Gene Annotation Tools Research Articles

Gene Enrichment and Pathway Analysis for Ketosis Resistance in Dairy Cattle: A GWAS-Based Approach

Ketosis in dairy cattle is a common metabolic disorder that arises during the transition period from late gestation to early lactation. It is primarily caused by an imbalance between energy intake and expenditure, leading to an excessive accumulation of ketone bodies. This condition can significantly affect cattle health and productivity. Recent advances in genomic research, especially genome-wide association studies (GWAS), offer an opportunity to explore the genetic factors that contribute to ketosis resistance. The aim of this study is to comprehensively review and analyze existing GWAS data using gene enrichment analysis to identify potential functional candidate gene pathways associated with ketosis resistance in dairy cattle. In this study, data obtained from seven different studies were examined and 640 non-repetitive genes were obtained after filtering. Using Enrichr, an online tool for gene annotation, pathway analysis was performed with human homologs of the identified genes. Our findings highlight the acylglycerol homeostasis pathway, the regulation of triglyceride metabolism, and the role of chylomicrons in maintaining metabolic balance during ketosis. Additionally, immune response pathways were found to be linked to the genes associated with ketosis, offering insights into the intricate interplay between metabolic and immune pathways in ketosis. This study emphasizes the importance of understanding genetic factors in developing breeding strategies aimed at enhancing metabolic health and productivity in dairy cattle. Future research should focus on validating these candidate genes and exploring their mechanistic roles to facilitate targeted interventions and improve resistance to ketosis in dairy herds.

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle

With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

Elucidating hippocampal proteome dynamics in moderate hepatic encephalopathy rats: insights from high-resolution mass spectrometry.

Hepatic encephalopathy (HE) is a debilitating neurological disorder associated with liver failure and characterized by impaired brain function. Decade-long studies have led to significant advances in our understanding of HE; however, effective therapeutic management of HE is lacking, and HE continues to be a significant cause of morbidity and mortality in patients, underscoring the need for continued research into its pathophysiology and treatment. Accordingly, the present study provides a comprehensive overview aimed at elucidating the molecular underpinnings of HE and identifying potential therapeutic targets. A moderate-grade HE model was induced in rats using thioacetamide, which simulates the liver damage observed in patients, and its impact on cognitive function, neuronal arborization, and cellular morphology was also evaluated. We employed label-free LC-MS/MS proteomics to quantitatively profile hippocampal proteins to explore the molecular mechanism of HE pathogenesis; 2175 proteins were identified, 47 of which exhibited significant alterations in moderate-grade HE. The expression of several significantly upregulated proteins, such as FAK1, CD9 and Tspan2, was further validated at the transcript and protein levels, confirming the mass spectrometry results. These proteins have not been previously reported in HE. Utilizing Metascape, a tool for gene annotation and analysis, we further studied the biological pathways integral to brain function, including gliogenesis, the role of erythrocytes in maintaining blood-brain barrier integrity, the modulation of chemical synaptic transmission, astrocyte differentiation, the regulation of organ growth, the response to cAMP, myelination, and synaptic function, which were disrupted during HE. The STRING database further elucidated the protein‒protein interaction patterns among the differentially expressed proteins. This study provides novel insights into the molecular mechanisms driving HE and paves the way for identifying novel therapeutic targets for improved disease management.

Exploring the mechanism of Jingshen Xiaoke decoction in treating T2DM mice based on network pharmacology and molecular docking.

Jingshen Xiaoke decoction (JS) was prepared by studying the classic prescriptions of famous scholars in the past dynasties to prevent and treat diabetes. The related mechanism of JS against hyperlipidemia has yet to be revealed. To investigate the mechanism of action of JS in treating diabetes mellitus by using bioinformatics methods. A database was used to search the active ingredients and targets of the JS and targets for type 2 diabetes mellitus (T2DM). The protein interaction between the intersection targets, and the constructed the PPI network diagram was analyzed using the STRING database. Furthermore, the gene annotation tool DAVID was used to enrich the intersecting targets for the Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway. Finally, Maestro software was used for molecular docking to verify the binding ability of the active ingredients to the core target genes. A total of 45 active ingredients in JS were screened out corresponding to 239 effective targets, of which 64 targets were potential targets for treating T2DM. The analysis of PPI network diagram analysis revealed that the ingredients' active components are quercetin, β-sitosterol, stigmasterol, luteolin, and 7-Methoxy-2-methyl isoflavone. GO functional enrichment analysis indicated 186 biological processes (BP), 23 molecular functions (MF) and 13 cellular components (CC). KEGG pathway enrichment analysis revealed the enrichment of 59 signal pathways. The molecular docking results demonstrated that the active ingredients and core targets had a good docking affinity with a binding activity less than -7 kcal/mol. Finally, the western blotting illustrated that JS could up-regulate the liver PI3K/AKT-signaling pathway. JS can regulate glucolipid metabolism, reduce the inflammatory response, improve insulin resistance and modulate the immune response through PI3K/AKT signaling pathway treating of T2DM and its complications effects.

Galba: genome annotation with miniprot and AUGUSTUS

BackgroundThe Earth Biogenome Project has rapidly increased the number of available eukaryotic genomes, but most released genomes continue to lack annotation of protein-coding genes. In addition, no transcriptome data is available for some genomes.ResultsVarious gene annotation tools have been developed but each has its limitations. Here, we introduce GALBA, a fully automated pipeline that utilizes miniprot, a rapid protein-to-genome aligner, in combination with AUGUSTUS to predict genes with high accuracy. Accuracy results indicate that GALBA is particularly strong in the annotation of large vertebrate genomes. We also present use cases in insects, vertebrates, and a land plant. GALBA is fully open source and available as a docker image for easy execution with Singularity in high-performance computing environments.ConclusionsOur pipeline addresses the critical need for accurate gene annotation in newly sequenced genomes, and we believe that GALBA will greatly facilitate genome annotation for diverse organisms.

In silico analysis of HOX-associated transcription factors as potential regulators of oral cancer

The objective of this study was identification of the transcription factor binding sites (TFBS) in the promoter of HOX genes and elucidation of the comprehensive interaction of transcription factors (TFs)/genes with HOX. Promoter sequences of HOXA3, HOXA5, HOXA9, HOXA10, HOXA13, HOXB5, HOXC10, HOXC12, and HOXD10 were analyzed to predict the TFBS and their targets using TRANSFAC, TRRUST, and Harmonizome. Functional analysis of the processed data sets was carried out using DAVID and GATHER gene annotation tools. A network of regulatory interactions was constructed using NetworkAnalyst and a comprehensive illustration of the TF-gene network was constructed with HOX as a central hub using the Encyclopedia of DNA Elements chromatin immunoprecipitation sequencing data. Further, the enriched network was constructed to elucidate the roles of these genes in the various pathways. Binding sites for E2F1, HNF3α, SP3, and KLF6 were common to promoter regions of all of the HOX genes. The functional annotation and pathway analysis elucidated the regulatory activity of a distinct set of TF-genes in interaction with HOX. A P value ≤.05 and false discovery rate ≤0.01 were considered statistically significant. We have confirmed that the predicted TFBSs in the HOX gene promoters function in transcriptional regulation by modulating target gene activity. TF-gene interactions are crucial to understanding oral carcinogenesis.

Prediction of Monophyletic Groups Based on Gene Order and Sequence Similarity in Organelle DNA

Organelle genomics has become its own field of study. Much information can be gleaned from the study of cell organelles. The differences in the genomes of organelles, such as the mitochondrion and the chloroplast are amenable to phylogenetic and cladistic studies. These differences include the genome sequence, GC%, genome length and gene order. The conserved nature of the organelle genomes and the gene inventory of both mitochondrial and chloroplast genomes also make this easier to accomplish. This paper includes a review of existing organelle genome software. These include gene annotation and genome visualization tools as well as organelle gene databases for both mitochondrion and plastid. A new R tool, available on github, called “Organelle DNA Lineages”, or ODL, was written to compare and classify organelle genomes based on their genome sequence and gene order. The software was run on the mitochondrial genomes of a set of 51 cephalopod species, delineating ten separate monophyletic groups, including argonauts, nautiluses, octopuses, cuttlefish, and six squid groups. This new tool can help enrich and expand the field of organelle genomics.

DROPA: DRIP-seq optimized peak annotator

BackgroundR-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique.ResultsHere, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%. A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others.ConclusionsDROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced.

Protein interaction network analysis of TGF-β signalling pathway enabled EMT process to anticipate the anticancer activity of curcumin

TGF-β signalling is a key mediator of epithelial to mesenchymal transition (EMT) process and its up-regulation is identified as a hallmark of metastasis. Since TGF-β signalling pathway is known as a key therapeutic target in the treatment of EMT enabled cancer and the study aims at identification of key EMT genes by gene annotation tools and protein interaction network (PIN) to analyse the regulatory dynamics of an interactome. Meanwhile, the potency of curcumin against TGF-β signalling was evaluated by network pharmacology approach. Resultantly, 15 genes were identified as key regulators of TGF-β signalling pathway and seven were shortlisted as leading curcumin targets. Cumulatively, both approaches have justified the role of targets. Thus, curcumin was subjected to molecular docking with targets using AutoDock Vina. Wherein, curcumin has shown significant binding energy with targets EP300 and JUN (-7.1 and -6.4 kcal/mol) respectively indicating the potential anticancer property.

Identification of Genes for Wheat Fungal Resistance Using Bioinformatics Techniques

For the majority of world populations, wheat (Triticum aestivum L.) would be the first essential and economic cereal grain crop. Pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development. Multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. Our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. In addition the introduction for restriction fragment length polymorphism (RFLP) for such genes in the new primer database. Approximately 138 sequences of DNA were recovered from the wheat genome by aligning 3845 anti-fungal amino acids through tblastn tool. The NCBI blastn online tool used to detect sequences with functional genes, where 92 genes have been detected. The total number of nucleotides was 48385, where the smallest DNA sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. The wheat chromosomes 3D, and 4B have the highest number of sequences (9) followed by chromosomes 3B (7) and 3A(6), where wheat genomes A, B and D have 30, 35 and 27 genes, respectively. Five different amino acids motifs have been revealed among studied wheat amino acid sequences. The gene annotation tools used to infer studied amino acid gene annotation. Amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (STK), thaumatin, and cysteine-rich repeats representing 2, 9, 8, 19, 23 genes respectively, in addition to 31 hypothetical genes. The proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics.

Expression Profiles of Selenium-Related Genes in Human Chondrocytes Exposed to T-2 Toxin and Deoxynivalenol.

The combination of excess mycotoxin exposure and selenium deficiency has been widely considered as a cause of Kashin-Beck disease (KBD). The present study aimed to investigate the expression profiles of selenium-related genes in human chondrocytes after exposure to T-2 toxin and deoxynivalenol (DON) and to preliminarily identify the potential biological functions of the identified genes. Gene expression profiling was performed on human chondrocytes treated with 0.01μg/ml T-2 toxin and 1.0μg/ml DON by using Affymetrix Human Gene Arrays. The 1660 selenium-related genes were derived from the Comparative Toxicogenomics Database. Gene-term enrichment analysis was conducted by the DAVID gene annotation tool. Our results showed that 69 and 191 selenium-related genes were differentially expressed after T-2 toxin and DON treatment, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these identified genes were involved in various biological functions, such as the GO terms in response to oxidative stress, cell cycle arrest, and apoptotic process and the KEGG metabolic, FoxO signaling, and p53 signaling pathways. Our results may help explain the mechanisms of interaction between mycotoxins and selenium following human chondrocyte damage and reveal the potential roles of environmental risk factors in cartilage lesions during KBD development.

AGORA: organellar genome annotation from the amino acid and nucleotide references.

Next-generation sequencing (NGS) technologies have led to the accumulation of high-throughput sequence data from various organisms in biology. To apply gene annotation of organellar genomes for various organisms, more optimized tools for functional gene annotation are required. Almost all gene annotation tools are mainly focused on the chloroplast genome of land plants or the mitochondrial genome of animals. We have developed a web application AGORA for the fast, user-friendly and improved annotations of organellar genomes. Annotator for Genes of Organelle from the Reference sequence Analysis (AGORA) annotates genes based on a basic local alignment search tool (BLAST)-based homology search and clustering with selected reference sequences from the NCBI database or user-defined uploaded data. AGORA can annotate the functional genes in almost all mitochondrion and plastid genomes of eukaryotes. The gene annotation of a genome with an exon-intron structure within a gene or inverted repeat region is also available. It provides information of start and end positions of each gene, BLAST results compared with the reference sequence and visualization of gene map by OGDRAW. Users can freely use the software, and the accessible URL is https://bigdata.dongguk.edu/gene_project/AGORA/. The main module of the tool is implemented by the python and php, and the web page is built by the HTML and CSS to support all browsers. Supplementary data are available at Bioinformatics online.

RNA-Seq Analyses of the Role of miR-21 in Acute Pancreatitis

Background/Aims: Our previous study demonstrated that a deficiency of microRNA 21 (miR-21) protects mice from acute pancreatitis, yet the underlying molecular networks associated with miR-21 in pancreatitis and pancreatitis-associated lung injury remain unexplored. Methods: We used next generation sequencing to analyze gene expression profiles of pancreatic tissues from wild-type (WT) and miR-21 knockout (KO) mice treated with caerulein by using a 1-day treatment protocol. The Database for Annotation, Visualization, and Integrated Discovery gene annotation tool and Ingenuity Pathway Analysis were used to analyze the molecular pathways, while quantitative real-time PCR, western blotting, and immunohistochemistry were used to explore the molecular mechanisms. Results: We identified 152 differentially expressed genes (DEGs) in pancreata between WT and KO mice treated with caerulein. Cellular biogenesis and metabolism were the major pathways affected between WT and KO mice, whereas cell death and inflammatory response discriminated between WT and KO mice under acute pancreatitis. We validated 16 DEGs, consisting of 6 upregulated genes and 10 downregulated genes, involved in pancreatic injury. In particular, the upregulation of Pias3 and downregulation of Hmgb1 in KO pancreata coincided with a reduced severity of pancreatitis. In addition, we found Hmgb1 stimulation resulted in the overexpression of miR-21 in peripheral blood mononuclear cells, and deletion of miR-21 led to a reduction of caerulein-induced acute pancreatitis-associated lung injury by repressing Hmgb1 expression. Conclusion: Our data support the hypothesis that miR-21 modulates the inflammatory response during acute pancreatitis through the upregulation of Pias3 and downregulation of Hmgb1. Our findings further underscore a role for miR-21 in the promotion of acute pancreatitis.

Next-Generation Sequencing and the Crustacean Immune System: The Need for Alternatives in Immune Gene Annotation.

Next-generation sequencing has been a huge benefit to investigators studying non-model species. High-throughput gene expression studies, which were once restricted to animals with extensive genomic resources, can now be applied to any species. Transcriptomic studies using RNA-Seq can discover hundreds of thousands of transcripts from any species of interest. The power and limitation of these techniques is the sheer size of the dataset that is acquired. Parsing these large datasets is becoming easier as more bioinformatic tools are available for biologists without extensive computer programming expertise. Gene annotation and physiological pathway tools such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology enable the application of the vast amount of information acquired from model organisms to non-model species. While noble in nature, utilization of these tools can inadvertently misrepresent transcriptomic data from non-model species via annotation omission. Annotation followed by molecular pathway analysis highlights pathways that are disproportionately affected by disease, stress, or the physiological condition being examined. Problems occur when gene annotation procedures only recognizes a subset, often 50% or less, of the genes differently expressed from a non-model organisms. Annotated transcripts normally belong to highly conserved metabolic or regulatory genes that likely have a secondary or tertiary role, if any at all, in immunity. They appear to be disproportionately affected simply because conserved genes are most easily annotated. Evolutionarily induced specialization of physiological pathways is a driving force of adaptive evolution, but it results in genes that have diverged sufficiently to prevent their identification and annotation through conventional gene or protein databases. The purpose of this manuscript is to highlight some of the challenges faced when annotating crustacean immune genes by using an American lobster (Homarus americanus) transcriptome as an example. Immune genes have evolved rapidly over time, facilitating speciation and adaption to highly divergent ecological niches. Complete and proper annotation of immune genes from invertebrates has been challenging. Modulation of the crustacean immune system occurs in a variety of physiological responses including biotic and abiotic stressors, molting and reproduction. A simple method for the identification of a greater number of potential immune genes is proposed, along with a short introductory primer on crustacean immune response. The intended audience is not the advanced bioinformatic user, but those investigating physiological responses who require rudimentary understanding of crustacean immunological principles, but where immune gene regulation is not their primary interest.

Target identification in Fusobacterium nucleatum by subtractive genomics approach and enrichment analysis of host-pathogen protein-protein interactions.

BackgroundFusobacterium nucleatum, a well studied bacterium in periodontal diseases, appendicitis, gingivitis, osteomyelitis and pregnancy complications has recently gained attention due to its association with colorectal cancer (CRC) progression. Treatment with berberine was shown to reverse F. nucleatum-induced CRC progression in mice by balancing the growth of opportunistic pathogens in tumor microenvironment. Intestinal microbiota imbalance and the infections caused by F. nucleatum might be regulated by therapeutic intervention. Hence, we aimed to predict drug target proteins in F. nucleatum, through subtractive genomics approach and host-pathogen protein-protein interactions (HP-PPIs). We also carried out enrichment analysis of host interacting partners to hypothesize the possible mechanisms involved in CRC progression due to F. nucleatum.ResultsIn subtractive genomics approach, the essential, virulence and resistance related proteins were retrieved from RefSeq proteome of F. nucleatum by searching against Database of Essential Genes (DEG), Virulence Factor Database (VFDB) and Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) tool respectively. A subsequent hierarchical screening to identify non-human homologous, metabolic pathway-independent/pathway-specific and druggable proteins resulted in eight pathway-independent and 27 pathway-specific druggable targets. Co-aggregation of F. nucleatum with host induces proinflammatory gene expression thereby potentiates tumorigenesis. Hence, proteins from IBDsite, a database for inflammatory bowel disease (IBD) research and those involved in colorectal adenocarcinoma as interpreted from The Cancer Genome Atlas (TCGA) were retrieved to predict drug targets based on HP-PPIs with F. nucleatum proteome. Prediction of HP-PPIs exhibited 186 interactions contributed by 103 host and 76 bacterial proteins. Bacterial interacting partners were accounted as putative targets. And enrichment analysis of host interacting partners showed statistically enriched terms that were in positive correlation with CRC, atherosclerosis, cardiovascular, osteoporosis, Alzheimer’s and other diseases.ConclusionSubtractive genomics analysis provided a set of target proteins suggested to be indispensable for survival and pathogenicity of F. nucleatum. These target proteins might be considered for designing potent inhibitors to abrogate F. nucleatum infections. From enrichment analysis, it was hypothesized that F. nucleatum infection might enhance CRC progression by simultaneously regulating multiple signaling cascades which could lead to up-regulation of proinflammatory responses, oncogenes, modulation of host immune defense mechanism and suppression of DNA repair system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0700-0) contains supplementary material, which is available to authorized users.

Full characterization of the integrative and conjugative element carrying the metallo-β-lactamase bla SPM-1 and bicyclomycin bcr1 resistance genes found in the pandemic Pseudomonas aeruginosa clone SP/ST277.

This study aimed to characterize the genomic context of the bla SPM-1 gene in Brazilian strains belonging to the pandemic Pseudomonas aeruginosa clone SP/ST277. WGS of clone SP/ST277 strains was performed using a Nextera paired-end library in an Illumina HiSeq 2500 sequencer. bla SPM-1 context was assessed by de novo assembly and gene prediction and annotation tools. bla SPM-1 was screened in P. aeruginosa genomes through BlastN, and comparative genomics were performed. The metallo-β-lactamase bla SPM-1 has been disseminated by the pandemic Brazilian P. aeruginosa clone SP/ST277. In spite of its association with the CR4 element and with the Tn4371 element, the overall bla SPM-1 genomic context remains uncharacterized and its determination is valuable to understanding gene dispersion dynamics and the consequent emergence of carbapenem resistance. In this study, bla SPM-1 and its surrounding sequences (CR4-groEL-bla SPM-1-CR4-groEL) were found in the variable region of an ICE-like element resembling Tn4371 (where ICE stands for integrative and conjugative element). This element, named ICETn4371 6061, had 46 ORFs, including the bicyclomycin resistance bcr1 gene. An integrase gene and a set of conjugative transfer genes were identified. Gene content and order were shared with other Tn4371-ICEs, presenting remarkable amino acid identities. bla SPM-1 and surrounding sequences were missing in ICETn4371 6061 of PS600-MA, another isolate belonging to clone SP/ST277, indicating their mobilization. Eight/nine P. aeruginosa genomes assigned to clone SP/ST277, by in silico MLST, harboured bla SPM-1 inserted into ICETn4371 6061. The presence of bla SPM-1 in a Tn4371-ICE with intact integration/conjugation modules demonstrated that, besides gene dispersion by clonal expansion of the pandemic SP/ST277 lineage, bla SPM-1 may be spread through ICE conjugation.

An in silico analytical study of lung cancer and smokers datasets from gene expression omnibus (GEO) for prediction of differentially expressed genes.

Smoking is the leading cause of lung cancer development and several genes have been identified as potential biomarker for lungs cancer. Contributing to the present scientific knowledge of biomarkers for lung cancer two different data sets, i.e. GDS3257 and GDS3054 were downloaded from NCBI׳s GEO database and normalized by RMA and GRMA packages (Bioconductor). Diffrentially expressed genes were extracted by using and were R (3.1.2); DAVID online tool was used for gene annotation and GENE MANIA tool was used for construction of gene regulatory network. Nine smoking independent gene were found whereas average expressions of those genes were almost similar in both the datasets. Five genes among them were found to be associated with cancer subtypes. Thirty smoking specific genes were identified; among those genes eight were associated with cancer sub types. GPR110, IL1RN and HSP90AA1 were found directly associated with lung cancer. SEMA6A differentially expresses in only non-smoking lung cancer samples. FLG is differentially expressed smoking specific gene and is related to onset of various cancer subtypes. Functional annotation and network analysis revealed that FLG participates in various epidermal tissue developmental processes and is co-expressed with other genes. Lung tissues are epidermal tissues and thus it suggests that alteration in FLG may cause lung cancer. We conclude that smoking alters expression of several genes and associated biological pathways during development of lung cancers.

697. New Insights Into the Mechanisms of Argonaute Protein Competition and Implications for RNAi Gene Therapies

RNA interference (RNAi) has become an established tool for basic gene annotation and a promising option for therapeutic concepts that require specific inhibition of dysregulated or exogenous genes. However, we and others accumulated strong evidence that overexpression of short hairpin RNAs (shRNAs, an expressable and widely used form of RNAi trigger) can cause cytotoxicity leading up to organ damage and lethality in shRNA-treated animals. A meticulous dissection of the underlying mechanisms is critical as it will ultimately guide the design of safer RNAi therapies and thus foster their clinical translation. One informative finding was that co-expression of Ago2 (Slicer, as it cleaves targeted mRNA), a key component of the RNA-induced silencing complex RISC, increases shRNA efficiency in cells and animals, implying that Ago2 is a rate-limiting factor whose saturation may be involved in cytotoxicity. Moreover, over-expression of the three other mammalian Ago proteins – Ago1, Ago3 or Ago4 (all slicing-incompetent) – dampens RNAi potency, underscoring that a delicate balance of all four Ago variants is key to efficient and safe gene silencing. Here, we investigated three hypotheses that could explain how relative Ago1-4 levels may affect the functionality of shRNA-loaded RISC and hence the strength of target knockdown: over-abundance of non-Slicer Ago proteins could (i) dysregulate Ago2 expression (transcription and/or translation), (ii) re-localize intracellular Ago2 away from sites of RNAi activity, or (iii) quantitatively sequester shRNAs into slicing-incompetent RISC. Using codon-optimized Ago variants that are distinguishable by real-time PCR from the endogenous counterparts, as well as Western blotting, we could eliminate model (i). Likewise, wide-field and confocal microscopy analyses revealed no evidence for Ago2 re-localization upon Ago1/3/4 over-expression and vice versa. We therefore focused on hypothesis (iii) and fused the Ago1-4 cDNAs with GFP or RFP tags that enabled their specific immunoprecipitation, alone or in combination, using the TRAP system. Indeed, we found that Slicer and non-Slicers compete for shRNA loading and mRNA knockdown in a dose-dependent manner, readily explaining all prior observations of RNAi enhancement by Ago2 over-expression, or shRNA inhibition by Ago1/3/4, respectively. Importantly, the combination of GFP/RFP-Ago fusion constructs with the co-precipitation protocols newly established during this work should be widely useful to address other vital questions in the RNAi field, such as the selective processing of different cellular small RNAs by the four Ago proteins. Moreover, our notion that over-expression of non-Slicers has no adverse effect on endogenous Ago2 levels or localization paves the way for novel strategies to fine-tune therapeutic RNAi in a safe and potent manner. As a prototype, we present a new shRNA/Ago co-expression system whereby Ago1/2 levels can be regulated independently through exogenous macrolides, permitting a tight control of target knockdown efficiencies and providing an original avenue to reduce toxicity in future RNAi gene therapy applications.

Dynamic Regulation of Schwann Cell Enhancers after Peripheral Nerve Injury

Myelination of the peripheral nervous system is required for axonal function and long term stability. After peripheral nerve injury, Schwann cells transition from axon myelination to a demyelinated state that supports neuronal survival and ultimately remyelination of axons. Reprogramming of gene expression patterns during development and injury responses is shaped by the actions of distal regulatory elements that integrate the actions of multiple transcription factors. We used ChIP-seq to measure changes in histone H3K27 acetylation, a mark of active enhancers, to identify enhancers in myelinating rat peripheral nerve and their dynamics after demyelinating nerve injury. Analysis of injury-induced enhancers identified enriched motifs for c-Jun, a transcription factor required for Schwann cells to support nerve regeneration. We identify a c-Jun-bound enhancer in the gene for Runx2, a transcription factor induced after nerve injury, and we show that Runx2 is required for activation of other induced genes. In contrast, enhancers that lose H3K27ac after nerve injury are enriched for binding sites of the Sox10 and early growth response 2 (Egr2/Krox20) transcription factors, which are critical determinants of Schwann cell differentiation. Egr2 expression is lost after nerve injury, and many Egr2-binding sites lose H3K27ac after nerve injury. However, the majority of Egr2-bound enhancers retain H3K27ac, indicating that other transcription factors maintain active enhancer status after nerve injury. The global epigenomic changes in H3K27ac deposition pinpoint dynamic changes in enhancers that mediate the effects of transcription factors that control Schwann cell myelination and peripheral nervous system responses to nerve injury.

CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.