Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD

Dissecting the Spatiotemporal Regulation of Kinetochore Interactions

Kinetochores are conserved protein complexes that bind the centromeres in replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we investigated the phospho-regulated dynamic interaction between the conserved kinetochore protein Cnn1CENP-T, the centromere region and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization is unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain that associates with centromere region, and a N-terminal Spc24/25-interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that a previously established Ndc80 binding site in the N-terminus of Cnn1, Cnn160-84, should be extended to include flanking residues, Cnn125-91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74 based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that in G1 through metaphase, Cnn1 localizes via the histone-fold domain or a N-terminal Spc24/25- interaction sequence because deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. Endogenous expression of the N-terminal region is sufficient to localize to the kinetochore demonstrating the availability to bind to Cnn1 and indicating the presence of the Cnn1-kinetchore linkages throughout the cell cycle. At anaphase onset (when Mps1 kinase activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/25-interaction sequence confirming previous studies using full-length Cnn1. In sum, we provide the first in vivo evidence of Cnn1 pre-anaphase linkages with the kinetochore and enrichment of the linkages during anaphase with this interaction sequence. Cse4 is a centromere-specific nucleosomal protein that has a similar motif organization to Cnn1 in that it has a histone-fold and a N-terminal tail sequence. Shugoshin (Sgo1) is also reported to localize to the centromere and pericentromere and is a key component in tension-sensing as a result of bi-polar attachment. We demonstrate a novel interaction between Sgo1 and Cse4 (yeast ortholog of CENP-A) at the centromere using yeast two-hybrid, live cell imaging and co-immunoprecipitation assays. We mapped this interaction to the first 132 residues of Sgo1 and the Cse4 N-terminal tail. Using mutational analysis, we identified several regions including a basic patch, S105 site and a putative coiled-coil region in Cse4 involved in the Sgo1 interaction. In sum, the identification of the Cse4-Sgo1 interaction is a key determinant in recruiting Sgo1 to the centromere and controlling the tension-sensing mechanism

Identification of Functional Genetic Variants Associated with Alcohol Dependence and Related Phenotypes Using a High-Throughput Assay

Background: Genome-wide association studies (GWAS) of alcohol dependence (AD) and related phenotypes have identified multiple loci, but the functional variants underlying the loci have in most cases not been identified. Noncoding variants can influence phenotype by affecting gene expression; for example, variants in the 3' untranslated regions (3'UTR) can affect gene expression posttranscriptionally. Methods: We adapted a high-throughput assay known as PASSPORT-seq (parallel assessment of polymorphisms in miRNA target sites by sequencing) to identify among variants associated with AD and related phenotypes those that cause differential expression in neuronal cell lines. Based upon meta-analyses of alcohol-related traits in African American and European Americans in the Collaborative Study on the Genetics of Alcoholism, we tested 296 single nucleotide polymorphisms (SNPs with meta-analysis p values ≤ 0.001) that were located in 3'UTRs. Results: We identified 60 SNPs that affected gene expression (false discovery rate [FDR] < 0.05) in SH-SY5Y cells and 92 that affected expression in SK-N-BE(2) cells. Among these, 30 SNPs altered RNA levels in the same direction in both cell lines. Many of these SNPs reside in the binding sites of miRNAs and RNA-binding proteins and are expression quantitative trait loci of genes including KIF6,FRMD4A,CADM2,ADD2,PLK2, and GAS7. Conclusion: The SNPs identified in the PASSPORT-seq assay are functional variants that might affect the risk for AD and related phenotypes. Our study provides insights into gene regulation in AD and demonstrates the value of PASSPORT-seq as a tool to screen genetic variants in GWAS loci for one potential mechanism of action

Budding yeast CENP-A^Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

<p>Shugoshin is an evolutionarily conserved protein, which is involved in tension sensing on mitotic chromosomes, kinetochore biorientation, and protection of centromeric (<i>CEN</i>) cohesin for faithful chromosome segregation. Interaction of the C-terminus of Sgo1 with phosphorylated histone H2A regulates its association with <i>CEN</i> and pericentromeric (peri-<i>CEN</i>) chromatin, whereas mutations in histone H3 selectively compromise the association of Sgo1 with peri-<i>CEN</i> but not <i>CEN</i> chromatin. Given that histone H3 is absent from <i>CEN</i> and is replaced by a histone H3 variant CENP-A^Cse4, we investigated if CENP-A^Cse4 interacts with Sgo1 and promotes its association with the <i>CEN</i> chromatin. In this study, we found that Sgo1 interacts with CENP-A^Cse4 <i>in vivo</i> and <i>in vitro</i>. The N-terminus coiled-coil domain of Sgo1 without the C-terminus (<i>sgo1-NT</i>) is sufficient for its interaction with CENP-A^Cse4, association with <i>CEN</i> but not the peri-<i>CEN</i>, and this <i>CEN</i> association is cell cycle dependent with maximum enrichment in mitosis. In agreement with the role of CENP-A^Cse4 in <i>CEN</i> maintenance of Sgo1, depletion of CENP-A^Cse4 results in the loss of Sgo1 and <i>sgo1-NT</i> from the <i>CEN</i> chromatin. The N-terminus of Sgo1 is required for genome stability as a mutant lacking the N-terminus (<i>sgo1-CT</i>) exhibits increased chromosome missegregation when compared to a <i>sgo1-NT</i> mutant. In summary, our results define a novel role for the N-terminus of Sgo1 in CENP-A^Cse4 mediated recruitment of Sgo1 to <i>CEN</i> chromatin for faithful chromosome segregation.</p

Functional 3’-UTR Variants Identify Regulatory Mechanisms Impacting Alcohol Use Disorder and Related Traits

Although genome-wide association studies (GWAS) have identified loci associated with alcohol consumption and alcohol use disorder (AUD), they do not identify which variants are functional. To approach this, we evaluated the impact of variants in 3' untranslated regions (3'-UTRs) of genes in loci associated with substance use and neurological disorders using a massively parallel reporter assay (MPRA) in neuroblastoma and microglia cells. Functionally impactful variants explained a higher proportion of heritability of alcohol traits than non-functional variants. We identified genes whose 3'UTR activities are associated with AUD and alcohol consumption by combining variant effects from MPRA with GWAS results. We examined their effects by evaluating gene expression after CRISPR inhibition of neuronal cells and stratifying brain tissue samples by MPRA-derived 3'-UTR activity. A pathway analysis of differentially expressed genes identified inflammation response pathways. These analyses suggest that variation in response to inflammation contributes to the propensity to increase alcohol consumption

Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD

Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD