Gene-edited Mtsoc1 triple mutant Medicago plants do not flower.

Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. SOC1-like genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model Arabidopsis thaliana (Arabidopsis), where SOC1 promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. Medicago truncatula (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one SOC1 gene, multiple gene duplication events have given rise to three MtSOC1 paralogs within the Medicago genus in legumes: one Fabaceae group A SOC1 gene, MtSOC1a, and two tandemly repeated Fabaceae group B SOC1 genes, MtSOC1b and MtSOC1c. Previously, we showed that MtSOC1a has unique functions in floral promotion in Medicago. The Mtsoc1a Tnt1 retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, Mtsoc1b Tnt1 single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of Mtsoc1a Mtsoc1b Mtsoc1c triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three MtSOC1 genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single Mtsoc1a mutant and the Arabidopsis soc1 mutant; implicating multiple MtSOC1 genes in critical overlapping roles in the transition to flowering in Medicago.

piRNA generation is associated with the pioneer round of translation in stem cells.

Much insight has been gained on how stem cells maintain genomic integrity, but less attention has been paid to how they maintain their transcriptome. Here, we report that the PIWI protein SMEDWI-1 plays a role in the filtering of dysfunctional transcripts from the transcriptome of planarian stem cells. SMEDWI-1 accomplishes this through association with the ribosomes during the pioneer round of translation, and processing of poorly translated transcripts into piRNAs. This results in the removal of such transcripts from the cytoplasmic pool and at the same time creates a dynamic pool of small RNAs for post-transcriptional surveillance through the piRNA pathway. Loss of SMEDWI-1 results in elevated levels of several non-coding transcripts, including rRNAs, snRNAs and pseudogene mRNAs, while reducing levels of several coding transcripts. In the absence of SMEDWI-1, stem cell colonies are delayed in their expansion and a higher fraction of descendants exit the stem cell state, indicating that this transcriptomic sanitation mediated by SMEDWI-1 is essential to maintain stem cell health. This study presents a new model for the function of PIWI proteins in stem cell maintenance, that complements their role in transposon repression, and proposes a new biogenesis pathway for piRNAs in stem cells.

Chromatin analysis of adult pluripotent stem cells reveals a unique stemness maintenance strategy.

Many highly regenerative organisms maintain adult pluripotent stem cells throughout their life, but how the long-term maintenance of pluripotency is accomplished is unclear. To decipher the regulatory logic of adult pluripotent stem cells, we analyzed the chromatin organization of stem cell genes in the planarian Schmidtea mediterranea. We identify a special chromatin state of stem cell genes, which is distinct from that of tissue-specific genes and resembles constitutive genes. Where tissue-specific promoters have detectable transcription factor binding sites, the promoters of stem cell-specific genes instead have sequence features that broadly decrease nucleosome binding affinity. This genic organization makes pluripotency-related gene expression the default state in these cells, which is maintained by the activity of chromatin remodelers ISWI and SNF2 in the stem cells.

Insights in piRNA targeting rules.

PIWI-interacting RNAs (piRNAs) play an important role in the defense against transposons in the germline and stem cells of animals. To what extent other transcripts are also regulated by piRNAs is an ongoing topic of debate. The amount of sequence complementarity between piRNA and target that is required for effective downregulation of the targeted transcript is guiding in this discussion. Over the years, various methods have been applied to infer targeting requirements from the collections of piRNAs and potential target transcripts, and recent structural studies of the PIWI proteins have provided an additional perspective. In this review, I summarize the findings from these studies and propose a set of requirements that can be used to predict targets to the best of our current abilities. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA-Based Catalysis > RNA-Mediated Cleavage.

MtING2 encodes an ING domain PHD finger protein which affects Medicago growth, flowering, global patterns of H3K4me3, and gene expression.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that >7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

NODeJ: an ImageJ plugin for 3D segmentation of nuclear objects.

BACKGROUND: The three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization. RESULTS: We present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest. CONCLUSION AND AVAILABILITY: NODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis . The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE .

PIWI-mediated control of tissue-specific transposons is essential for somatic cell differentiation.

PIWI proteins are known as mediators of transposon silencing in animal germlines but are also found in adult pluripotent stem cells of highly regenerative animals, where they are essential for regeneration. Study of the nuclear PIWI protein SMEDWI-2 in the planarian somatic stem cell system reveals an intricate interplay between transposons and cell differentiation in which a subset of transposons is inevitably activated during cell differentiation, and the PIWI protein is required to regain control. Absence of SMEDWI-2 leads to tissue-specific transposon derepression related to cell-type-specific chromatin remodeling events and in addition causes reduced accessibility of lineage-specific genes and defective cell differentiation, resulting in fatal tissue dysfunction. Finally, we show that additional PIWI proteins provide a stem-cell-specific second layer of protection in planarian neoblasts. These findings reveal a far-reaching role of PIWI proteins and PIWI-interacting RNAs (piRNAs) in stem cell biology and cell differentiation.

A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes.

The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya - either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals. Studies of Gcna-mutant C. elegans and mice indicate that GCNA has functioned in reproduction for at least 600 million years. Homology to IDR-containing proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome.

Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment.

Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage, including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings indicate that planarian neoblasts comprise two major and functionally distinct cellular compartments.

Piwi and potency: PIWI proteins in animal stem cells and regeneration.

PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a possible correlation between the differentiation potential of a cell and its PIWI levels.

MUT-14 and SMUT-1 DEAD box RNA helicases have overlapping roles in germline RNAi and endogenous siRNA formation.

More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs.

Whole-body acoel regeneration is controlled by Wnt and Bmp-Admp signaling.

Whole-body regeneration is widespread in the Metazoa, yet little is known about how underlying molecular mechanisms compare across phyla. Acoels are an enigmatic phylum of invertebrate worms that can be highly informative about many questions in bilaterian evolution, including regeneration. We developed the three-banded panther worm, Hofstenia miamia, as a new acoelomorph model system for molecular studies of regeneration. Hofstenia were readily cultured, with accessible embryos, juveniles, and adults for experimentation. We developed molecular resources and tools for Hofstenia, including a transcriptome and robust systemic RNAi. We report the identification of molecular mechanisms that promote whole-body regeneration in Hofstenia. Wnt signaling controls regeneration of the anterior-posterior axis, and Bmp-Admp signaling controls regeneration of the dorsal-ventral axis. Perturbation of these pathways resulted in regeneration-abnormal phenotypes involving axial feature duplication, such as the regeneration of two heads following Wnt perturbation or the regeneration of ventral cells in place of dorsal ones following bmp or admp RNAi. Hofstenia regenerative mechanisms are strikingly similar to those guiding regeneration in planarians. However, phylogenetic analyses using the Hofstenia transcriptome support an early branching position for acoels among bilaterians, with the last common ancestor of acoels and planarians being the ancestor of the Bilateria. Therefore, these findings identify similar whole-body regeneration mechanisms in animals separated by more than 550 million years of evolution.

Differential impact of the HEN1 homolog HENN-1 on 21U and 26G RNAs in the germline of Caenorhabditis elegans.

RNA interference (RNAi)-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.

The role of small non-coding RNAs in genome stability and chromatin organization.

Small non-coding RNAs make up much of the RNA content of a cell and have the potential to regulate gene expression on many different levels. Initial discoveries in the 1990s and early 21st century focused on determining mechanisms of post-transcriptional regulation mediated by small-interfering RNAs (siRNAs) and microRNAs (miRNAs). More recent research, however, has identified new classes of RNAs and new regulatory mechanisms, expanding the known regulatory potential of small non-coding RNAs to encompass chromatin regulation. In this Commentary, we provide an overview of these chromatin-related mechanisms and speculate on the extent to which they are conserved among eukaryotes.

The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1alpha and IL-18 production by a murine and human keratinocyte cell line.

Assessment of allergenic potency of low molecular weight compounds is generally performed using animal models, such as the guinea pig maximisation test and the murine local lymph node assay (LLNA). Progress in unravelling the mechanisms of skin sensitisation, including effects on the production of cytokines by the different cell types of the skin, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitisation testing. The aim of the present study was to establish an in vitro method to assess the potency of allergens, on the basis of their induction of cytokine production by murine and human keratinocytes. In the present study we used test systems comprised of the murine epidermal keratinocyte cell line HEL-30 and the human keratinocyte cell line HaCaT. We exposed these cell lines to the allergens ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), and phthalic anhydride (PA). IL-1alpha and IL-18 dose-response data were evaluated by non-linear regression analysis and at a stimulation index of 3 of cytokine production of treatment versus control, the corresponding allergen concentration was calculated. For HEL-30, for both cytokines DNCB showed the strongest potency followed in this order by PA, benzocaine, and DEA. This classification was similar to our previous findings obtained in the LLNA. For HaCaT, unfortunately, such ranking proved to be much less feasible. In conclusion, to assess the potency of allergens the murine keratinocyte cell line HEL-30 may be a useful in vitro test system, alternative to in vivo models, although this requires further testing using a much wider range of compounds.