Multi-animal-model study reveals mutations in neural plasticity and nociception genes linked to excessive alcohol drinking.

BACKGROUND: The basis for familial alcohol use disorder (AUD) remains an enigma due to various biological and societal confounds. The present study used three of the most adopted and documented rat models, combining the alcohol-preferring/non-alcohol-preferring (P/NP) lines and high alcohol-drinking/low alcohol-drinking (HAD/LAD) replicated lines, of AUD as examined through the lens of whole genomic analyses. METHODS: We used complete genome sequencing of the P/NP lines and previously published sequences of the HAD/LAD replicates to enhance the discovery of variants associated with AUD and to remove confounding with genetic background and random genetic drift. Specifically, we used high-order statistical methods to search for genetic variants whose frequency changes in whole sets of gene ontologies corresponded with phenotypic changes in the direction of selection, that is, ethanol-drinking preference. RESULTS: Our first finding was that in addition to variants causing translational changes, the principal genetic changes associated with drinking predisposition were silent mutations and mutations in the 3' untranslated regions (3'UTR) of genes. Neither of these types of mutations alters the amino acid sequence of the translated protein but they influence both the rate and conformation of gene transcription, including its stability and posttranslational events that alter gene efficacy. This finding argues for refocusing human genomic studies on changes in gene efficacy. Among the key ontologies identified were the central genes associated with the Na+ voltage-gated channels of neurons and glia (including the Scn1a, Scn2a, Scn2b, Scn3a, Scn7a, and Scn9a subtypes) and excitatory glutamatergic secretion (including Grm2 and Myo6), both of which are essential in neuroplasticity. In addition, we identified "Nociception or Sensory Perception of Pain," which contained variants in nociception (Arrb1, Ccl3, Ephb1) and enlist sodium (Scn1a, Scn2a, Scn2b, Scn3a, Scn7a), pain activation (Scn9a), and potassium channel (Kcna1) genes. CONCLUSION: The multi-model analyses used herein reduced the confounding effects of random drift and the "founders" genetic background. The most differentiated bidirectionally selected genes across all three animal models were Scn9a, Scn1a, and Kcna, all of which are annotated in the nociception ontology. The complexity of neuroplasticity and nociception adds strength to the hypothesis that neuroplasticity and pain (physical or psychological) are prominent phenotypes genetically linked to the development of AUD.

Structural Properties of CXCL4L1 and Its Recognition of the CXCR3 N-Terminus.

Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

CIS-Acting Allele-Specific Expression Differences Induced by Alcohol and Impacted by Sex as Well as Parental Genotype of Origin.

BACKGROUND: Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high- and low-alcohol drinking (HAD and LAD) rat lines. Here, we report allele-specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. METHODS: The HAD2 and LAD2 rats, which have been sequenced, were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2, and for the other 5 litters, the sire was a LAD2. From these 10 litters, 2 males and 2 females were picked from each F1 litter (N = 40 total). The F1 pups were divided, balancing for sex and direction of cross, into an alcohol (15%) versus a water control group. Alcohol drinking started in the middle of adolescence (~postnatal day 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA-sequencing and ASE analyses. RESULTS: Analyses revealed that adolescent ethanol (EtOH) drinking, individual EtOH drinking levels, parentage, and sex-of-animal affected ASEs of about 300 genes. The identified genes included those associated with EtOH metabolism (e.g., Aldh2); neuromodulatory function (e.g., Cckbr, Slc6a7, and Slc1a1); ion channel activity (e.g., Kcnc3); and other synaptic and epigenetic functions. CONCLUSIONS: These data indicate that EtOH drinking differentially amplified paternal versus maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to EtOH-induced changes in cis-regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene-by-environment interactions mediating a genetic predisposition for, and/or the active development of, AUDs.

Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics.

Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.

From the Cover: Zinc oxide Nanoparticles-Induced Reactive Oxygen Species Promotes Multimodal Cyto- and Epigenetic Toxicity.

In this study we evaluated and correlated the cytotoxic effects of zinc oxide nanoparticles (ZnO-NPs) to the epigenetic modifications, using human embryonic kidney (HEK-293) cells as a model system. Imaging of singlet and total reactive oxygen species (ROS) in ZnO-NPs-treated live cells was performed followed by the evaluation of its effects on cytoskeletal, mitochondrial, and nuclear integrity, and on the expression of ROS responsive genes. Next, we determined the global and locus-specific changes in DNA-methylation at the 3 global genomic repeat sequences namely LINE-1, subtelomeric D4Z4 and pericentromeric NBL2, and at the promoter of selected ROS responsive genes (AOX1, HMOX1, NCF2, SOD3). Our studies revealed severe actin depolymerization, increased release of mitochondrial cytochrome C, and nuclear enlargement in ZnO-NPs-treated cells. At the epigenetic level, we observed global reduction in 5-methylcytosine and increase in 5-hydroxymethylcytosine content. Additionally, we observed significant increase in the expression of Ten-Eleven Translocation (TET)-methylcytosine dioxygenase genes but not in the expression of DNA-methyltransferases (DNMTs). Based on our findings, we suggest that ZnO-NPs induce abundant increase in ROS to promote multimodal structural and functional anomalies in cells. Most importantly, ZnO-NP-induced ROS may promote global hypomethylation in cells by triggering the expression of TET-enzymes, avoiding DNMT interferences. Global DNA demethylation is considered to be the hallmark of the majority of cancers and once acquired this could be propagated to future progenies. The present study, hence, can be used as a platform for the assessment of epigenomic toxicity of ZnO-NPs in humans in the light of its use in commercial products.

Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum.

Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology.

High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

Optogenetic regulation of site-specific subtelomeric DNA methylation.

Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest.

Epigenetic regulation of the neural transcriptome and alcohol interference during development.

Alcohol intoxicated cells broadly alter their metabolites - among them methyl and acetic acid can alter the DNA and histone epigenetic codes. Together with the promiscuous effect of alcohol on enzyme activities (including DNA methyltransferases) and the downstream effect on microRNA and transposable elements, alcohol is well placed to affect intrinsic transcriptional programs of developing cells. Considering that the developmental consequences of early alcohol exposure so profoundly affect neural systems, it is not unfounded to reason that alcohol exploits transcriptional regulators to challenge canonical gene expression and in effect, intrinsic developmental pathways to achieve widespread damage in the developing nervous system. To fully evaluate the role of epigenetic regulation in alcohol-related developmental disease, it is important to first gather the targets of epigenetic players in neurodevelopmental models. Here, we attempt to review the cellular and genomic windows of opportunity for alcohol to act on intrinsic neurodevelopmental programs. We also discuss some established targets of fetal alcohol exposure and propose pathways for future study. Overall, this review hopes to illustrate the known epigenetic program and its alterations in normal neural stem cell development and further, aims to depict how alcohol, through neuroepigenetics, may lead to neurodevelopmental deficits observed in fetal alcohol spectrum disorders.

Environmental alterations of epigenetics prior to the birth.

The etiology of many brain diseases remains allusive to date after intensive investigation of genomic background and symptomatology from the day of birth. Emerging evidences indicate that a third factor, epigenetics prior to the birth, can exert profound influence on the development and functioning of the brain and over many neurodevelopmental syndromes. This chapter reviews how aversive environmental exposure to parents might predispose or increase vulnerability of offspring to neurodevelopmental deficit through alteration of epigenetics. These epigenetic altering environmental factors will be discussed in the category of addictive agents, nutrition or diet, prescriptive medicine, environmental pollutant, and stress. Epigenetic alterations induced by these aversive environmental factors cover all aspects of epigenetics including DNA methylation, histone modification, noncoding RNA, and chromatin modification. Next, the mechanisms how these environmental inputs influence epigenetics will be discussed. Finally, how environmentally altered epigenetic marks affect neurodevelopment is exemplified by the alcohol-induced fetal alcohol syndrome. It is hoped that a thorough understanding of the nature of prenatal epigenetic inputs will enable researchers with a clear vision to better unravel neurodevelopmental deficit, late-onset neuropsychiatric diseases, or idiosyncratic mental disorders.

Implications of genomic signatures in the differential vulnerability to fetal alcohol exposure in C57BL/6 and DBA/2 mice.

Maternal alcohol consumption inflicts a multitude of phenotypic consequences that range from undetectable changes to severe dysmorphology. Using tightly controlled murine studies that deliver precise amounts of alcohol at discrete developmental stages, our group and other labs demonstrated in prior studies that the C57BL/6 and DBA/2 inbred mouse strains display differential susceptibility to the teratogenic effects of alcohol. Since the phenotypic diversity extends beyond the amount, dosage and timing of alcohol exposure, it is likely that an individual's genetic background contributes to the phenotypic spectrum. To identify the genomic signatures associated with these observed differences in alcohol-induced dysmorphology, we conducted a microarray-based transcriptome study that also interrogated the genomic signatures between these two lines based on genetic background and alcohol exposure. This approach is called a gene x environment (GxE) analysis; one example of a GxE interaction would be a gene whose expression level increases in C57BL/6, but decreases in DBA/2 embryos, following alcohol exposure. We identified 35 candidate genes exhibiting GxE interactions. To identify cis-acting factors that mediated these interactions, we interrogated the proximal promoters of these 35 candidates and found 241 single nucleotide variants (SNVs) in 16 promoters. Further investigation indicated that 186 SNVs (15 promoters) are predicted to alter transcription factor binding. In addition, 62 SNVs created, removed or altered the placement of a CpG dinucleotide in 13 of the proximal promoters, 53 of which overlapped putative transcription factor binding sites. These 53 SNVs are also our top candidates for future studies aimed at examining the effects of alcohol on epigenetic gene regulation.

ENU mutagenesis reveals that Notchless homolog 1 (Drosophila) affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development.

BACKGROUND: Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by Trp53 and Wnt3 demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (l11Jus1 and l11Jus4), which fall into the same complementation group, survive through implantation but fail prior to gastrulation. RESULTS: Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the Notchless homolog 1 (Drosophila) (Nle1) gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the Nle1 mutant mice is much more severe than single Notch receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple Notch downstream target genes, as well as select members of the Wnt pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the Notch pathway or in Notch downstream target genes. However, our data reveal that Cdkn1a, a NOTCH target, was upregulated in Nle1 mutants, while several members of the Wnt pathway are downregulated. In addition, we found that Nle1 mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts. CONCLUSIONS: Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals.

Identification of 129S6/SvEvTac-specific polymorphisms on mouse chromosome 11.

Polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) can be associated with phenotypic traits and be used as markers for disease diagnosis. Identification of these genetic variations within laboratory mice is crucial to improve our understanding of the genetic background of the mice used for research. As part of a positional cloning project, we sequenced six genes (Mettl16, Evi2a, Psmd11, Cct6d, Rffl, and Ap2b1) within a 6.8-Mb domain of mmu chr 11 in the C57BL/6J and 129S6/SvEvTac inbred strains. Although 129S6/SvEvTac is widely used in the mouse community, there is very little current (or projected future) sequence information available for this strain. We identified 6 Indels and 21 novel SNPs and confirmed genotype information for 114 additional SNPs in these 6 genes. Mettl16 and Ap2b1 contained the largest numbers of variants between the C57BL/6J and 129S6/SvEvTac strains. In addition, we found five new SNPs between 129S6/SvEvTac and 129S1/SvImJ within the Ap2b1 locus. Although we did not detect differences between C57BL/6J and 129S6/SvEvTac within Evi2a, this locus contains a relatively high SNP density compared with the surrounding sequence. Our study highlights the genetic differences among three inbred mouse strains (C57BL/6J, 129S6/SvEvTac, and 129S1/SvImJ) and provides valuable sequence information that can be used to track alleles in genomics-based studies.