Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state

In the metazoan germline, piwi proteins and associated piwi-interacting RNAs (piRNAs) provide a defense system against the expression of transposable elements. In the cytoplasm, piRNA sequences guide piwi complexes to destroy complementary transposon transcripts by endonucleolytic cleavage. However, some piwi family members are nuclear, raising the possibility of alternative pathways for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromosomes. Knockdown of Piwi in the germline increases expression of transposable elements that are targeted by piRNAs, whereas protein-coding genes remain largely unaffected. Derepression of transposons upon Piwi depletion correlates with increased occupancy of Pol II on their promoters. Expression of piRNAs that target a reporter construct results in a decrease in Pol II occupancy and an increase in repressive H3K9me3 marks and heterochromatin protein 1 (HP1) on the reporter locus. Our results indicate that Piwi identifies targets complementary to the associated piRNA and induces transcriptional repression by establishing a repressive chromatin state when correct targets are found

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity

FMRFamide-like peptides expand the behavioral repertoire of a densely connected nervous system

Animals, including humans, can adapt to environmental stress through phenotypic plasticity. The free-living nematode Caenorhabditis elegans can adapt to harsh environments by undergoing a whole-animal change, involving exiting reproductive development and entering the stress-resistant dauer larval stage. The dauer is a dispersal stage with dauer-specific behaviors for finding and stowing onto carrier animals, but how dauers acquire these behaviors, despite having a physically limited nervous system of 302 neurons, is poorly understood. We compared dauer and reproductive development using whole-animal RNA sequencing at fine time points and at sufficient depth to measure transcriptional changes within single cells. We detected 8,042 genes differentially expressed during dauer and reproductive development and observed striking up-regulation of neuropeptide genes during dauer entry. We knocked down neuropeptide processing using sbt-1 mutants and demonstrate that neuropeptide signaling promotes the decision to enter dauer rather than reproductive development. We also demonstrate that during dauer neuropeptides modulate the dauer-specific nictation behavior (carrier animal-hitchhiking) and are necessary for switching from repulsion to CO_2 (a carrier animal cue) in nondauers to CO_2 attraction in dauers. We tested individual neuropeptides using CRISPR knockouts and existing strains and demonstrate that the combined effects of flp-10 and flp-17 mimic the effects of sbt-1on nictation and CO_2 attraction. Through meta-analysis, we discovered similar up-regulation of neuropeptides in the dauer-like infective juveniles of diverse parasitic nematodes, suggesting the antiparasitic target potential of SBT-1. Our findings reveal that, under stress, increased neuropeptide signaling in C. elegans enhances their decision-making accuracy and expands their behavioral repertoire

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites

Neonatal brain tissue classification with morphological adaptation and unified segmentation

Measuring the distribution of brain tissue types (tissue classification) in neonates is necessary for studying typical and atypical brain development, such as that associated with preterm birth, and may provide biomarkers for neurodevelopmental outcomes. Compared with magnetic resonance images of adults, neonatal images present specific challenges that require the development of specialized, population-specific methods. This paper introduces MANTiS (Morphologically Adaptive Neonatal Tissue Segmentation), which extends the unified segmentation approach to tissue classification implemented in Statistical Parametric Mapping (SPM) software to neonates. MANTiS utilizes a combination of unified segmentation, template adaptation via morphological segmentation tools and topological filtering, to segment the neonatal brain into eight tissue classes: cortical gray matter, white matter, deep nuclear gray matter, cerebellum, brainstem, cerebrospinal fluid (CSF), hippocampus and amygdala. We evaluated the performance of MANTiS using two independent datasets. The first dataset, provided by the NeoBrainS12 challenge, consisted of coronal T2-weighted images of preterm infants (born ≤30 weeks’ gestation) acquired at 30 weeks’ corrected gestational age (n= 5), coronal T2-weighted images of preterm infants acquired at 40 weeks’ corrected gestational age (n= 5) and axial T2-weighted images of preterm infants acquired at 40 weeks’ corrected gestational age (n= 5). The second dataset, provided by the Washington University NeuroDevelopmental Research (WUNDeR) group, consisted of T2-weighted images of preterm infants (born <30 weeks’ gestation) acquired shortly after birth (n= 12), preterm infants acquired at term-equivalent age (n= 12), and healthy term-born infants (born ≥38 weeks’ gestation) acquired within the first nine days of life (n= 12). For the NeoBrainS12 dataset, mean Dice scores comparing MANTiS with manual segmentations were all above 0.7, except for the cortical gray matter for coronal images acquired at 30 weeks. This demonstrates that MANTiS’ performance is competitive with existing techniques. For the WUNDeR dataset, mean Dice scores comparing MANTiS with manually edited segmentations demonstrated good agreement, where all scores were above 0.75, except for the hippocampus and amygdala. The results show that MANTiS is able to segment neonatal brain tissues well, even in images that have brain abnormalities common in preterm infants. MANTiS is available for download as an SPM toolbox from http://developmentalimagingmcri.github.io/mantis

The Nrf1 CNC-bZIP Protein Is Regulated by the Proteasome and Activated by Hypoxia

BACKGROUND: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. PRINCIPAL FINDINGS: We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. CONCLUSIONS: Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status

Genetic Interaction of Centrosomin and Bazooka in Apical Domain Regulation in Drosophila Photoreceptor

Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis. mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs). photoreceptor

Single nucleotide polymorphism discovery in elite north american potato germplasm

BACKGROUND: Current breeding approaches in potato rely almost entirely on phenotypic evaluations; molecular markers, with the exception of a few linked to disease resistance traits, are not widely used. Large-scale sequence datasets generated primarily through Sanger Expressed Sequence Tag projects are available from a limited number of potato cultivars and access to next generation sequencing technologies permits rapid generation of sequence data for additional cultivars. When coupled with the advent of high throughput genotyping methods, an opportunity now exists for potato breeders to incorporate considerably more genotypic data into their decision-making. RESULTS: To identify a large number of Single Nucleotide Polymorphisms (SNPs) in elite potato germplasm, we sequenced normalized cDNA prepared from three commercial potato cultivars: 'Atlantic', 'Premier Russet' and 'Snowden'. For each cultivar, we generated 2 Gb of sequence which was assembled into a representative transcriptome of (~)28-29 Mb for each cultivar. Using the Maq SNP filter that filters read depth, density, and quality, 575,340 SNPs were identified within these three cultivars. In parallel, 2,358 SNPs were identified within existing Sanger sequences for three additional cultivars, 'Bintje', 'Kennebec', and 'Shepody'. Using a stringent set of filters in conjunction with the potato reference genome, we identified 69,011 high confidence SNPs from these six cultivars for use in genotyping with the Infinium platform. Ninety-six of these SNPs were used with a BeadXpress assay to assess allelic diversity in a germplasm panel of 248 lines; 82 of the SNPs proved sufficiently informative for subsequent analyses. Within diverse North American germplasm, the chip processing market class was most distinct, clearly separated from all other market classes. The round white and russet market classes both include fresh market and processing cultivars. Nevertheless, the russet and round white market classes are more distant from each other than processing are from fresh market types within these two groups. CONCLUSIONS: The genotype data generated in this study, albeit limited in number, has revealed distinct relationships among the market classes of potato. The SNPs identified in this study will enable high-throughput genotyping of germplasm and populations, which in turn will enable more efficient marker-assisted breeding efforts in potato

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt

The inflammasome activates caspase-1 and the release of interleukin-1β (IL-1β) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2−/−/ApcMin/+ than in APCMin/+ mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1β and were primarily mediated by a non–bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK–mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2−/− mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers