagating cells and mediates androgen-independent growth of PCa, which extends current understanding of piRNAs in cancer biology and provides a potential approach for CRPC treatment.

GLIS1, Correlated with Immune Infiltrates, Is a Potential Prognostic Biomarker in Prostate Cancer.

Prostate cancer (PCa) is a prevalent malignant disease and the primary reason for cancer-related mortality among men globally. GLIS1 (GLIS family zinc finger 1) is a key regulator in various pathologies. However, the expression pattern, clinical relevance, and immunomodulatory function of GLIS1 in PCa remain unclear. In this study, GLIS1 was discovered to serve as a key gene in PCa by integrating mRNA and miRNA expression profiles from GEO database. We systematically explored the expression and prognostic values of GLIS1 in cancers using multiple databases. Additionally, we examined the functions of GLIS1 and the relationship between GLIS1 expression levels and immune infiltration in PCa. Results showed that GLIS1 was differentially expressed between normal and tumor tissues in various cancer types and was significantly low-expressed in PCa. Low GLIS1 expression was associated with poor PCa prognosis. GLIS1 was also involved in the activation, proliferation, differentiation, and migration of immune cells, and its expression showed a positive correlation with the infiltration of various immune cells. Moreover, GLIS1 expression was positively associated with various chemokines/chemokine receptors, indicating the involvement in regulating immune cell migration. In summary, GLIS1 is a potential prognostic biomarker and a therapeutic target to modulate anti-tumor immune response in PCa.

Initial characterization of gap phase introduction in every cell cycle of C. elegans embryogenesis.

Early embryonic cell cycles usually alternate between S and M phases without any gap phase. When the gap phases are developmentally introduced in various cell types remains poorly defined especially during embryogenesis. To establish the cell-specific introduction of gap phases in embryo, we generate multiple fluorescence ubiquitin cell cycle indicators (FUCCI) in C. elegans. Time-lapse 3D imaging followed by lineal expression profiling reveals sharp and differential accumulation of the FUCCI reporters, allowing the systematic demarcation of cell cycle phases throughout embryogenesis. Accumulation of the reporters reliably identifies both G1 and G2 phases only in two embryonic cells with an extended cell cycle length, suggesting that the remaining cells divide either without a G1 phase, or with a brief G1 phase that is too short to be picked up by our reporters. In summary, we provide an initial picture of gap phase introduction in a metazoan embryo. The newly developed FUCCI reporters pave the way for further characterization of developmental control of cell cycle progression.

Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation.

The invariant development and transparent body of the nematode Caenorhabditis elegans enables complete delineation of cell lineages throughout development. Despite extensive studies of cell division, cell migration and cell fate differentiation, cell morphology during development has not yet been systematically characterized in any metazoan, including C. elegans. This knowledge gap substantially hampers many studies in both developmental and cell biology. Here we report an automatic pipeline, CShaper, which combines automated segmentation of fluorescently labeled membranes with automated cell lineage tracing. We apply this pipeline to quantify morphological parameters of densely packed cells in 17 developing C. elegans embryos. Consequently, we generate a time-lapse 3D atlas of cell morphology for the C. elegans embryo from the 4- to 350-cell stages, including cell shape, volume, surface area, migration, nucleus position and cell-cell contact with resolved cell identities. We anticipate that CShaper and the morphological atlas will stimulate and enhance further studies in the fields of developmental biology, cell biology and biomechanics.

Multilevel regulation of muscle-specific transcription factor hlh-1 during Caenorhabditis elegans embryogenesis.

hlh-1 is a myogenic transcription factor required for body-wall muscle specification during embryogenesis in Caenorhabditis elegans. Despite its well-known role in muscle specification, comprehensive regulatory control upstream of hlh-1 remains poorly defined. Here, we first established a statistical reference for the spatiotemporal expression of hlh-1 at single-cell resolution up to the second last round of divisions for most of the cell lineages (from 4- to 350-cell stage) using 13 wild-type embryos. We next generated lineal expression of hlh-1 after RNA interference (RNAi) perturbation of 65 genes, which were selected based on their degree of conservation, mutant phenotypes, and known roles in development. We then compared the expression profiles between wild-type and RNAi embryos by clustering according to their lineal expression patterns using mean-shift and density-based clustering algorithms, which not only confirmed the roles of existing genes but also uncovered the potential functions of novel genes in muscle specification at multiple levels, including cellular, lineal, and embryonic levels. By combining the public data on protein-protein interactions, protein-DNA interactions, and genetic interactions with our RNAi data, we inferred regulatory pathways upstream of hlh-1 that function globally or locally. This work not only revealed diverse and multilevel regulatory mechanisms coordinating muscle differentiation during C. elegans embryogenesis but also laid a foundation for further characterizing the regulatory pathways controlling muscle specification at the cellular, lineal (local), or embryonic (global) level.

Comparative mitochondrial genomics reveals a possible role of a recent duplication of NADH dehydrogenase subunit 5 in gene regulation.

Mitochondrial genome (mtDNA) carries not only well-conserved protein coding, tRNA and rRNA genes, but also highly variable non-coding regions (NCRs). However, the NCRs show poor conservation across species, making their function and evolution elusive. Identification and functional characterization of NCRs across species would be critical for addressing these questions. To this end, we devised a computational pipeline and performed de novo assembly and annotation of mtDNA from 19 Caenorhabditis species using next-generation sequencing (NGS) data. The mtDNAs for 14 out of the 19 species are reported for the first time. Comparison of the 19 genomes reveals species-specific sampling of partial displacement-loop (D-loop) sequence as a novel NCR inserted into a unique tRNA cluster, suggesting an important role of the D-loop and the tRNA cluster in shaping NCR evolution. Intriguingly, RNA-Seq analysis suggests that a novel NCR resulting from a recent duplication of NADH dehydrogenase subunit 5 (ND5) could be utilized as a 3' UTR for up-regulation of its upstream gene. The expression analysis shows a species- and sex-specific expression of mitochondrial genes encoded by mtDNA and nucleus, respectively. Our analyses provide important insights into the function and evolution of mitochondrial NCRs and pave the way for further studying the function and evolution of mitochondrial genome.

Establishment of Signaling Interactions with Cellular Resolution for Every Cell Cycle of Embryogenesis.

Intercellular signaling interactions play a key role in breaking fate symmetry during animal development. Identification of signaling interactions at cellular resolution is technically challenging, especially in a developing embryo. Here, we develop a platform that allows automated inference and validation of signaling interactions for every cell cycle of Caenorhabditis elegans embryogenesis. This is achieved by the generation of a systems-level cell contact map, which consists of 1114 highly confident intercellular contacts, by modeling analysis and is validated through cell membrane labeling coupled with cell lineage analysis. We apply the map to identify cell pairs between which a Notch signaling interaction takes place. By generating expression patterns for two ligands and two receptors of the Notch signaling pathway with cellular resolution using the automated expression profiling technique, we are able to refine existing and identify novel Notch interactions during C. elegans embryogenesis. Targeted cell ablation followed by cell lineage analysis demonstrates the roles of signaling interactions during cell division in breaking fate symmetry. Finally, we describe the development of a website that allows online access to the cell-cell contact map for mapping of other signaling interactions by the community. The platform can be adapted to establish cellular interactions from any other signaling pathway.

Genomic basis of recombination suppression in the hybrid between Caenorhabditis briggsae and C. nigoni.

DNA recombination is required for effective segregation and diversification of genomes and for the successful completion of meiosis. Recent studies in various species hybrids have demonstrated a genetic link between DNA recombination and speciation. Consistent with this, we observed a striking suppression of recombination in the hybrids between two nematodes, the hermaphroditic Caenorhabditis briggsae and the gonochoristic C. nigoni. To unravel the molecular basis underlying the recombination suppression in their hybrids, we generated a C. nigoni genome with chromosome-level contiguity and produced an improved C. briggsae genome with resolved gaps up to 2.8 Mb. The genome alignment reveals not only high sequence divergences but also pervasive intra- and inter-chromosomal sequence re-arrangements between the two species, which are plausible culprits for the observed suppression. Comparison of recombination boundary sequences suggests that recombination in the hybrid requires extensive sequence homology, which is rarely seen between the two genomes. The new genomes and genomic libraries form invaluable resources for studying genome evolution, hybrid incompatibilities and sex evolution for this pair of model species.

Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division.

Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

Specific down-regulation of spermatogenesis genes targeted by 22G RNAs in hybrid sterile males associated with an X-Chromosome introgression.

Hybrid incompatibility (HI) prevents gene flow between species, thus lying at the heart of speciation genetics. One of the most common HIs is male sterility. Two superficially contradictory observations exist for hybrid male sterility. First, an introgression on the X Chromosome is more likely to produce male sterility than on autosome (so-called large-X theory); second, spermatogenesis genes are enriched on the autosomes but depleted on the X Chromosome (demasculinization of X Chromosome). Analysis of gene expression in Drosophila hybrids suggests a genetic interaction between the X Chromosome and autosomes that is essential for male fertility. However, the prevalence of such an interaction and its underlying mechanism remain largely unknown. Here we examine the interaction in nematode species by contrasting the expression of both coding genes and transposable elements (TEs) between hybrid sterile males and its parental nematode males. We use two lines of hybrid sterile males, each carrying an independent introgression fragment from Caenorhabditis briggsae X Chromosome in an otherwise Caenorhabditis nigoni background, which demonstrate similar defects in spermatogenesis. We observe a similar pattern of down-regulated genes that are specific for spermatogenesis between the two hybrids. Importantly, the down-regulated genes caused by the X Chromosome introgressions show a significant enrichment on the autosomes, supporting an epistatic interaction between the X Chromosome and autosomes. We investigate the underlying mechanism of the interaction by measuring small RNAs and find that a subset of 22G RNAs specifically targeting the down-regulated spermatogenesis genes is significantly up-regulated in hybrids, suggesting that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction.

Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis.

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development.

Systems-level quantification of division timing reveals a common genetic architecture controlling asynchrony and fate asymmetry.

Coordination of cell division timing is crucial for proper cell fate specification and tissue growth. However, the differential regulation of cell division timing across or within cell types during metazoan development remains poorly understood. To elucidate the systems-level genetic architecture coordinating division timing, we performed a high-content screening for genes whose depletion produced a significant reduction in the asynchrony of division between sister cells (ADS) compared to that of wild-type during Caenorhabditis elegans embryogenesis. We quantified division timing using 3D time-lapse imaging followed by computer-aided lineage analysis. A total of 822 genes were selected for perturbation based on their conservation and known roles in development. Surprisingly, we find that cell fate determinants are not only essential for establishing fate asymmetry, but also are imperative for setting the ADS regardless of cellular context, indicating a common genetic architecture used by both cellular processes. The fate determinants demonstrate either coupled or separate regulation between the two processes. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Our quantitative dataset with cellular resolution provides a resource for future analyses of the genetic control of spatial and temporal coordination during metazoan development.