Lactation-associated macrophages exist in murine mammary tissue and human milk.

Macrophages are involved in immune defense, organogenesis and tissue homeostasis. Macrophages contribute to the different phases of mammary gland remodeling during development, pregnancy and involution postlactation. Less is known about the dynamics of mammary gland macrophages in the lactation stage. Here, we describe a macrophage population present during lactation in mice. By multiparameter flow cytometry and single-cell RNA sequencing, we identified a lactation-induced CD11c+CX3CR1+Dectin-1+ macrophage population (liMac) that was distinct from the two resident F4/80hi and F4/80lo macrophage subsets present pregestationally. LiMacs were predominantly monocyte-derived and expanded by proliferation in situ concomitant with nursing. LiMacs developed independently of IL-34, but required CSF-1 signaling and were partly microbiota-dependent. Locally, they resided adjacent to the basal cells of the alveoli and extravasated into the milk. We found several macrophage subsets in human milk that resembled liMacs. Collectively, these findings reveal the emergence of unique macrophages in the mammary gland and milk during lactation.

Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Human pluripotent stem cell-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

Induction of the nuclear receptor PPAR-γ by the cytokine GM-CSF is critical for the differentiation of fetal monocytes into alveolar macrophages.

Tissue-resident macrophages constitute heterogeneous populations with unique functions and distinct gene-expression signatures. While it has been established that they originate mostly from embryonic progenitor cells, the signals that induce a characteristic tissue-specific differentiation program remain unknown. We found that the nuclear receptor PPAR-γ determined the perinatal differentiation and identity of alveolar macrophages (AMs). In contrast, PPAR-γ was dispensable for the development of macrophages located in the peritoneum, liver, brain, heart, kidneys, intestine and fat. Transcriptome analysis of the precursors of AMs from newborn mice showed that PPAR-γ conferred a unique signature, including several transcription factors and genes associated with the differentiation and function of AMs. Expression of PPAR-γ in fetal lung monocytes was dependent on the cytokine GM-CSF. Therefore, GM-CSF has a lung-specific role in the perinatal development of AMs through the induction of PPAR-γ in fetal monocytes.

De-obstruction of bladder outlet in humans reverses organ remodelling by normalizing the expression of key transcription factors.

BACKGROUND: Benign prostatic hyperplasia in elderly males often causes bladder outlet obstruction termed benign prostatic obstruction (BPO). BPO induces lower urinary tract symptoms and quantifiable urodynamic alterations in bladder function. When conservative medical treatments are exhausted, surgical interventions like transurethral resection of the prostate (TURP) are employed for bladder outlet de-obstruction. Elucidating the molecular changes in the human bladder resulting from BPO and their reversal post-de-obstruction is pivotal for defining the "point of no return", when the organ deterioration becomes irreversible. In this study we carried out a comprehensive molecular and urodynamic characterization of the bladders in men with BPO before TURP and 3 months after the relief of obstruction. METHODS: We report integrated transcriptome and proteome analysis of bladder samples from male patients with BPO before and 3 months after de-obstruction surgery (TURP). mRNA and protein profiles were correlated with urodynamic findings, specifically voiding detrusor pressure (PdetQmax) before TURP. We delineated the molecular classifiers of each group, pointing at the different pre-TURP bladder status. RESULTS: Age-matched patients with BPO without DO were divided into two groups based on the PdetQmax values recorded by UDI before de-obstruction: high and medium pressure (HP and MP) groups. Three months after de-obstruction surgery, the voiding parameters PdetQmax, Qmax and RV were significantly improved in both groups, without notable inter-group differences in the values after TURP. Patients with high PdetQmax showed less advanced remodeling and inflammatory changes than those with lower values. We detected significant dysregulation of gene expression, which was at least partially reversed by de-obstruction in both patients' groups. Transcription factor SOX21 and its target thrombospondin 4 (THBS4) demonstrated normalization post-TURP. CONCLUSIONS: Our findings reveal substantial yet incomplete reversal of cell signalling pathways three months after TURP, consistent with improved urodynamic parameters. We propose a set of biomarker genes, indicative of BPO, and possibly contributing to the bladder changes. This study unveils the stages of progressive obstruction-induced bladder decompensation and offers insights into selecting an optimal intervention point to mitigate loss of contractility.

The Pattern of RNA Editing Changes in Pleural Mesothelioma upon Epithelial-Mesenchymal Transition.

Pleural mesothelioma (PM) is a cancer where epithelioid, biphasic and sarcomatoid histotypes are observed. Sarcomatoid PM is characterized by mesenchymal features. Multi-omics have been used to characterize the epithelial-to-mesenchymal (EMT) phenotype at the molecular level. We contribute to this effort by including the analysis of RNA editing. We extracted samples with the highest vs. lowest Epithelial score from two PM cohorts and observed increased RNA editing in introns and decreased RNA editing in 3'UTR upon EMT. The same was observed in primary PM primary cultures stratified by transcriptomics analysis into two groups, one of them enriched with mesenchymal features. Our data demonstrate that, as has been observed in other cancer types, RNA editing associates to EMT phenotype in PM.

Phenotype of Mrps5-Associated Phylogenetic Polymorphisms Is Intimately Linked to Mitoribosomal Misreading.

We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.

A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers.

The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g., adapter ligation bias) and lacks absolute transcript quantitation as well as tailor-made quality controls. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs, we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability and coupled with a novel panel of exogenous small RNA spike-in controls to enable quality control and absolute quantitation, thus ensuring comparability of data across independent NGS experiments. The established microRNA Next-Generation-Sequencing Discovery Assay (miND) was validated for its relative accuracy, precision, analytical measurement range and sequencing bias and was considered fit-for-purpose for microRNA biomarker discovery. Summarized, all these criteria were met, and thus, our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.

Integrated mRNA-miRNA transcriptome analysis of bladder biopsies from patients with bladder pain syndrome identifies signaling alterations contributing to the disease pathogenesis.

BACKGROUND: Interstitial cystitis, or bladder pain syndrome (IC/BPS), is a chronic bladder disorder characterized by lower abdominal pain associated with the urinary bladder and accompanied by urinary frequency and urgency in the absence of identifiable causes. IC/PBS can be separated into the classic Hunner's ulcerative type and the more prevalent non-ulcerative disease. Our aim was to unravel the biological processes and dysregulated cell signaling pathways leading to the bladder remodeling in non-ulcerative bladder pain syndrome (BPS) by studying the gene expression changes in the patients' biopsies. METHODS: We performed paired microRNA (miRNA) and mRNA expression profiling in the bladder biopsies of BPS patients with non-Hunner interstitial cystitis phenotype, using comprehensive Next-generation sequencing (NGS) and studied the activated pathways and altered biological processes based on the global gene expression changes. Paired mRNA-miRNA transcriptome analysis delineated the regulatory role of the dysregulated miRNAs by identifying their targets in the disease-induced pathways. RESULTS: EIF2 Signaling and Regulation of eIF4 and p70S6K Signaling, activated in response to cellular stress, were among the most significantly regulated processes during BPS. Leukotriene Biosynthesis nociceptive pathway, important in inflammatory diseases and neuropathic pain, was also significantly activated. The biological processes identified using Gene Ontology over-representation analysis were clustered into six main functional groups: cell cycle regulation, chemotaxis of immune cells, muscle development, muscle contraction, remodeling of extracellular matrix and peripheral nervous system organization and development. Compared to the Hunner's ulcerative type IC, activation of the immune pathways was modest in non-ulcerative BPS, limited to neutrophil chemotaxis and IFN-γ-mediated signaling. We identified 62 miRNAs, regulated and abundant in BPS and show that they target the mRNAs implicated in eIF2 signalling pathway. CONCLUSIONS: The bladders of non-ulcerative BPS patients recruited in this study had alterations consistent with a strong cell proliferative response and an up-regulation of smooth muscle contractility, while the contribution of inflammatory processes was modest. Pathway analysis of the integrated mRNA-miRNA NGS dataset pinpointed important regulatory miRNAs whose dysregulation might contribute to the pathogenesis. Observed molecular changes in the peripheral nervous system organization and development indicate the potential role of local bladder innervation in the pain perceived in this type of BPS.

Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression.

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

Mutant MRPS5 affects mitoribosomal accuracy and confers stress-related behavioral alterations.

The 1555 A to G substitution in mitochondrial 12S A-site rRNA is associated with maternally transmitted deafness of variable penetrance in the absence of otherwise overt disease. Here, we recapitulate the suggested A1555G-mediated pathomechanism in an experimental model of mitoribosomal mistranslation by directed mutagenesis of mitoribosomal protein MRPS5. We first establish that the ratio of cysteine/methionine incorporation and read-through of mtDNA-encoded MT-CO1 protein constitute reliable measures of mitoribosomal misreading. Next, we demonstrate that human HEK293 cells expressing mutant V336Y MRPS5 show increased mitoribosomal mistranslation. As for immortalized lymphocytes of individuals with the pathogenic A1555G mutation, we find little changes in the transcriptome of mutant V336Y MRPS5 HEK cells, except for a coordinated upregulation of transcripts for cytoplasmic ribosomal proteins. Homozygous knock-in mutant Mrps5 V338Y mice show impaired mitochondrial function and a phenotype composed of enhanced susceptibility to noise-induced hearing damage and anxiety-related behavioral alterations. The experimental data in V338Y mutant mice point to a key role of mitochondrial translation and function in stress-related behavioral and physiological adaptations.

Effects of a male meiotic driver on male and female transcriptomes in the house mouse.

Not all genetic loci follow Mendel's rules, and the evolutionary consequences of this are not yet fully known. Genomic conflict involving multiple loci is a likely outcome, as restoration of Mendelian inheritance patterns will be selected for, and sexual conflict may also arise when sexes are differentially affected. Here, we investigate effects of the t haplotype, an autosomal male meiotic driver in house mice, on genome-wide gene expression patterns in males and females. We analysed gonads, liver and brain in adult same-sex sibling pairs differing in genotype, allowing us to identify t-associated differences in gene regulation. In testes, only 40% of differentially expressed genes mapped to the approximately 708 annotated genes comprising the t haplotype. Thus, much of the activity of the t haplotype occurs in trans, and as upregulation. Sperm maturation functions were enriched among both cis and trans acting t haplotype genes. Within the t haplotype, we observed more downregulation and differential exon usage. In ovaries, liver and brain, the majority of expression differences mapped to the t haplotype, and were largely independent of the differences seen in the testis. Overall, we found widespread transcriptional effects of this male meiotic driver in the house mouse genome.

Gene expression profiling of the canine placenta during normal and antigestagen-induced luteolysis.

The domestic dog is the only domestic animal species that does not produce steroids in the placenta and instead relies on luteal steroids throughout pregnancy. Nevertheless, the canine placenta is highly responsive to steroids, and withdrawal of progesterone (P4) affects the feto-maternal unit, initializing the parturition cascade. Similar effects can be observed during antigestagen-induced abortion. Here, aiming to provide new insights into mechanisms involved in the termination of canine pregnancy, next generation sequencing (NGS, RNA-seq) was applied. Placental transcriptomes derived from natural prepartum and antigestagen-induced abortions were analyzed and compared with fully developed mid-gestation placentas. The contrast "prepartum luteolysis over mid-gestation" revealed 1973 differentially expressed genes (DEG). Terms associated with apoptosis, impairment of vascular function and activation of signaling of several cytokines (e.g., IL-8, IL-3, TGF-ß) were overrepresented at natural luteolysis. When compared with mid-term, antigestagen treatment revealed 135 highly regulated DEG that were involved in the induced luteolysis and showed similar associations with functional terms and expression patterns as during natural luteolysis. The contrast "antigestagen-induced luteolysis over prepartum luteolysis" revealed that, although similar changes occur in both conditions, they are more pronounced during natural prepartum. Among P4-regulated DEG were those related to immune system and cortisol metabolism. It appears that, besides inducing placental PGF2α output, both natural and induced P4 withdrawal is associated with disruption of the feto-maternal interface, leading to impaired vascular functions, apoptosis and controlled modulation of the immune response. The time-related maturation of the feto-maternal interface needs to be considered because it may be clinically relevant.

Non-Coding Transcript Heterogeneity in Mesothelioma: Insights from Asbestos-Exposed Mice.

Mesothelioma is an aggressive, rapidly fatal cancer and a better understanding of its molecular heterogeneity may help with making more efficient therapeutic strategies. Non-coding RNAs represent a larger part of the transcriptome but their contribution to diseases is not fully understood yet. We used recently obtained RNA-seq data from asbestos-exposed mice and performed data mining of publicly available datasets in order to evaluate how non-coding RNA contribute to mesothelioma heterogeneity. Nine non-coding RNAs are specifically elevated in mesothelioma tumors and contribute to human mesothelioma heterogeneity. Because some of them have known oncogenic properties, this study supports the concept of non-coding RNAs as cancer progenitor genes.

Transcriptome analysis reveals differences in mechanisms regulating cessation of luteal function in pregnant and non-pregnant dogs.

BACKGROUND: In the domestic dog, corpora lutea (CL) are the only source of progesterone (P4), both in pregnant and non-pregnant cycles because there is no placental steroidogenesis. The absence of an endogenous luteolysin in absence of pregnancy results in long-lasting physiological pseudopregnancy, strongly contrasting with the acute luteolysis observed prepartum. The underlying biological mechanisms and the involvement of P4 signalling remain, however, not fully understood. Therefore, here, next-generation sequencing (RNA-Seq) was performed on CL from the late luteal phase and compared with normally luteolyzing CL collected at the prepartum P4 decrease. RESULTS: The contrast "luteal regression over luteolysis" yielded 1595 differentially expressed genes (DEG). The CL in late luteal regression were predominantly associated with functional terms linked to extracellular matrix (p = 5.52e-05). Other terms related to transcriptional activity (p = 2.45e-04), and steroid hormone signalling (p = 2.29e-04), which were more highly represented in late regression than during luteolysis. The prepartum luteolysis was associated with immune inflammatory responses (p = 2.87e-14), including acute-phase reaction (p = 4.10e-06). Immune system-related events were also more highly represented in CL derived from normal luteolysis (p = 7.02e-04), compared with those from dogs in which luteolysis was induced with an antigestagen (1480 DEG in total). Additionally, the withdrawal of P4 at mid-gestation resulted in 92 DEG; over-represented terms enriched in antigestagen-treated dogs were related to the inflammatory response (p = 0.005) or response to IL1 (p = 7.29e-05). Terms related to proliferation, e.g., centrosome organization (p = 0.002) and steroid metabolic processes (p = 0.001), prevailed at mid-gestation. Thereby, our results revealed the nature of luteotropic effects of P4 within canine CL. It appears that, even though they result in diminished steroidogenic output, the effect of antigestagens is more related to the withdrawal of P4 support than to the PGF2alpha-related inflammatory reaction observed at physiological parturition. CONCLUSIONS: We report the differential gene expression associated with maintenance and cessation of luteal function in pregnant and non-pregnant dogs. Based on the differentially expressed genes, we indicate functional pathways and gene networks that are potentially involved in the underlying endocrine and molecular mechanisms. This study establishes future research directions that may be helpful in understanding some of the clinical conditions, such as luteal insufficiency, associated with negative pregnancy outcome in dogs.

SUSHI: an exquisite recipe for fully documented, reproducible and reusable NGS data analysis.

BACKGROUND: Next generation sequencing (NGS) produces massive datasets consisting of billions of reads and up to thousands of samples. Subsequent bioinformatic analysis is typically done with the help of open source tools, where each application performs a single step towards the final result. This situation leaves the bioinformaticians with the tasks to combine the tools, manage the data files and meta-information, document the analysis, and ensure reproducibility. RESULTS: We present SUSHI, an agile data analysis framework that relieves bioinformaticians from the administrative challenges of their data analysis. SUSHI lets users build reproducible data analysis workflows from individual applications and manages the input data, the parameters, meta-information with user-driven semantics, and the job scripts. As distinguishing features, SUSHI provides an expert command line interface as well as a convenient web interface to run bioinformatics tools. SUSHI datasets are self-contained and self-documented on the file system. This makes them fully reproducible and ready to be shared. With the associated meta-information being formatted as plain text tables, the datasets can be readily further analyzed and interpreted outside SUSHI. CONCLUSION: SUSHI provides an exquisite recipe for analysing NGS data. By following the SUSHI recipe, SUSHI makes data analysis straightforward and takes care of documentation and administration tasks. Thus, the user can fully dedicate his time to the analysis itself. SUSHI is suitable for use by bioinformaticians as well as life science researchers. It is targeted for, but by no means constrained to, NGS data analysis. Our SUSHI instance is in productive use and has served as data analysis interface for more than 1000 data analysis projects. SUSHI source code as well as a demo server are freely available.

MicroRNA MiR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting WNT2 signaling pathway.

MicroRNA miR-199a-5p impairs tight junction formation, leading to increased urothelial permeability in bladder pain syndrome. Now, using transcriptome analysis in urothelial TEU-2 cells, we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF, and WNT signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder, and we altered its levels in bladder smooth muscle cells (SMCs) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size, and up-regulated miR-199a-5p targets, including WNT2. Overexpression of WNT2 protein or treating SMCs with recombinant WNT2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of WNT2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM α-actin, and SM myosin heavy chain mRNA and protein levels. These changes as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of the smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor A downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of WNT-dependent inhibitory Krüppel-like transcription factor 4 in miR-199a-5p-overexpressing cells. In contrast, Krüppel-like transcription factor 4 was induced in antimiR-expressing cells following the activation of WNT2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the WNT2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, which is relevant for organ remodeling.

Field Trial and Molecular Characterization of RNAi-Transgenic Tomato Plants That Exhibit Resistance to Tomato Yellow Leaf Curl Geminivirus.

RNA interference (RNAi) is a widely used approach to generate virus-resistant transgenic crops. However, issues of agricultural importance like the long-term durability of RNAi-mediated resistance under field conditions and the potential side effects provoked in the plant by the stable RNAi expression remain poorly investigated. Here, we performed field trials and molecular characterization studies of two homozygous transgenic tomato lines, with different selection markers, expressing an intron-hairpin RNA cognate to the Tomato yellow leaf curl virus (TYLCV) C1 gene. The tested F6 and F4 progenies of the respective kanamycin- and basta-resistant plants exhibited unchanged field resistance to TYLCV and stably expressed the transgene-derived short interfering RNA (siRNAs) to represent 6 to 8% of the total plant small RNAs. This value outnumbered the average percentage of viral siRNAs in the nontransformed plants exposed to TYLCV-infested whiteflies. As a result of the RNAi transgene expression, a common set of up- and downregulated genes was revealed in the transcriptome profile of the plants selected from either of the two transgenic events. A previously unidentified geminivirus causing no symptoms of viral disease was detected in some of the transgenic plants. The novel virus acquired V1 and V2 genes from TYLCV and C1, C2, C3, and C4 genes from a distantly related geminivirus and, thereby, it could evade the repressive sequence-specific action of transgene-derived siRNAs. Our findings shed light on the mechanisms of siRNA-directed antiviral silencing in transgenic plants and highlight the applicability limitations of this technology as it may alter the transcriptional pattern of nontarget genes.

Diurnal changes in the histone H3 signature H3K9ac|H3K27ac|H3S28p are associated with diurnal gene expression in Arabidopsis.

Post-translational chromatin modifications are an important regulatory mechanism in light signalling and circadian clock function. The regulation of diurnal transcript level changes requires fine-tuning of the expression of generally active genes depending on the prevailing environmental conditions. We investigated the association of histone modifications H3K4me3, H3K9ac, H3K9me2, H3S10p, H3K27ac, H3K27me3 and H3S28p with diurnal changes in transcript expression using chromatin immunoprecipitations followed by sequencing (ChIP-Seq) in fully expanded leaves 6 of Arabidopsis thaliana grown in short-day optimal and water-deficit conditions. We identified a differential H3K9ac, H3K27ac and H3S28p signature between end-of-day and end-of-night that is correlated with changes in diurnal transcript levels. Genes with this signature have particular over-represented promoter elements and encode proteins that are significantly enriched for transcription factors, circadian clock and starch catabolic process. Additional activating modifications were prevalent in optimally watered (H3S10p) and in water-deficit (H3K4me3) plants. The data suggest a mechanism for diurnal transcript level regulation in which reduced binding of repressive transcription factors facilitates activating H3K9ac, H3K27ac and H3S28p chromatin modifications. The presence of activating chromatin modification patterns on genes only at times of the day when their expression is required can explain why some genes are differentially inducible during the diurnal cycle.

RIP-chip-SRM--a new combinatorial large-scale approach identifies a set of translationally regulated bantam/miR-58 targets in C. elegans.

MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression. As miRNAs are involved in a wide range of biological processes and diseases, much effort has been invested in identifying their mRNA targets. Here, we present a novel combinatorial approach, RIP-chip-SRM (RNA-binding protein immunopurification + microarray + targeted protein quantification via selected reaction monitoring), to identify de novo high-confidence miRNA targets in the nematode Caenorhabditis elegans. We used differential RIP-chip analysis of miRNA-induced silencing complexes from wild-type and miRNA mutant animals, followed by quantitative targeted proteomics via selected reaction monitoring to identify and validate mRNA targets of the C. elegans bantam homolog miR-58. Comparison of total mRNA and protein abundance changes in mir-58 mutant and wild-type animals indicated that the direct bantam/miR-58 targets identified here are mainly regulated at the level of protein abundance, not mRNA stability.