Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood.

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.

A standard knockout procedure alters expression of adjacent loci at the translational level.

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head' orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5' untranslated region (5' UTR). In the 'tail-to-tail' arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3' UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing.

In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription­replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model.

STUDY QUESTION: Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted reproductive technologies (ART)? SUMMARY ANSWER: Our results demonstrate dose-dependent and sex-specific effects of folic acid supplementation in ART and provide evidence that moderate dose supplements may be optimal for both sexes. WHAT IS KNOWN ALREADY: Children conceived using ART are at an increased risk for growth and genomic imprinting disorders, often associated with DNA methylation defects. Folic acid supplementation is recommended during pregnancy to prevent adverse offspring outcomes; however, the effects of folic acid supplementation in ART remain unclear. STUDY DESIGN, SIZE, DURATION: Outbred female mice were fed three folic acid-supplemented diets, control (rodent daily recommended intake or DRI; CD), moderate (4-fold DRI; 4FASD) or high (10-fold DRI; 10FASD) dose, for six weeks prior to ART and throughout gestation. Mouse ART involved a combination of superovulation, in vitro fertilisation, embryo culture and embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Midgestation embryos and placentas (n = 74-99/group) were collected; embryos were assessed for developmental delay and gross morphological abnormalities and embryos and placentas were examined for epigenetic defects. We assessed methylation at four imprinted genes (Snrpn, Kcnq1ot1, Peg1 and H19) in matched midgestation embryos and placentas (n = 31-32/group) using bisulfite pyrosequencing. In addition, we examined genome-wide DNA methylation patterns in placentas (n = 6 normal placentas per sex/group) and embryos (n = 6 normal female embryos/group; n = 3 delayed female embryos/group) using reduced representation bisulfite sequencing (RRBS). MAIN RESULTS AND THE ROLE OF CHANCE: Moderate, but not high dose supplementation, was associated with a decrease in the proportion of developmentally delayed embryos. Although moderate dose folic acid supplementation reduced DNA methylation variance at certain imprinted genes in embryonic and placental tissues, high dose supplementation exacerbated the negative effects of ART at imprinted loci. Furthermore, folic acid supplements resolved female-biased aberrant imprinted gene methylation. Supplementation was more effective at correcting ART-induced genome-wide methylation defects in male versus female placentas; however, folic acid supplementation also led to additional methylation perturbations which were more pronounced in males. LARGE-SCALE DATA: The RRBS data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE123143. LIMITATIONS REASONS FOR CAUTION: Although the combination of mouse ART utilised in this study consisted of techniques commonly used in human fertility clinics, there may be species differences. Therefore, human studies, designed to determine the optimal levels of folic acid supplementation for ART pregnancies, and taking into account foetal sex, are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our findings support moderation in the dose of folic acid supplements taken during ART. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Canadian Institutes of Health Research (FDN-148425). The authors declare no conflict of interest.

Chemically Controlled Epigenome Editing through an Inducible dCas9 System.

Although histone modifications are associated with gene activities, studies of their causal relationships have been difficult. For this purpose, we developed an inducible system integrating dCas9-based targeting and chemically induced proximity technologies to allow small molecule induced recruitment of P300 acetyltransferase and the acetylation of H3K27 at precise gene loci in cells. Employing the new technique, we elucidated the temporal order of histone acetylation and gene activation, as well as the stability of the installed histone modification.

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Genomewide Association Study of Alcohol Dependence Identifies Risk Loci Altering Ethanol-Response Behaviors in Model Organisms.

BACKGROUND: Alcohol dependence (AD) shows evidence for genetic liability, but genes influencing risk remain largely unidentified. METHODS: We conducted a genomewide association study in 706 related AD cases and 1,748 unscreened population controls from Ireland. We sought replication in 15,496 samples of European descent. We used model organisms (MOs) to assess the role of orthologous genes in ethanol (EtOH)-response behaviors. We tested 1 primate-specific gene for expression differences in case/control postmortem brain tissue. RESULTS: We detected significant association in COL6A3 and suggestive association in 2 previously implicated loci, KLF12 and RYR3. None of these signals are significant in replication. A suggestive signal in the long noncoding RNA LOC339975 is significant in case:control meta-analysis, but not in a population sample. Knockdown of a COL6A3 ortholog in Caenorhabditis elegans reduced EtOH sensitivity. Col6a3 expression correlated with handling-induced convulsions in mice. Loss of function of the KLF12 ortholog in C. elegans impaired development of acute functional tolerance (AFT). Klf12 expression correlated with locomotor activation following EtOH injection in mice. Loss of function of the RYR3 ortholog reduced EtOH sensitivity in C. elegans and rapid tolerance in Drosophila. The ryanodine receptor antagonist dantrolene reduced motivation to self-administer EtOH in rats. Expression of LOC339975 does not differ between cases and controls but is reduced in carriers of the associated rs11726136 allele in nucleus accumbens (NAc). CONCLUSIONS: We detect association between AD and COL6A3, KLF12, RYR3, and LOC339975. Despite nonreplication of COL6A3, KLF12, and RYR3 signals, orthologs of these genes influence behavioral response to EtOH in MOs, suggesting potential involvement in human EtOH response and AD liability. The associated LOC339975 allele may influence gene expression in human NAc. Although the functions of long noncoding RNAs are poorly understood, there is mounting evidence implicating these genes in multiple brain functions and disorders.

Recessive mutations in the cancer gene Ataxia Telangiectasia Mutated (ATM), at a locus previously associated with metformin response, cause dysglycaemia and insulin resistance.

AIM: To investigate glucose and insulin metabolism in participants with ataxia telangiectasia in the absence of a diagnosis of diabetes. METHODS: A standard oral glucose tolerance test was performed in participants with ataxia telangiectasia (n = 10) and in a control cohort (n = 10). Serial glucose and insulin measurements were taken to permit cohort comparisons of glucose-insulin homeostasis and indices of insulin secretion and sensitivity. RESULTS: During the oral glucose tolerance test, the 2-h glucose (6.75 vs 4.93 mmol/l; P = 0.029), insulin concentrations (285.6 vs 148.5 pmol/l; P = 0.043), incremental area under the curve for glucose (314 vs 161 mmol/l/min; P = 0.036) and incremental area under the curve for insulin (37,720 vs 18,080 pmol/l/min; P = 0.03) were higher in participants with ataxia telangiectasia than in the controls. There were no significant differences between groups in fasting glucose, insulin concentrations or insulinogenic index measurement (0.94 vs 0.95; P = 0.95). The Matsuda index, reflecting whole-body insulin sensitivity, was lower in participants with ataxia telangiectasia (5.96 vs 11.03; P = 0.019) than in control subjects. CONCLUSIONS: Mutations in Ataxia Telangiectasia Mutated (ATM) that cause ataxia telangiectasia are associated with elevated glycaemia and low insulin sensitivity in participants without diabetes. This indicates a role of ATM in glucose and insulin metabolic pathways.

Exposure to anticancer drugs can result in transgenerational genomic instability in mice.

The genetic effects of human exposure to anticancer drugs remain poorly understood. To establish whether exposure to anticancer drugs can result not only in mutation induction in the germ line of treated animals, but also in altered mutation rates in their offspring, we evaluated mutation rates in the offspring of male mice treated with three commonly used chemotherapeutic agents: cyclophosphamide, mitomycin C, and procarbazine. The doses of paternal exposure were approximately equivalent to those used clinically. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat locus Ms6-hm was established in DNA samples extracted from sperm and bone marrow of the offspring of treated males. After paternal exposure to any one of these three drugs, expanded simple tandem repeat mutation frequencies were significantly elevated in the germ line (sperm) and bone marrow of their offspring. This observed transgenerational instability was attributed to elevated mutation rates at the alleles derived from both the exposed fathers and from the nonexposed mothers, thus implying a genome-wide destabilization. Our results suggest that paternal exposure to a wide variety of mutagens can result in transgenerational instability manifesting in their offspring. Our data also raise important issues concerning delayed transgenerational effects in the children of survivors of anticancer therapy.

Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement.

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10(-19) ≤ P ≤ 4.5 × 10(-18)) and D6D (FADS2) (6.05 × 10(-8) ≤ P ≤ 4.20 × 10(-7)) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10(-16)). The significance of haplotype association with D5D activity (P = 2.19 × 10(-17)) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10(-28)) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10(-9)) and decreased D6D activity (P = 9.16 × 10(-6)) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.

Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents.

OBJECTIVES: To establish an experimental platform in Staphylococcus aureus for identifying genetic loci that determine intrinsic antibiotic susceptibility and/or that have the potential to contribute to acquired antibiotic resistance. A near-saturation S. aureus transposon (Tn) library was screened for mutants exhibiting altered susceptibility to the antistaphylococcal agents daptomycin, vancomycin and nisin. METHODS: S. aureus SH1000 was mutagenized with Tn InsTet(G+)2(Cm) by electroporation of transposomes. Approximately 20500 transposants were screened for increased or reduced susceptibility to the three antistaphylococcal agents and Tn insertion sites were mapped by DNA sequencing in mutants of interest. RESULTS: Transposants exhibiting hypersusceptibility or reduced susceptibility were identified for all three antibacterial agents; mapping of Tn insertion sites in these mutants identified genetic determinants of intrinsic susceptibility and potential contributors to acquired resistance, respectively. Tn insertions in the dlt operon caused cross-hypersusceptibility to vancomycin, daptomycin and nisin. Daptomycin hypersusceptibility was also associated with disruption of genes directing lipoteichoic acid and riboflavin biosynthesis, apparent inactivation of a putative membrane protein encoded by SAOUHSC_00957 and truncation of the cell-division gene ezrA. Tn-mediated disruption of the vraDE- and SAOUHSC_02953/4-encoded ABC transporters conferred hypersusceptibility to nisin. Reduced susceptibility to both daptomycin and vancomycin was associated with Tn insertions in rpsU and upstream of yycFG. Several loci were associated with reduced susceptibility to nisin, including two genes encoding putative glycosyltransferases. CONCLUSIONS: Tn library screening identified both known and novel modulators of antibacterial susceptibility in S. aureus and therefore represents a useful approach towards delineating the staphylococcal resistome.

Regulation of mouse Cyp24a1 expression via promoter-proximal and downstream-distal enhancers highlights new concepts of 1,25-dihydroxyvitamin D(3) action.

CYP24A1 functions in vitamin D target tissues to degrade 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Thus, the concentration of this enzyme and the regulation of its expression is a primary determinant of the overall biological activity of 1,25(OH)(2)D(3) within cells. The principle regulator of CYP24A1 expression is 1,25(OH)(2)D(3) itself, which functions through the vitamin D receptor to upregulate the transcriptional activity of the Cyp24a1 gene. In this report, we explore the mechanism of this regulation using recently developed ChIP-chip and ChIP-seq techniques that permit an unbiased search for enhancer elements that participate in this transcriptional control. Our studies both confirm a regulatory region defined earlier and located proximal to the transcriptional start site (TSS) of mouse Cyp24a1 (-160 and -265nt) and identify a novel intergenic region located downstream of the transcription unit that contains two enhancers (+35 and +37kb) that facilitate 1,25(OH)(2)D(3)-dependent upregulation of Cyp24a1 expression. Interestingly, while C/EBPß also binds under basal conditions to a site located immediately upstream of the Cyp24a1 promoter (-345nt), occupancy by this factor is strikingly increased following 1,25(OH)(2)D(3) treatment. The locations and activities of these regulatory regions that mediate 1,25(OH)(2)D(3) actions were confirmed in mice in vivo. We conclude that the mechanism through which 1,25(OH)(2)D(3) induces the CYP24A1 enzyme, thereby autoregulating its own destruction, involves both promoter-proximal as well as downstream-distal enhancers. These findings highlight new concepts regarding the molecular mechanism of action of 1,25(OH)(2)D(3) and other hormonal regulators.

Omics data integration identifies ELOVL7 and MMD gene regions as novel loci for adalimumab response in patients with Crohn's disease.

Response to anti-TNF therapy is of pivotal importance in patients with Crohn's disease (CD). Here we integrated our and previously reported PBMC derived transcriptomic and genomic data for identification of biomarkers for discrimination between responders and non-responders to anti-TNF therapy. CD patients, who were naïve with respect to the treatment with biologicals, were enrolled in the study. DNA and RNA were extracted from peripheral blood mononuclear cells. RNA-seq was performed using BGISEQ-500. Genotyping was performed using Infinium Global Screening Array. Association regressions were carried out with 12 week response to adalimumab as an outcome variable. RNA-seq analysis confirmed 7 out of 65 previously suggested genes involved in anti-TNF response. Subsequently, analysis of single nucleotide variants in regions of confirmed genes identified 5 variants near MMD and two in ELOVL7 intronic regions associated with treatment response to anti-TNF. Functional analysis has shown that rs1465352, rs4422035 and rs78620886 are listed at H3K9ac_Pro histone modification epigenetic mark. The present study confirmed MMD and ELOVL7 involvement in anti-TNF response and revealed that the regulation of MMD and ELOVL7 gene regions in ADA response may be a part of a complex interplay extending from genetic to epigenetic and to transcriptomic level.

Association between mercury in cord serum and sex-specific DNA methylation in cord tissues.

Prenatal exposure to mercury in utero causes abnormal foetal growth and adverse outcomes. DNA methylation is currently considered a possible mechanism through which this occurs. However, few studies have investigated the association between prenatal exposure to mercury and DNA methylation in detail. This study aimed to clarify the relationship between prenatal exposure to total mercury (Hg) and DNA methylation and its associations with sex-specific characteristics in male and female offspring. In a birth cohort study known as the Chiba study of Mother and Child Health, the DNA methylation status in cord tissue and Hg concentrations in cord serum were examined. A total of 67 participants (27 males and 40 females) were analysed based on Spearman's correlations, adjusted by a false discovery rate of the sex of each offspring. Only one methylated locus was positively correlated with Hg concentrations in cord serum in male offspring, but not in female offspring, and was annotated to the haloacid dehalogenase-like hydrolase domain-containing protein 1 (HDHD1) gene on chromosome X. This locus was located in the intron of the HDHD1 gene body and is a binding site for the zinc finger protein CCCTC-binding factor. One of the other loci, located in HDHD1, was highly methylated in the group with higher mercury concentrations, and this locus was in the gene body of HDHD1. Our results suggest that prenatal exposure to Hg might affect the epigenetic status of male foetuses.

In Vitro Mouse Lymphoma Cell (L5178Y Tk+/- -3.7.2.C) Forward Mutation Assay.

The in vitro mouse lymphoma cell assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk+/- -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions and multilocus, chromosomal rearrangements, mitotic recombination and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA; small and large colony mutants. The induction of small colony mutants is associated with chemicals inducing gross chromosomal aberrations, whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development Test Guideline 490 is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

Gene Variants at Loci Related to Blood Pressure Account for Variation in Response to Antihypertensive Drugs Between Black and White Individuals.

Selection of antihypertensive treatment according to self-defined ethnicity is recommended by some guidelines but might be better guided by individual genotype rather than ethnicity or race. We compared the extent to which variation in blood pressure response across different ethnicities may be explained by genetic factors: genetically defined ancestry and gene variants at loci known to be associated with blood pressure. We analyzed data from 5 trials in which genotyping had been performed (n=4696) and in which treatment responses to ß-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blocker, thiazide or thiazide-like diuretic and calcium channel blocker were available. Genetically defined ancestry for proportion of African ancestry was computed using the 1000 genomes population database as a reference. Differences in response to the thiazide diuretic hydrochlorothiazide, the ß-blockers atenolol and metoprolol, the angiotensin-converting enzyme inhibitor lisinopril, and the angiotensin receptor blocker candesartan were more closely associated to genetically defined ancestry than self-defined ethnicity in admixed subjects. A relatively small number of gene variants related to loci associated with drug-signaling pathways (KCNK3, SULT1C3, AMH, PDE3A, PLCE1, PRKAG2) with large effect size (-3.5 to +3.5 mm Hg difference in response per allele) and differing allele frequencies in black versus white individuals explained a large proportion of the difference in response to candesartan and hydrochlorothiazide between these groups. These findings suggest that a genomic precision medicine approach can be used to individualize antihypertensive treatment within and across populations without recourse to surrogates of genetic structure such as self-defined ethnicity.

Limbic circuitry activation in ethanol withdrawal is regulated by a chromosome 1 locus.

Physiological dependence and associated withdrawal episodes are thought to constitute a motivational force sustaining alcohol use/abuse and contributing to relapse in alcoholics. Although no animal model exactly duplicates alcoholism, models for specific factors, including the withdrawal syndrome, are useful for identifying potential genetic and neural determinants of liability in humans. We previously identified highly significant quantitative trait loci (QTLs) with large effects on predisposition to withdrawal after chronic and acute alcohol exposure in mice and mapped these loci to the same region of chromosome 1 (Alcdp1 and Alcw1, respectively). The present studies utilize a novel Alcdp1/Alcw1 congenic model (in which an interval spanning Alcdp1 and Alcw1 from the C57BL/6J donor strain [build GRCm38 150.3-174.6 Mb] has been introgressed onto a uniform inbred DBA/2J genetic background) known to demonstrate significantly less severe chronic and acute withdrawal compared to appropriate background strain animals. Here, using c-Fos induction as a high-resolution marker of neuronal activation, we report that male Alcdp1/Alcw1 congenic animals demonstrate significantly less alcohol withdrawal-associated neural activation compared to appropriate background strain animals in the prelimbic and cingulate cortices of the prefrontal cortex as well as discrete regions of the extended amygdala (i.e., basolateral) and extended basal ganglia (i.e., dorsolateral striatum, and caudal substantia nigra pars reticulata). These studies are the first to begin to elucidate circuitry by which this confirmed addiction-relevant QTL could influence behavior. This circuitry overlaps limbic circuitry involved in stress, providing additional mechanistic information. Alcdp1/Alcw1 maps to a region syntenic with human chromosome 1q, where multiple studies find significant associations with risk for alcoholism.

Importance of Adult Dmbx1 in Long-Lasting Orexigenic Effect of Agouti-Related Peptide.

Dmbx1 is a brain-specific homeodomain transcription factor expressed primarily during embryogenesis, and its systemic disruption (Dmbx1(-/-)) in the ICR mouse strain resulted in leanness associated with impaired long-lasting orexigenic effect of agouti-related peptide (AgRP). Because spatial and temporal expression patterns of Dmbx1 change dramatically during embryogenesis, it remains unknown when and where Dmbx1 plays a critical role in energy homeostasis. In the present study, the physiological roles of Dmbx1 were examined by its conditional disruption (Dmbx1(loxP/loxP)) in the C57BL/6 mouse strain. Although Dmbx1 disruption in fetal brain resulted in neonatal lethality, its disruption by synapsin promoter-driven Cre recombinase, which eliminated Dmbx1 expression postnatally, exempted the mice (Syn-Cre;Dmbx1(loxP/loxP) mice) from lethality. Syn-Cre;Dmbx1(loxP/loxP) mice show mild leanness and impaired long-lasting orexigenic action of AgRP, demonstrating the physiological relevance of Dmbx1 in the adult. Visualization of Dmbx1-expressing neurons in adult brain using the mice harboring tamoxifen-inducible Cre recombinase in the Dmbx1 locus (Dmbx1(CreERT2/+) mice) revealed Dmbx1 expression in small numbers of neurons in restricted regions, including the lateral parabrachial nucleus (LPB). Notably, c-Fos expression in LPB was increased at 48 hours after AgRP administration in Dmbx1(loxP/loxP) mice but not in Syn-Cre;Dmbx1(loxP/loxP) mice. These c-Fos-positive neurons in LPB did not coincide with neurons expressing Dmbx1 or melanocortin 4 receptor but did coincide with those expressing calcitonin gene-related peptide. Accordingly, Dmbx1 in the adult LPB is required for the long-lasting orexigenic effect of AgRP via the neural circuitry involving calcitonin gene-related peptide neurons.

Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila.

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

System for stable ß-estradiol-inducible gene expression in the moss Physcomitrella patens.

Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with ß-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing ß-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This ß-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.