mEnrich-seq: methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome.

Metagenomics has enabled the comprehensive study of microbiomes. However, many applications would benefit from a method that sequences specific bacterial taxa of interest, but not most background taxa. We developed mEnrich-seq (in which 'm' stands for methylation and seq for sequencing) for enriching taxa of interest from metagenomic DNA before sequencing. The core idea is to exploit the self versus nonself differentiation by natural bacterial DNA methylation and rationally choose methylation-sensitive restriction enzymes, individually or in combination, to deplete host and background taxa while enriching targeted taxa. This idea is integrated with library preparation procedures and applied in several applications to enrich (up to 117-fold) pathogenic or beneficial bacteria from human urine and fecal samples, including species that are hard to culture or of low abundance. We assessed 4,601 bacterial strains with mapped methylomes so far and showed broad applicability of mEnrich-seq. mEnrich-seq provides microbiome researchers with a versatile and cost-effective approach for selective sequencing of diverse taxa of interest.

Navigating the pitfalls of mapping DNA and RNA modifications.

Chemical modifications to nucleic acids occur across the kingdoms of life and carry important regulatory information. Reliable high-resolution mapping of these modifications is the foundation of functional and mechanistic studies, and recent methodological advances based on next-generation sequencing and long-read sequencing platforms are critical to achieving this aim. However, mapping technologies may have limitations that sometimes lead to inconsistent results. Some of these limitations are technical in nature and specific to certain types of technology. Here, however, we focus on common (yet not always widely recognized) pitfalls that are shared among frequently used mapping technologies and discuss strategies to help technology developers and users mitigate their effects. Although the emphasis is primarily on DNA modifications, RNA modifications are also discussed.

Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution.

The discovery of N6-methyldeoxyadenine (6mA) across eukaryotes led to a search for additional epigenetic mechanisms. However, some studies have highlighted confounding factors that challenge the prevalence of 6mA in eukaryotes. We developed a metagenomic method to quantitatively deconvolve 6mA events from a genomic DNA sample into species of interest, genomic regions, and sources of contamination. Applying this method, we observed high-resolution 6mA deposition in two protozoa. We found that commensal or soil bacteria explained the vast majority of 6mA in insect and plant samples. We found no evidence of high abundance of 6mA in Drosophila, Arabidopsis, or humans. Plasmids used for genetic manipulation, even those from Dam methyltransferase mutant Escherichia coli, could carry abundant 6mA, confounding the evaluation of candidate 6mA methyltransferases and demethylases. On the basis of this work, we advocate for a reassessment of 6mA in eukaryotes.

Sex-Determining Region Y Controls the Effects of Fetal Alcohol Exposure on Proopiomelanocortin Gene Expression.

Fetal alcohol exposure (FAE) causes various neurodevelopmental deficits in offspring, including reduced expression of the stress regulatory proopiomelanocortin (Pomc) gene and an elevated stress response for multiple generations via the male germline. Male germline-specific effects of FAE on the Pomc gene raises the question if the sex-determining region Y (SRY) may have a role in regulating Pomc gene expression. Using a transgenerational model of FAE in Fischer 344 rats, we determined the role of SRY in the regulation of the Pomc gene. FAEs, like on the Pomc gene, reduced Sry gene expression in sperm and the mediobasal hypothalamus (MBH) in male adult offspring. Fetal alcohol-induced inhibition of Sry gene expression was associated with increased Sry promoter DNA methylation. Additionally, fetal alcohol effects on the Sry gene persisted for three generations in the male germline but not in the female germline. Sry gene knockdown reduced the Pomc gene expression. Sry recruitment onto the Pomc promoter was found to be reduced in the hypothalamus of fetal alcohol-exposed rats compared to control rats. Pomc promoter luciferase activity was increased following Sry overexpression. A site-directed mutagenesis study revealed that SRY binding sites are required for POMC promoter transcription activity. Overall, these findings suggest that SRY plays a stimulatory role in the regulation of Pomc gene expression and may potentially contribute to the fetal alcohol-induced changes in the level of Pomc gene expression for multiple generations.

Discovering multiple types of DNA methylation from bacteria and microbiome using nanopore sequencing.

Bacterial DNA methylation occurs at diverse sequence contexts and plays important functional roles in cellular defense and gene regulation. Existing methods for detecting DNA modification from nanopore sequencing data do not effectively support de novo study of unknown bacterial methylomes. In this work, we observed that a nanopore sequencing signal displays complex heterogeneity across methylation events of the same type. To enable nanopore sequencing for broadly applicable methylation discovery, we generated a training dataset from an assortment of bacterial species and developed a method, named nanodisco ( https://github.com/fanglab/nanodisco ), that couples the identification and fine mapping of the three forms of methylation into a multi-label classification framework. We applied it to individual bacteria and the mouse gut microbiome for reliable methylation discovery. In addition, we demonstrated the use of DNA methylation for binning metagenomic contigs, associating mobile genetic elements with their host genomes and identifying misassembled metagenomic contigs.

Non-Invasive microRNA Profiling in Saliva can Serve as a Biomarker of Alcohol Exposure and Its Effects in Humans.

Alcohol Use Disorder (AUD) is one of the most prevalent mental disorders worldwide. Considering the widespread occurrence of AUD, a reliable, cheap, non-invasive biomarker of alcohol consumption is desired by healthcare providers, clinicians, researchers, public health and criminal justice officials. microRNAs could serve as such biomarkers. They are easily detectable in saliva, which can be sampled from individuals in a non-invasive manner. Moreover, microRNAs expression is dynamically regulated by environmental factors, including alcohol. Since excessive alcohol consumption is a hallmark of alcohol abuse, we have profiled microRNA expression in the saliva of chronic, heavy alcohol abusers using microRNA microarrays. We observed significant changes in salivary microRNA expression caused by excessive alcohol consumption. These changes fell into three categories: downregulated microRNAs, upregulated microRNAs, and microRNAs upregulated de novo. Analysis of these combinatorial changes in microRNA expression suggests dysregulation of specific biological pathways leading to impairment of the immune system and development of several types of epithelial cancer. Moreover, some of the altered microRNAs are also modulators of inflammation, suggesting their contribution to pro-inflammatory mechanisms of alcohol actions. Establishment of the cellular source of microRNAs in saliva corroborated these results. We determined that most of the microRNAs in saliva come from two types of cells: leukocytes involved in immune responses and inflammation, and buccal cells, involved in development of epithelial, oral cancers. In summary, we propose that microRNA profiling in saliva can be a useful, non-invasive biomarker allowing the monitoring of alcohol abuse, as well as alcohol-related inflammation and early detection of cancer.

Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis.

Clostridioides (formerly Clostridium) difficile is a leading cause of healthcare-associated infections. Although considerable progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we perform a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observe a high level of epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity, the corresponding gene of which is highly conserved across our dataset and in all of the approximately 300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacts sporulation, a key step in C. difficile disease transmission, and these results are consistently supported by multiomics data, genetic experiments and a mouse colonization model. Further experimental and transcriptomic analyses suggest that epigenetic regulation is associated with cell length, biofilm formation and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen. This study also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomic studies.

Fetal alcohol spectrum disorders and their transmission through genetic and epigenetic mechanisms.

Fetal alcohol spectrum disorders (FASD) are a group of related conditions that arise from prenatal exposure to maternal consumption of the teratogen, ethanol. It has been estimated that roughly 1% of children in the US suffer from FASD (Sampson etal., 1997), though in some world populations, such as inhabitants of some poorer regions of South Africa, the rate can climb to as high as 20% (May etal., 2013). FASD are the largest cause of mental retardation in U.S. neonates, and ironically, are entirely preventable. FASD have been linked to major changes in the hypothalamic-pituitary-adrenal (HPA) axis, resulting in lifelong impairments through mental disorders, retardation, and sensitivity to stress. FASD are linked to an impaired immune system which consequently leads to an elevated risk of cancer and other diseases. FASD arise from a complex interplay of genetic and epigenetic factors. Here, we review current literature on the topic to tease apart what is known in these areas particularly emphasizing HPA axis dysfunction and how this ties into new studies of transgenerational inheritance in FASD.

Seasonal variation of urinary microRNA expression in male goats (Capra hircus) as assessed by next generation sequencing.

Testosterone plays a key role in preparation of a male domesticated goat (Capra hircus) to breeding season including changes in the urogenital tract of a male goat (buck). microRNAs are important regulators of cellular metabolism, differentiation and function. They are powerful intermediaries of hormonal activity in the body, including the urogenital tract. We investigated seasonal changes in expression of microRNAs in goat buck urine and their potential consequences using next generation sequencing (microRNA-Seq). We determined the location of each microRNA gene in the goat genome. Testosterone was measured by radioimmunoassay and the androgen receptor binding sites (ARBS) in the promoters of the microRNA genes were determined by MatInspector. The overall impact of regulated microRNAs on cellular physiology was assessed by mirPath. We observed high testosterone levels during the breeding season and changes in the expression of forty microRNAs. Nineteen microRNAs were upregulated, while twenty-one were downregulated. We identified several ARBS in the promoters of regulated microRNAs. Notably, the mostly inhibited microRNA, miR-1246, has a unique set of several gene copy variants associated with a cluster of androgen receptor binding sites. Concomitant changes in regulated microRNA expression could promote transcription, proliferation and differentiation of urogenital tract cells. Together, these findings indicate that in a domesticated goat (Capra hircus), there are specific changes in the microRNA expression profile in buck urine during breeding season, which could be attributable to high testosterone levels during breeding, and could help in preparation of the urogenital tract for high metabolic demands of that season.

Translational regulation of Anopheles gambiae mRNAs in the midgut during Plasmodium falciparum infection.

BACKGROUND: Malaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors. Malaria parasites in the midgut are surrounded by a hostile environment rich in digestive enzymes, while a rapidly responding immune system recognizes Plasmodium ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized. RESULTS: In this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion. CONCLUSIONS: We analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal.

Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression.

Juvenile hormone (JH) plays crucial roles in many aspects of insect life. The Methoprene-tolerant (Met) gene product, a member of the bHLH-PAS family of transcriptional regulators, has been demonstrated to be a key component of the JH signaling pathway. However, the molecular function of Met in JH-induced signal transduction and gene regulation remains to be fully elucidated. Here we show that a transcriptional coactivator of the ecdysteroid receptor complex, FISC, acts as a functional partner of Met in mediating JH-induced gene expression. Met and FISC appear to use their PAS domains to form a dimer only in the presence of JH or JH analogs. In newly emerged adult female mosquitoes, expression of some JH responsive genes is considerably dampened when Met or FISC is depleted by RNAi. Met and FISC are found to be associated with the promoter of the early trypsin gene (AaET) when transcription of this gene is activated by JH. A juvenile hormone response element (JHRE) has been identified in the AaET upstream regulatory region and is bound in vitro by the Met-FISC complex present in the nuclear protein extracts of previtellogenic adult female mosquitoes. In addition, the Drosophila homologs of Met and FISC can also use this mosquito JHRE to activate gene transcription in response to JH in a cell transfection assay. Together, the evidence indicates that Met and FISC form a functional complex on the JHRE in the presence of JH and directly activate transcription of JH target genes.

Direct sequencing and expression analysis of a large number of miRNAs in Aedes aegypti and a multi-species survey of novel mosquito miRNAs.

BACKGROUND: MicroRNAs (miRNAs) are a novel class of gene regulators whose biogenesis involves hairpin structures called precursor miRNAs, or pre-miRNAs. A pre-miRNA is processed to make a miRNA:miRNA* duplex, which is then separated to generate a mature miRNA and a miRNA*. The mature miRNAs play key regulatory roles during embryonic development as well as other cellular processes. They are also implicated in control of viral infection as well as innate immunity. Direct experimental evidence for mosquito miRNAs has been recently reported in anopheline mosquitoes based on small-scale cloning efforts. RESULTS: We obtained approximately 130, 000 small RNA sequences from the yellow fever mosquito, Aedes aegypti, by 454 sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. We also performed bioinformatics analysis on the Ae. aegypti genome assembly to identify evidence for additional miRNAs. The combination of these approaches uncovered 98 different pre-miRNAs in Ae. aegypti which could produce 86 distinct miRNAs. Thirteen miRNAs, including eight novel miRNAs identified in this study, are currently only found in mosquitoes. We also identified five potential revisions to previously annotated miRNAs at the miRNA termini, two cases of highly abundant miRNA* sequences, 14 miRNA clusters, and 17 cases where more than one pre-miRNA hairpin produces the same or highly similar mature miRNAs. A number of miRNAs showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these miRNAs. Northern blots also revealed several miRNAs that showed stage-specific expression. Detailed expression analysis of eight of the 13 mosquito-specific miRNAs in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. Four of the 13 miRNAs are specific to certain lineage(s) within mosquitoes. CONCLUSION: This study provides the first systematic analysis of miRNAs in Ae. aegypti and offers a substantially expanded list of miRNAs for all mosquitoes. New insights were gained on the evolution of conserved and lineage-specific miRNAs in mosquitoes. The expression profiles of a few miRNAs suggest stage-specific functions and functions related to embryonic development or blood feeding. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases.