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Sequencing of the human genome has permitted the development of genome-wide association studies (GWAS) to analyze the genetics of a number of complex disorders such as depression, anxiety and substance abuse. Thanks to their ability to analyze huge cohort sizes, these studies have successfully identified thousands of loci associated with a broad spectrum of complex diseases. Disconcertingly, the majority of these GWAS hits occur in non-coding regions of the genome, much of which controls the cell-type-specific expression of genes essential to health. In contrast to gene coding sequences, it is a challenge to understand the function of this non-coding regulatory genome using conventional biochemical techniques in cell lines. The current commentary scrutinizes the field of complex genetics from the standpoint of the large-scale whole-genome functional analysis of the promoters and cis-regulatory elements using chromatin markers. We contrast these large scale quantitative techniques against comparative genomics and in vivo analyses including CRISPR/CAS9 genome editing to determine the functional characteristics of these elements and to understand how polymorphic variation and epigenetic changes within these elements might contribute to complex disease and drug response. Most importantly, we suggest that, although the role of chromatin markers will continue to be important in identifying and characterizing enhancers, more emphasis must be placed on their analysis in relevant in-vivo models that take account of the appropriate cell-type-specific roles of these elements. It is hoped that offering these insights might refocus progress in analyzing the data tsunami of non-coding GWAS and whole-genome sequencing \"hits\" that threatens to overwhelm progress in the field.

Integrases, such as that of the Streptomyces temperate bacteriophage ΦC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ΦC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a \"subunit rotation\" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining ait sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ΦC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid \"phes\" recombination sites, each of which comprises a ΦC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive \"360° rotation\" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory \"gating\" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

The extremotolerant isolate H45 was one of several actinomycetes isolated from a high-altitude Atacama Desert soil collected in northwest Chile. The isolate was identified as a new Lentzea sp. using a combination of chemotaxonomic, morphological and phylogenetic properties. Large scale fermentation of the strain in two different media followed by chromatographic purification led to the isolation of six new diene and monoene glycosides named lentzeosides A-F, together with the known compound (Z)-3-hexenyl glucoside. The structures of the new compounds were confirmed by HRESIMS and NMR analyses. Compounds 1-6 displayed moderate inhibitory activity against HIV integrase.

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n = 115,865; p = 0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found the evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans from our UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co-expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men.

Integrases, such as that of the Streptomyces temperate bacteriophage ...C31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ...C31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a \"subunit rotation\" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ...C31 integrase-mediated recombination in a topologically constrained experimental system using hybrid \"phes\" recombination sites, each of which comprises a ...C31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180... rotation. However, multiple processive \"360... rotation\" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory \"gating\" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round. (ProQuest: ... denotes formulae/symbols omitted.)

Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase quality of life and our resilience to future pandemics. In the current study we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p=4.69x10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer whereas the obesity associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing suggesting a possible mechanism. We discuss our findings within the context of the known function and regulation of BDNF in obesity and anxiety whilst exploring the validity of interrogating GWAS data using comparative genomics and functional analysis using CRISPR genome editing in mice. Competing Interest Statement The authors have declared no competing interest.

Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Towards addressing the hypothesis that genetic and epigenetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we isolated the human CNR1 promotor (CNR1prom) and demonstrate its activity in primary cells and transgenic mice. We also provide evidence of CNR1prom in CB1 autoregulation and its repression by DNA-methylation. We further characterised a conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. Deletion of ECR1 from mice using CRISPR genome editing significantly reduced CNR1 expression in the hippocampus. These mice also displayed reduced ethanol intake and hypothermia response to CB1 agonism. Moreover, human specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in hippocampal cells than did the ancestral T-allele. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity. In the context of the known roles of CB1 the current study suggests a mechanism through which ECR1(C) may be neuroprotective in the hippocampus against stress. The cell-specific approaches used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important step in gaining a mechanistic understanding of cannabinoid pharmacogenetics.

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n=115,865; p=0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men that was consistent with these previous studies. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men. Footnotes * This version of the manuscript contains more conclusive GAL5.1 CRISPR mouse knockout anxiety data as well as more robust PKC data.

Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock. These profiles can be difficult and expensive to obtain for thousands of samples (e.g. for accurate association of microbial profiles with traits), therefore the objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeKI or PstI, and two bioinformatic pipelines (reference-based and reference-free) were compared to bacterial 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n = 236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to RE-RRS. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of laboratory and analytical method, and the relationship with methane production. The results suggested that the best method was PstI RE-RRS analyzed with the reference-free approach, which accounted for 53.3±5.9% of reads, and had repeatabilities of 0.49±0.07 and 0.50±0.07 for the first two principal components (PC1 and PC2), phenotypic correlations with methane yield of 0.43±0.06 and 0.46±0.06 for PC1 and PC2, and explained 41±8% of the variation in methane yield. These results were significantly better than for bacterial 16S rRNA gene sequencing of the same samples (p<0.05) except for the correlation between PC2 and methane yield. A Sensitivity study suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, meaning the current work using 118 samples/lane and future proposed 384 samples/lane are well within that threshold. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples.

Producing animal protein while reducing the animal's impact on the environment, e.g., through improved feed efficiency and lowered methane emissions, has gained interest in recent years. Genetic selection is one possible path to reduce the environmental impact of livestock production, but these traits are difficult and expensive to measure on many animals. The rumen microbiome may serve as a proxy for these traits due to its role in feed digestion. Restriction enzyme-reduced representation sequencing (RE-RRS) is a high-throughput and cost-effective approach to rumen metagenome profiling, but the systematic (e.g., sequencing) and biological factors influencing the resulting reference based (RB) and reference free (RF) profiles need to be explored before widespread industry adoption is possible. Metagenome profiles were generated by RE-RRS of 4,479 rumen samples collected from 1,708 sheep, and assigned to eight groups based on diet, age, time off feed, and country (New Zealand or Australia) at the time of sample collection. Systematic effects were found to have minimal influence on metagenome profiles. Diet was a major driver of differences between samples, followed by time off feed, then age of the sheep. The RF approach resulted in more reads being assigned per sample and afforded greater resolution when distinguishing between groups than the RB approach. Normalizing relative abundances within the sampling Cohort abolished structures related to age, diet, and time off feed, allowing a clear signal based on methane emissions to be elucidated. Genus-level abundances of rumen microbes showed low-to-moderate heritability and repeatability and were consistent between diets. Variation in rumen metagenomic profiles was influenced by diet, age, time off feed and genetics. Not accounting for environmental factors may limit the ability to associate the profile with traits of interest. However, these differences can be accounted for by adjusting for Cohort effects, revealing robust biological signals. The abundances of some genera were consistently heritable and repeatable across different environments, suggesting that metagenomic profiles could be used to predict an individual's future performance, or performance of its offspring, in a range of environments. These results highlight the potential of using rumen metagenomic profiles for selection purposes in a practical, agricultural setting.