Browsing by Author "Lossie, Amy C."

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    Cis‐acting allele specific expression (ASE) differences induced by alcohol and impacted by sex as well as parental genotype of origin
    (Wiley, 2018) Lo, Chiao-Ling; Lumeng, Lawrence; Bell, Richard L.; Liang, Tiebing; Lossie, Amy C.; Muir, Williams M.; Zhou, Feng C.; Anatomy and Cell Biology, School of Medicine
    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, two males and two females were picked from each F1 litter (N = 40 total). The F1‐pups were divided, with balancing for sex and direction of cross, into an alcohol (15%) vs a water control group. Alcohol‐drinking started in the middle of adolescence (~PND 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 drinking, individual ethanol drinking levels, parentage, and sex‐of‐animal affected ASEs of about 300 genes. The identified genes included those associated with ethanol metabolism (e.g., Aldh2); neuromodulatory function [e.g., Cckbr, Slc6a7, and Slc1a1]; ion channel activity (e.g., Kcnc3); as well as other synaptic and epigenetic function. Conclusion These data indicate that ethanol drinking differentially amplified paternal vs maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to ethanol‐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, alcohol use disorders.
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    Cloning, Annotation and Developmental Expression of the Chicken Intestinal MUC2 Gene
    (Public Library of Science, 2013) Jiang, Zhengyu; Applegate, Todd J.; Lossie, Amy C.; Medicine, School of Medicine
    Intestinal mucin 2 (MUC2) encodes a heavily glycosylated, gel-forming mucin, which creates an important protective mucosal layer along the gastrointestinal tract in humans and other species. This first line of defense guards against attacks from microorganisms and is integral to the innate immune system. As a first step towards characterizing the innate immune response of MUC2 in different species, we report the cloning of a full-length, 11,359 bp chicken MUC2 cDNA, and describe the genomic organization and functional annotation of this complex, 74.5 kb locus. MUC2 contains 64 exons and demonstrates distinct spatiotemporal expression profiles throughout development in the gastrointestinal tract; expression increases with gestational age and from anterior to posterior along the gut. The chicken protein has a similar domain organization as the human orthologue, with a signal peptide and several von Willebrand domains in the N-terminus and the characteristic cystine knot at the C-terminus. The PTS domain of the chicken MUC2 protein spans ∼1600 amino acids and is interspersed with four CysD motifs. However, the PTS domain in the chicken diverges significantly from the human orthologue; although the chicken domain is shorter, the repetitive unit is 69 amino acids in length, which is three times longer than the human. The amino acid composition shows very little similarity to the human motif, which potentially contributes to differences in the innate immune response between species, as glycosylation across this rapidly evolving domain provides much of the musical barrier. Future studies of the function of MUC2 in the innate immune response system in chicken could provide an important model organism to increase our understanding of the biological significance of MUC2 in host defense and highlight the potential of the chicken for creating new immune-based therapies.
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    ENU mutagenesis reveals that Notchless homolog 1 (Drosophila) affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development
    (Springer Nature, 2012-12-12) Lossie, Amy C.; Lo, Chiao-Ling; Baumgarner, Katherine M.; Cramer, Melissa J.; Garner, Joseph P.; Justice, Monica J.; Medicine, School of Medicine
    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.
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    High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model
    (Plos, 2016-08) Lo, Chiao-Ling; Lossie, Amy C.; Liang, Tiebing; Liu, Yunlong; Xuei, Xiaoling; Lumeng, Lawrence; Zhou, Feng C.; Muir, William M.; Department of Anatomy & Cell Biology, IU School of Medicine
    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.
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    Implications of genomic signatures in the differential vulnerability to fetal alcohol exposure in C57BL/6 and DBA/2 mice
    (Frontiers Media, 2014-06-11) Lossie, Amy C.; Muir, William M.; Lo, Chiao-Ling; Timm, Floyd; Liu, Yunlong; Gray, Whitney; Zhou, Feng C.; Medicine, School of Medicine
    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.
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    A snapshot of the hepatic transcriptome: ad libitum alcohol intake suppresses expression of cholesterol synthesis genes in alcohol-preferring (P) rats
    (PLoS, 2014-12-26) Klein, Jonathon D.; Sherrill, Jeremy B.; Morello, Gabriella M.; San Miguel, Phillip J.; Ding, Zhenming; Liangpunsakul, Suthat; Liang, Tiebing; Muir, William M.; Lumeng, Lawrence; Lossie, Amy C.; Department of Psychiatry, IU School of Medicine
    Research is uncovering the genetic and biochemical effects of consuming large quantities of alcohol. One prime example is the J- or U-shaped relationship between the levels of alcohol consumption and the risk of atherosclerotic cardiovascular disease. Moderate alcohol consumption in humans (about 30 g ethanol/d) is associated with reduced risk of coronary heart disease, while abstinence and heavier alcohol intake is linked to increased risk. However, the hepatic consequences of moderate alcohol drinking are largely unknown. Previous data from alcohol-preferring (P) rats showed that chronic consumption does not produce significant hepatic steatosis in this well-established model. Therefore, free-choice alcohol drinking in P rats may mimic low risk or nonhazardous drinking in humans, and chronic exposure in P animals can illuminate the molecular underpinnings of free-choice drinking in the liver. To address this gap, we captured the global, steady-state liver transcriptome following a 23 week free-choice, moderate alcohol consumption regimen (∼ 7.43 g ethanol/kg/day) in inbred alcohol-preferring (iP10a) rats. Chronic consumption led to down-regulation of nine genes in the cholesterol biosynthesis pathway, including HMG-CoA reductase, the rate-limiting step for cholesterol synthesis. These findings corroborate our phenotypic analyses, which indicate that this paradigm produced animals whose hepatic triglyceride levels, cholesterol levels and liver histology were indistinguishable from controls. These findings explain, at least in part, the J- or U-shaped relationship between cardiovascular risk and alcohol intake, and provide outstanding candidates for future studies aimed at understanding the mechanisms that underlie the salutary cardiovascular benefits of chronic low risk and nonhazardous alcohol intake.
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    A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells.
    (PLOS, 2016) Chowdhury, Basudev; Seetharam, Arun; Wang, Zhiping; Liu, Yunlong; Lossie, Amy C.; Thimmapuram, Jyothi; Irudayaraj, Joseph; Department of Medical & Molecular Genetics, IU School of Medicine
    Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based “simulated” microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations.