Serotonin Transporter (SLC6A4) and FK506-Binding Protein 5 (FKBP5) Genotype and Methylation Relationships with Response to Meditation in Veterans with PTSD.

Meditation-based interventions are novel and effective non-pharmacologic treatments for veterans with PTSD. We examined relationships between treatment response, early life trauma exposure, DNA polymorphisms, and methylation in the serotonin transporter (SLC6A4) and FK506-binding protein 5 (FKBP5) genes. DNA samples and clinical outcomes were examined in 72 veterans with PTSD who received meditation-based therapy in two separate studies of mindfulness-based stress reduction (MBSR) and Transcendental Meditation (TM). The PTSD Checklist was administered to assess symptoms at baseline and after 9 weeks of meditation intervention. We examined the SLC6A4 promoter (5HTTLPR_L/S insertion/deletion + rs25531_A/G) polymorphisms according to previously defined gene expression groups, and the FKBP5 variant rs1360780 previously associated with PTSD disease risk. Methylation for CpG sites of SLC6A4 (28 sites) and FKBP5 (45 sites) genes was quantified in DNA samples collected before and after treatment. The 5HTTLPR LALA high expression genotype was associated with greater symptom improvement in participants exposed to early life trauma (p = 0.015). Separately, pre to post-treatment change of DNA methylation in a group of nine FKBP5 CpG sites was associated with greater symptom improvement (OR = 2.8, 95% CI 1.1-7.1, p = 0.027). These findings build on a wealth of existing knowledge regarding epigenetic and genetic relationships with PTSD disease risk to highlight the potential importance of SLC6A4 and FKBP5 for treatment mechanisms and as biomarkers of symptom improvement.

Using the Daphnia magna Transcriptome to Distinguish Water Source: Wetland and Stormwater Case Studies.

A major challenge in ecotoxicology is accurately and sufficiently measuring chemical exposures and biological effects given the presence of complex and dynamic contaminant mixtures in surface waters. It is impractical to quantify all chemicals in such matrices over space and time, and even if it were practical, concomitant biological effects would not be elucidated. Our study examined the performance of the Daphnia magna transcriptome to detect distinct responses across three water sources in Minnesota: laboratory (well) waters, wetland waters, and storm waters. Pyriproxyfen was included as a gene expression and male neonate production positive control to examine whether gene expression resulting from exposure to this well-studied juvenoid hormone analog can be detected in complex matrices. Laboratory-reared (<24 h) D. magna were exposed to a water source and/or pyriproxyfen for 16 days to monitor phenotypic changes or 96 h to examine gene expression responses using Illumina HiSeq 2500 (10 million reads per library, 50-bp paired end [2 × 50]). The results indicated that a unique gene expression profile was produced for each water source. At 119 ng/L pyriproxyfen (~25% effect concentration) for male neonate production, as expected, the Doublesex1 gene was up-regulated. In descending order, gene expression patterns were most discernable with respect to pyriproxyfen exposure status, season of stormwater sample collection, and wetland quality, as indicated by the index of biological integrity. However, the biological implications of the affected genes were not broadly clear given limited genome resources for invertebrates. Our study provides support for the utility of short-term whole-organism transcriptomic testing in D. magna to discern sample type, but highlights the need for further work on invertebrate genomics. Environ Toxicol Chem 2022;41:2107-2123. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF.

Enterococcus faecalis is a common commensal bacterium in animal gastrointestinal (GI) tracts and a leading cause of opportunistic infections of humans in the modern health care setting. E. faecalis OG1RF is a plasmid-free strain that contains few mobile elements yet retains the robust survival characteristics, intrinsic antibiotic resistance, and virulence traits characteristic of most E. faecalis genotypes. To facilitate interrogation of the core enterococcal genetic determinants for competitive fitness in the GI tract, biofilm formation, intrinsic antimicrobial resistance, and survival in the environment, we generated an arrayed, sequence-defined set of chromosomal transposon insertions in OG1RF. We used an orthogonal pooling strategy in conjunction with Illumina sequencing to identify a set of mutants with unique, single Himar-based transposon insertions. The mutants contained insertions in 1,926 of 2,651 (72.6%) annotated open reading frames and in the majority of hypothetical protein-encoding genes and intergenic regions greater than 100 bp in length, which could encode small RNAs. As proof of principle of the usefulness of this arrayed transposon library, we created a minimal input pool containing 6,829 mutants chosen for maximal genomic coverage and used an approach that we term SMarT (sequence-defined mariner technology) transposon sequencing (TnSeq) to identify numerous genetic determinants of bile resistance in E. faecalis OG1RF. These included several genes previously associated with bile acid resistance as well as new loci. Our arrayed library allows functional screening of a large percentage of the genome with a relatively small number of mutants, reducing potential effects of bottlenecking, and enables immediate recovery of mutants following competitions. IMPORTANCE The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.

The lung tissue microbiota of mild and moderate chronic obstructive pulmonary disease.

BACKGROUND: Oral taxa are often found in the chronic obstructive pulmonary disease (COPD) lung microbiota, but it is not clear if this is due to a physiologic process such as aspiration or experimental contamination at the time of specimen collection. METHODS: Microbiota samples were obtained from nine subjects with mild or moderate COPD by swabbing lung tissue and upper airway sites during lung lobectomy. Lung specimens were not contaminated with upper airway taxa since they were obtained surgically. The microbiota were analyzed with 16S rRNA gene qPCR and 16S rRNA gene hypervariable region 3 (V3) sequencing. Data analyses were performed using QIIME, SourceTracker, and R. RESULTS: Streptococcus was the most common genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways. Each subject's own bronchial and lung tissue microbiota were more similar to each other than were the bronchial and lung tissue microbiota of two different subjects (permutation test, p = 0.0139), indicating more within-subject similarity than between-subject similarity at these two lung sites. Principal coordinate analysis of all subject samples revealed clustering by anatomic sampling site (PERMANOVA, p = 0.001), but not by subject. SourceTracker analysis found that the sources of the lung tissue microbiota were 21.1% (mean) oral microbiota, 8.7% nasal microbiota, and 70.1% unknown. An analysis using the neutral theory of community ecology revealed that the lung tissue microbiota closely reflects the bronchial, oral, and nasal microbiota (immigration parameter estimates 0.69, 0.62, and 0.74, respectively), with some evidence of ecologic drift occurring in the lung tissue. CONCLUSION: This is the first study to evaluate the mild-moderate COPD lung tissue microbiota without potential for upper airway contamination of the lung samples. In our small study of subjects with COPD, we found oral and nasal bacteria in the lung tissue microbiota, confirming that aspiration is a source of the COPD lung microbiota.

Development of the Intestinal RNA Virus Community of Healthy Broiler Chickens.

Several RNA viruses such as astrovirus, rotavirus, reovirus and parvovirus have been detected in both healthy and diseased commercial poultry flocks. The aim of this study was to characterize (a) the development of the RNA viral community in the small intestines of healthy broiler chickens from hatch through 6 weeks of age (market age) and (b) the contribution of the breeder source vs. bird age in development of the community structure. Intestinal tissue samples were harvested from breeders and their progeny, processed for viral RNA extraction and sequenced using Illumina Hiseq sequencing technology resulting in 100 bp PE reads. The results from this study indicated that the breeder source influenced the RNA viral community only at hatch but later environment i.e. bird age had the more significant effect. The most abundant RNA viral family detected at 2, 4 and 6 weeks of age was Astroviridae, which decreased in abundance with age while the abundance of Picornaviridae increased with age.

Histone H2AX and the small RNA pathway modulate both non-homologous end-joining and homologous recombination in plants.

Using a zinc finger nuclease (ZFN) that creates a site-specific DNA double-strand break (DSB) at the endogenous ADH1 locus, we provide genetic evidence that histone H2AX and DSB-induced small RNAs modulate DNA repair by both non-homologous end-joining (NHEJ) and homologous recombination (HR) in the model plant Arabidopsis thaliana.

Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host.

Comparison of tissue sample processing methods for harvesting the viral metagenome and a snapshot of the RNA viral community in a turkey gut.

RNA viruses have been associated with enteritis in poultry and have been isolated from diseased birds. The same viral agents have also been detected in healthy flocks bringing into question their role in health and disease. In order to understand better eukaryotic viruses in the gut, this project focused on evaluating alternative methods to purify and concentrate viral particles, which do not involve the use of density gradients, for generating viral metagenome data. In this study, the sequence outcomes of three tissue processing methods have been evaluated and a data analysis pipeline has been established for RNA viruses from the gastrointestinal tract. In addition, with the use of the best method and increased sequencing depth, a glimpse of the RNA viral community in the gastrointestinal tract of a clinically normal 5-week old turkey is presented. The viruses from the Reoviridae and Astroviridae families together accounted for 76.3% of total viruses identified. The rarefaction curve at the species level further indicated that majority of the species diversity was included with the increased sequencing depth, implying that viruses from other viral families were present in very low abundance.

Origin replication complex binding, nucleosome depletion patterns, and a primary sequence motif can predict origins of replication in a genome with epigenetic centromeres.

UNLABELLED: Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A "mini-ARS screen" identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion. IMPORTANCE: DNA replication machinery is highly conserved, yet the definition of exactly what specifies a replication origin differs in different species. Here, we utilized computational genomics to predict origin locations in Candida albicans by combining locations of binding sites for the conserved origin replication complex, necessary for replication initiation, together with chromatin organization patterns. We identified predicted sequences that exhibited bona fide origin function and developed a linear plasmid assay to delimit the DNA fragments necessary for origin function. Additionally, we found that a short AC-rich motif, which is enriched in predicted origins, is required for origin function. Thus, we demonstrated a new machine learning paradigm for identification of potential origins from a genome with no prior information. Furthermore, this work suggests that C. albicans has two different types of origins: "hard-wired" arm origins that rely upon specific sequence motifs and "epigenetic" centromeric origins that are recruited to kinetochores in a sequence-independent manner.

Silencing is noisy: population and cell level noise in telomere-adjacent genes is dependent on telomere position and sir2.

Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways.

Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFN-mediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications.

Transcription activator-like effector nucleases enable efficient plant genome engineering.

The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.

The three clades of the telomere-associated TLO gene family of Candida albicans have different splicing, localization, and expression features.

Candida albicans grows within a wide range of host niches, and this adaptability enhances its success as a commensal and as a pathogen. The telomere-associated TLO gene family underwent a recent expansion from one or two copies in other CUG clade members to 14 expressed copies in C. albicans. This correlates with increased virulence and clinical prevalence relative to those of other Candida clade species. The 14 expressed TLO gene family members have a conserved Med2 domain at the N terminus, suggesting a role in general transcription. The C-terminal half is more divergent, distinguishing three clades: clade α and clade ß have no introns and encode proteins that localize primarily to the nucleus; clade γ sometimes undergoes splicing, and the gene products localize within the mitochondria as well as the nuclei. Additionally, TLOα genes are generally expressed at much higher levels than are TLOγ genes. We propose that expansion of the TLO gene family and the predicted role of Tlo proteins in transcription regulation provide C. albicans with the ability to adapt rapidly to the broad range of different environmental niches within the human host.

A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon.

Ty1, the most abundant retrotransposon in Saccharomyces cerevisiae, integrates preferentially upstream of genes transcribed by RNA polymerase III (Pol III). Targeting is likely due to interactions between the Ty1 integration complex and a feature of chromatin characteristic of sites of Pol III transcription. To better understand Ty1 targeting determinants, >150,000 Ty1 insertions were mapped onto the S. cerevisiae genome sequence. Logistic regression was used to assess relationships between patterns of Ty1 integration and various genomic features, including genome-wide data sets of histone modifications and transcription-factor binding sites. Nucleosomes were positively associated with Ty1 insertions, and fine-scale mapping of insertions upstream of genes transcribed by Pol III indicated that Ty1 preferentially integrates into nucleosome-bound DNA near the H2A/H2B interface. Outside of genes transcribed by Pol III, Ty1 avoids coding sequences, a pattern that is not due to selection, but rather reflects a preference for nucleosome-rich sites flanking genes. Ty1 insertion sites were also mapped in four mutant lines that affect Ty1 transposition frequency or integration specificity (rrm3Δ, hos2Δ, rtt109Δ, and rad6Δ). Patterns of integration were largely preserved in the mutants, although significantly more insertions into coding sequences were observed in the rad6Δ strain, suggesting a loosening of target specificity in this mutant that lacks an enzyme involved in ubiquitinating H2A. Overall, our data suggest that nucleosomes are necessary for Ty1 integration, and that a secondary factor, likely a histone modification or nucleosome-bound factor enriched at sites of Pol III transcription, determines preferred target sites.

Putting genetic interactions in context through a global modular decomposition.

Genetic interactions provide a powerful perspective into gene function, but our knowledge of the specific mechanisms that give rise to these interactions is still relatively limited. The availability of a global genetic interaction map in Saccharomyces cerevisiae, covering ∼30% of all possible double mutant combinations, provides an unprecedented opportunity for an unbiased assessment of the native structure within genetic interaction networks and how it relates to gene function and modular organization. Toward this end, we developed a data mining approach to exhaustively discover all block structures within this network, which allowed for its complete modular decomposition. The resulting modular structures revealed the importance of the context of individual genetic interactions in their interpretation and revealed distinct trends among genetic interaction hubs as well as insights into the evolution of duplicate genes. Block membership also revealed a surprising degree of multifunctionality across the yeast genome and enabled a novel association of VIP1 and IPK1 with DNA replication and repair, which is supported by experimental evidence. Our modular decomposition also provided a basis for testing the between-pathway model of negative genetic interactions and within-pathway model of positive genetic interactions. While we find that most modular structures involving negative genetic interactions fit the between-pathway model, we found that current models for positive genetic interactions fail to explain 80% of the modular structures detected. We also find differences between the modular structures of essential and nonessential genes.

Access to DNA establishes a secondary target site bias for the yeast retrotransposon Ty5.

Integration sites for many retrotransposons and retroviruses are determined by interactions between retroelement-encoded integrases and specific DNA-bound proteins. The Saccharomyces retrotransposon Ty5 preferentially integrates into heterochromatin because of interactions between Ty5 integrase and the heterochromatin protein silent information regulator 4. We mapped over 14,000 Ty5 insertions onto the S. cerevisiae genome, 76% of which occurred in heterochromatin, which is consistent with the known target site bias of Ty5. Using logistic regression, associations were assessed between Ty5 insertions and various chromosomal features such as genome-wide distributions of nucleosomes and histone modifications. Sites of Ty5 insertion, regardless of whether they occurred in heterochromatin or euchromatin, were strongly associated with DNase hypersensitive, nucleosome-free regions flanking genes. Our data support a model wherein silent information regulator 4 tethers the Ty5 integration machinery to domains of heterochromatin, and then, specific target sites are selected based on DNA access, resulting in a secondary target site bias. For insertions in euchromatin, DNA access is the primary determinant of target site choice. One consequence of the secondary target site bias of Ty5 is that insertions in coding sequences occur infrequently, which may preserve genome integrity.

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.