Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

An encyclopedia of enhancer-gene regulatory interactions in the human genome.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

PARP1 Regulates Circular RNA Biogenesis though Control of Transcriptional Dynamics.

Circular RNAs (circRNAs) are a recently discovered class of RNAs derived from protein-coding genes that have important biological and pathological roles. They are formed through backsplicing during co-transcriptional alternative splicing; however, the unified mechanism that accounts for backsplicing decisions remains unclear. Factors that regulate the transcriptional timing and spatial organization of pre-mRNA, including RNAPII kinetics, the availability of splicing factors, and features of gene architecture, have been shown to influence backsplicing decisions. Poly (ADP-ribose) polymerase I (PARP1) regulates alternative splicing through both its presence on chromatin as well as its PARylation activity. However, no studies have investigated PARP1's possible role in regulating circRNA biogenesis. Here, we hypothesized that PARP1's role in splicing extends to circRNA biogenesis. Our results identify many unique circRNAs in PARP1 depletion and PARylation-inhibited conditions compared to the wild type. We found that while all genes producing circRNAs share gene architecture features common to circRNA host genes, genes producing circRNAs in PARP1 knockdown conditions had longer upstream introns than downstream introns, whereas flanking introns in wild type host genes were symmetrical. Interestingly, we found that the behavior of PARP1 in regulating RNAPII pausing is distinct between these two classes of host genes. We conclude that the PARP1 pausing of RNAPII works within the context of gene architecture to regulate transcriptional kinetics, and therefore circRNA biogenesis. Furthermore, this regulation of PARP1 within host genes acts to fine tune their transcriptional output with implications in gene function.

Polychlorinated biphenyls alter hepatic m6A mRNA methylation in a mouse model of environmental liver disease.

Exposure to polychlorinated biphenyls (PCBs) has been associated with liver injury in human cohorts and with nonalcoholic steatohepatitis (NASH) in mice fed a high fat diet (HFD). N (6)-methyladenosine (m6A) modification of mRNA regulates transcript fate, but the contribution of m6A modification on the regulation of transcripts in PCB-induced steatosis and fibrosis is unknown. This study tested the hypothesis that PCB and HFD exposure alters the levels of m6A modification in transcripts that play a role in NASH in vivo. Male C57Bl6/J mice were fed a HFD (12 wks) and administered a single oral dose of Aroclor1260, PCB126, or Aroclor1260 + PCB126. Genome-wide identification of m6A peaks was accomplished by m6A mRNA immunoprecipitation sequencing (m6A-RIP) and the mRNA transcriptome identified by RNA-seq. Exposure of HFD-fed mice to Aroclor1260 decreased the number of m6A peaks and m6A-containing genes relative to PCB vehicle control whereas PCB126 or the combination of Aroclor1260 + PCB126 increased m6A modification frequency. ∼41% of genes had one m6A peak and ∼49% had 2-4 m6A peaks. 117 m6A peaks were common in the four experimental groups. The Aroclor1260 + PCB126 exposure group showed the highest number (52) of m6A-peaks. qRT-PCR confirmed enrichment of m6A-containing fragments of the Apob transcript with PCB exposure. A1cf transcript abundance, m6A peak count, and protein abundance was increased with Aroclor1260 + PCB126 co-exposure. Irrespective of the PCB type, all PCB groups exhibited enriched pathways related to lipid/lipoprotein metabolism and inflammation through the m6A modification. Integrated analysis of m6A-RIP-seq and mRNA-seq identified 242 differentially expressed genes (DEGs) with increased or reduced number of m6A peaks. These data show that PCB exposure in HFD-fed mice alters the m6A landscape offering an additional layer of regulation of gene expression affecting a subset of gene responses in NASH.

Shoc2 controls ERK1/2-driven neural crest development by balancing components of the extracellular matrix.

The extracellular signal-regulated kinase (ERK1/2) pathway is essential in embryonic development. The scaffold protein Shoc2 is a critical modulator of ERK1/2 signals, and mutations in the shoc2 gene lead to the human developmental disease known as Noonan-like syndrome with loose anagen hair (NSLH). The loss of Shoc2 and the shoc2 NSLH-causing mutations affect the tissues of neural crest (NC) origin. In this study, we utilized the zebrafish model to dissect the role of Shoc2-ERK1/2 signals in the development of NC. These studies established that the loss of Shoc2 significantly altered the expression of transcription factors regulating the specification and differentiation of NC cells. Using comparative transcriptome analysis of NC-derived cells from shoc2 CRISPR/Cas9 mutant larvae, we found that Shoc2-mediated signals regulate gene programs at several levels, including expression of genes coding for the proteins of extracellular matrix (ECM) and ECM regulators. Together, our results demonstrate that Shoc2 is an essential regulator of NC development. This study also indicates that disbalance in the turnover of the ECM may lead to the abnormalities found in NSLH patients.

Multiomics analysis of the impact of polychlorinated biphenyls on environmental liver disease in a mouse model.

Exposure to high fat diet (HFD) and persistent organic pollutants including polychlorinated biphenyls (PCBs) is associated with liver injury in human populations and non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in animal models. Previously, exposure of HFD-fed male mice to the non-dioxin-like (NDL) PCB mixture Aroclor1260, dioxin-like (DL) PCB126, or Aroclor1260 + PCB126 co-exposure caused toxicant-associated steatohepatitis (TASH) and differentially altered the liver proteome. Here unbiased mRNA and miRNA sequencing (mRNA- and miRNA- seq) was used to identify biological pathways altered in these liver samples. Fewer transcripts and miRs were up- or down- regulated by PCB126 or Aroclor1260 compared to the combination, suggesting that crosstalk between the receptors activated by these PCBs amplifies changes in the transcriptome. Pathway enrichment analysis identified "positive regulation of Wnt/ß-catenin signaling" and "role of miRNAs in cell migration, survival, and angiogenesis" for differentially expressed mRNAs and miRNAs, respectively. We evaluated the five miRNAs increased in human plasma with PCB exposure and suspected TASH and found that miR-192-5p was increased with PCB exposure in mouse liver. Although we observed little overlap between differentially expressed mRNA transcripts and proteins, biological pathway-relevant PCB-induced miRNA-mRNA and miRNA-protein inverse relationships were identified that may explain protein changes. These results provide novel insights into miRNA and mRNA transcriptome changes playing direct and indirect roles in the functional protein pathways in PCB-related hepatic lipid accumulation, inflammation, and fibrosis in a mouse model of TASH and its relevance to human liver disease in exposed populations.

The induction of peripheral trained immunity in the pancreas incites anti-tumor activity to control pancreatic cancer progression.

Despite the remarkable success of immunotherapy in many types of cancer, pancreatic ductal adenocarcinoma has yet to benefit. Innate immune cells are critical to anti-tumor immunosurveillance and recent studies have revealed that these populations possess a form of memory, termed trained innate immunity, which occurs through transcriptomic, epigenetic, and metabolic reprograming. Here we demonstrate that yeast-derived particulate ß-glucan, an inducer of trained immunity, traffics to the pancreas, which causes a CCR2-dependent influx of monocytes/macrophages to the pancreas that display features of trained immunity. These cells can be activated upon exposure to tumor cells and tumor-derived factors, and show enhanced cytotoxicity against pancreatic tumor cells. In orthotopic models of pancreatic ductal adenocarcinoma, ß-glucan treated mice show significantly reduced tumor burden and prolonged survival, which is further enhanced when combined with immunotherapy. These findings characterize the dynamic mechanisms and localization of peripheral trained immunity and identify an application of trained immunity to cancer.

Combined exposure to polychlorinated biphenyls and high-fat diet modifies the global epitranscriptomic landscape in mouse liver.

Exposure to a single dose of polychlorinated biphenyls (PCBs) and a 12-week high-fat diet (HFD) results in nonalcoholic steatohepatitis (NASH) in mice by altering intracellular signaling and inhibiting epidermal growth factor receptor signaling. Post-transcriptional chemical modification (PTM) of RNA regulates biological processes, but the contribution of epitranscriptomics to PCB-induced steatosis remains unknown. This study tested the hypothesis that PCB and HFD exposure alters the global RNA epitranscriptome in male mouse liver. C57BL/6J male mice were fed a HFD for 12 weeks and exposed to a single dose of Aroclor 1260 (20 mg/kg), PCB 126 (20 µg/kg), both Aroclor 1260 and PCB 126 or vehicle control after 2 weeks on HFD. Chemical RNA modifications were identified at the nucleoside level by liquid chromatography-mass spectrometry. From 22 PTM global RNA modifications, we identified 10 significant changes in RNA modifications in liver with HFD and PCB 126 exposure. Only two modifications were significantly different from HFD control liver in all three PCB exposure groups: 2'-O-methyladenosine (Am) and N(6)-methyladenosine (m6A). Exposure to HFD + PCB 126 + Aroclor 1260 increased the abundance of N(6), O(2)-dimethyladenosine (m6Am), which is associated with the largest number of transcript changes. Increased m6Am and pseudouridine were associated with increased protein expression of the writers of these modifications: Phosphorylated CTD Interacting Factor 1 (PCIF1) and Pseudouridine Synthase 10 (PUS10), respectively, in HFD + PCB 126- + Aroclor 1260-exposed mouse liver. Increased N1-methyladenosine (m1A) and m6A were associated with increased transcript levels of the readers of these modifications: YTH N6-Methyladenosine RNA Binding Protein 2 (YTHDF2), YTH Domain Containing 2 (YTHDC2), and reader FMRP Translational Regulator 1 (FMR1) transcript and protein abundance. The results demonstrate that PCB exposure alters the global epitranscriptome in a mouse model of NASH; however, the mechanism for these changes requires further investigation.

seekCRIT: Detecting and characterizing differentially expressed circular RNAs using high-throughput sequencing data.

Over the past two decades, researchers have discovered a special form of alternative splicing that produces a circular form of RNA. Although these circular RNAs (circRNAs) have garnered considerable attention in the scientific community for their biogenesis and functions, the focus of current studies has been on the tissue-specific circRNAs that exist only in one tissue but not in other tissues or on the disease-specific circRNAs that exist in certain disease conditions, such as cancer, but not under normal conditions. This approach was conducted in the relative absence of methods that analyze a group of common circRNAs that exist in both conditions, but are more abundant in one condition relative to another (differentially expressed). Studies of differentially expressed circRNAs (DECs) between two conditions would serve as a significant first step in filling this void. Here, we introduce a novel computational tool, seekCRIT (seek for differentially expressed CircRNAs In Transcriptome), that identifies the DECs between two conditions from high-throughput sequencing data. Using rat retina RNA-seq data from ischemic and normal conditions, we show that over 74% of identifiable circRNAs are expressed in both conditions and over 40 circRNAs are differentially expressed between two conditions. We also obtain a high qPCR validation rate of 90% for DECs with a FDR of < 5%. Our results demonstrate that seekCRIT is a novel and efficient approach to detect DECs using rRNA depleted RNA-seq data. seekCRIT is freely downloadable at https://github.com/UofLBioinformatics/seekCRIT. The source code is licensed under the MIT License. seekCRIT is developed and tested on Linux CentOS-7.

A causal mediation model of ischemia reperfusion injury in the retina.

The goal of this study is to develop a model that explains the relationship between microRNAs, transcription factors, and their co-target genes. This relationship was previously reported in gene regulatory loops associated with 24 hour (24h) and 7 day (7d) time periods following ischemia-reperfusion injury in a rat's retina. Using a model system of retinal ischemia-reperfusion injury, we propose that microRNAs first influence transcription factors, which in turn act as mediators to influence transcription of genes via triadic regulatory loops. Analysis of the relative contributions of direct and indirect regulatory influences on genes revealed that a substantial fraction of the regulatory loops (69% for 24 hours and 77% for 7 days) could be explained by causal mediation. Over 40% of the mediated loops in both time points were regulated by transcription factors only, while about 20% of the loops were regulated entirely by microRNAs. The remaining fractions of the mediated regulatory loops were cooperatively mediated by both microRNAs and transcription factors. The results from these analyses were supported by the patterns of expression of the genes, transcription factors, and microRNAs involved in the mediated loops in both post-ischemic time points. Additionally, network motif detection for the mediated loops showed a handful of time specific motifs related to ischemia-reperfusion injury in a rat's retina. In summary, the effects of microRNAs on genes are mediated, in large part, via transcription factors.

Gene Expression and Silencing Studies in Phytophthora infestans Reveal Infection-Specific Nutrient Transporters and a Role for the Nitrate Reductase Pathway in Plant Pathogenesis.

To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity.

Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

Regulatory networks in retinal ischemia-reperfusion injury.

BACKGROUND: Retinal function is ordered by interactions between transcriptional and posttranscriptional regulators at the molecular level. These regulators include transcription factors (TFs) and posttranscriptional factors such as microRNAs (miRs). Some studies propose that miRs predominantly target the TFs rather than other types of protein coding genes and such studies suggest a possible interconnection of these two regulators in co-regulatory networks. RESULTS: Our lab has generated mRNA and miRNA microarray expression data to investigate time-dependent changes in gene expression, following induction of ischemia-reperfusion (IR) injury in the rat retina. Data from different reperfusion time points following retinal IR-injury were analyzed. Paired expression data for miRNA-target gene (TG), TF-TG, miRNA-TF were used to identify regulatory loop motifs whose expressions were altered by the IR injury paradigm. These loops were subsequently integrated into larger regulatory networks and biological functions were assayed. Systematic analyses of the networks have provided new insights into retinal gene regulation in the early and late periods of IR. We found both overlapping and unique patterns of molecular expression at the two time points. These patterns can be defined by their characteristic molecular motifs as well as their associated biological processes. We highlighted the regulatory elements of miRs and TFs associated with biological processes in the early and late phases of ischemia-reperfusion injury. CONCLUSIONS: The etiology of retinal ischemia-reperfusion injury is orchestrated by complex and still not well understood gene networks. This work represents the first large network analysis to integrate miRNA and mRNA expression profiles in context of retinal ischemia. It is likely that an appreciation of such regulatory networks will have prognostic potential. In addition, the computational framework described in this study can be used to construct miRNA-TF interactive systems networks for various diseases/disorders of the retina and other tissues.

Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway.

PURPOSE: Ischemia-reperfusion (IR) injury is involved in the pathology of many retinal disorders since it contributes to the death of retinal neurons and the subsequent decline in vision. We determined the molecular patterns of some of the principal molecules involved in necroptosis and investigated whether IR retinal injury is associated with the extracellular signal-regulated kinase-1/2- receptor-interacting protein kinase 3 (ERK1/2-RIP3) pathway. METHODS: The cellular and subcellular localization of molecules involved in the cell death pathway, including RAGE, ERK1/2, FLIP, and RIP3, was determined with immunohistochemistry of cryosections of IR-injured retina from 2-month-old Long Evans rats. The total and phosphorylated protein levels were analyzed with quantitative western blots. ERK1/2 activity was inhibited by intravitreal injection of U0126, a highly selective inhibitor of mitogen-activated protein kinase 1/2 (MEK1/2). RESULTS: The IR-injured rat retinas expressed two RAGE isoforms with different intracellular localizations at early time points after injury. At that time point, frame inhibition of ERK activation decreased RIP3 accumulation and cell death. FLIP was detected in the IR-injured rat retinas at early time points after ischemia reperfusion. CONCLUSIONS: We report that the necroptotic cell death mechanism is executed by an ERK1/2-RIP3 pathway in the retinal ganglion cells in early stages after IR injury. Inhibition of ERK1/2 activity increased retinal ganglion cell (RGC) survival indicating that targeting of this pathway within the initial 12 h after IR injury can be used to inhibit the necroptosis pathway. We also provide evidence that a novel RAGE isoform is expressed in the early stages in rat retinal RGCs.

Time-dependent Gene Profiling Indicates the Presence of Different Phases for Ischemia/Reperfusion Injury in Retina.

Ischemia/reperfusion (IR) injury has been associated with several retinal pathologies, and a few genes/gene products have been linked to IR injury. However, the big picture of temporal changes, regarding the affected gene networks, pathways, and processes remains to be determined. The purpose of the present study was to investigate initial, intermediate, and later stages to characterize the etiology of IR injury in terms of the pathways affected over time. Analyses indicated that at the initial stage, 0-hour reperfusion following the ischemic period, the ischemia-associated genes were related to changes in metabolism. In contrast, at the 24-hour time point, the signature events in reperfusion injury include enhanced inflammatory and immune responses as well as cell death indicating that this would be a critical period for the development of any interventional therapeutic strategies. Genes in the signal transduction pathways, particularly transmitter receptors, are downregulated at this time. Activation of the complement system pathway clearly plays an important role in the later stages of reperfusion injury. Together, these results demonstrate that the etiology of injury related to IR is characterized by the appearance of specific patterns of gene expression at any given time point during retinal IR injury. These results indicate that evaluation of treatment strategies with respect to time is very critical.

MicroRNAs in the Neural Retina.

The health and function of the visual system rely on a collaborative interaction between diverse classes of molecular regulators. One of these classes consists of transcription factors, which are known to bind to DNA and control the transcription activities of their target genes. For a long time, it was thought that the transcription factors were the only regulators of gene expression. More recently, however, a novel class of regulators emerged. This class consists of a large number of small noncoding endogenous RNAs, namely, miRNAs. The miRNAs compose an essential component of posttranscriptional gene regulation, since they ultimately control the fate of gene transcripts. The retina, as a part of the central nervous system, is a well-established model for unraveling the molecular mechanisms underlying neuronal and glial functions. Numerous recent efforts have been made towards identification of miRNAs and their inferred roles in the visual pathway. In this review, we summarize the current state of our knowledge regarding the expression and function of miRNA in the neural retina and we discuss their potential uses as biomarkers for some retinal disorders.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

The Cre/lox system: a practical tool to efficiently eliminate selectable markers in fungal endophytes.

Interspecific hybridization is a common evolutionary process for the many epichloid fungi that consequently possess multiple gene copies accumulated from their parental strains. Serial gene manipulations in such strains are impeded by the limited availability of selectable resistance marker genes. Therefore, we developed a method for marker elimination suitable for a range of filamentous fungi that allows the reuse of the same marker for successive manipulations, and can also generate gene knockout mutants free of any foreign genes. For epichloae, the complete elimination of the marker gene from the genome would mitigate public concerns and regulatory hurdles to the use of such fungal strains in field experiments.

Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid.

A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum,Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5-2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.