Exploring the Genetic Predisposition to Epigenetic Changes in Alzheimer's Disease.

Alzheimer's disease (AD) is a prevalent type of dementia in elderly populations with a significant genetic component. The accumulating evidence suggests that AD involves a reconfiguration of the epigenetic landscape, including DNA methylation, post-translational modification of histone proteins, and chromatin remodeling. Along with environmental factors, individual specific genetic features play a considerable role in the formation of epigenetic architecture. In this study, we attempt to identify the non-coding regulatory SNPs (rSNPs) able to affect the epigenetic mechanisms in AD. To this end, the multi-omics approach is used. The GEO (Gene Expression Omnibus) available data (GSE153875) for AD patients and controls are integrated to reveal the rSNPs that display allele-specific features in both ChIP-seq profiles of four histone modifications and RNA-seq. Furthermore, we analyze the presence of rSNPs in the promoters of genes reported to be differentially expressed between AD and the normal brain (AD-related genes) and involved in epigenetic regulation according to the EpiFactors database. We also searched for the rSNPs in the promoters of the genes coding for transcription regulators of the identified AD-related genes. These regulators were selected based on the corresponding ChIP-seq peaks (ENCODE) in the promoter regions of these genes. Finally, we formed a panel of rSNPs localized to the promoters of genes that contribute to the epigenetic landscape in AD and, thus, to the genetic predisposition for this disease.

SNPs in 3'UTR miRNA Target Sequences Associated with Individual Drug Susceptibility.

The complementary interaction of microRNAs (miRNAs) with their binding sites in the 3'untranslated regions (3'UTRs) of target gene mRNAs represses translation, playing a leading role in gene expression control. MiRNA recognition elements (MREs) in the 3'UTRs of genes often contain single nucleotide polymorphisms (SNPs), which can change the binding affinity for target miRNAs leading to dysregulated gene expression. Accumulated data suggest that these SNPs can be associated with various human pathologies (cancer, diabetes, neuropsychiatric disorders, and cardiovascular diseases) by disturbing the interaction of miRNAs with their MREs located in mRNA 3'UTRs. Numerous data show the role of SNPs in 3'UTR MREs in individual drug susceptibility and drug resistance mechanisms. In this review, we brief the data on such SNPs focusing on the most rigorously proven cases. Some SNPs belong to conventional genes from the drug-metabolizing system (in particular, the genes coding for cytochromes P450 (CYP 450), phase II enzymes (SULT1A1 and UGT1A), and ABCB3 transporter and their expression regulators (PXR and GATA4)). Other examples of SNPs are related to the genes involved in DNA repair, RNA editing, and specific drug metabolisms. We discuss the gene-by-gene studies and genome-wide approaches utilized or potentially utilizable to detect the MRE SNPs associated with individual response to drugs.

Regulatory SNPs: Altered Transcription Factor Binding Sites Implicated in Complex Traits and Diseases.

The vast majority of the genetic variants (mainly SNPs) associated with various human traits and diseases map to a noncoding part of the genome and are enriched in its regulatory compartment, suggesting that many causal variants may affect gene expression. The leading mechanism of action of these SNPs consists in the alterations in the transcription factor binding via creation or disruption of transcription factor binding sites (TFBSs) or some change in the affinity of these regulatory proteins to their cognate sites. In this review, we first focus on the history of the discovery of regulatory SNPs (rSNPs) and systematized description of the existing methodical approaches to their study. Then, we brief the recent comprehensive examples of rSNPs studied from the discovery of the changes in the TFBS sequence as a result of a nucleotide substitution to identification of its effect on the target gene expression and, eventually, to phenotype. We also describe state-of-the-art genome-wide approaches to identification of regulatory variants, including both making molecular sense of genome-wide association studies (GWAS) and the alternative approaches the primary goal of which is to determine the functionality of genetic variants. Among these approaches, special attention is paid to expression quantitative trait loci (eQTLs) analysis and the search for allele-specific events in RNA-seq (ASE events) as well as in ChIP-seq, DNase-seq, and ATAC-seq (ASB events) data.

A Panel of rSNPs Demonstrating Allelic Asymmetry in Both ChIP-seq and RNA-seq Data and the Search for Their Phenotypic Outcomes through Analysis of DEGs.

Currently, the detection of the allele asymmetry of gene expression from RNA-seq data or the transcription factor binding from ChIP-seq data is one of the approaches used to identify the functional genetic variants that can affect gene expression (regulatory SNPs or rSNPs). In this study, we searched for rSNPs using the data for human pulmonary arterial endothelial cells (PAECs) available from the Sequence Read Archive (SRA). Allele-asymmetric binding and expression events are analyzed in paired ChIP-seq data for H3K4me3 mark and RNA-seq data obtained for 19 individuals. Two statistical approaches, weighted z-scores and predicted probabilities, were used to improve the efficiency of finding rSNPs. In total, we identified 14,266 rSNPs associated with both allele-specific binding and expression. Among them, 645 rSNPs were associated with GWAS phenotypes; 4746 rSNPs were reported as eQTLs by GTEx, and 11,536 rSNPs were located in 374 candidate transcription factor binding motifs. Additionally, we searched for the rSNPs associated with gene expression using an SRA RNA-seq dataset for 281 clinically annotated human postmortem brain samples and detected eQTLs for 2505 rSNPs. Based on these results, we conducted Gene Ontology (GO), Disease Ontology (DO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and constructed the protein-protein interaction networks to represent the top-ranked biological processes with a possible contribution to the phenotypic outcome.

Genes associated with cognitive performance in the Morris water maze: an RNA-seq study.

Learning and memory are among higher-order cognitive functions that are based on numerous molecular processes including changes in the expression of genes. To identify genes associated with learning and memory formation, here, we used the RNA-seq (high-throughput mRNA sequencing) technology to compare hippocampal transcriptomes between mice with high and low Morris water maze (MWM) cognitive performance. We identified 88 differentially expressed genes (DEGs) and 24 differentially alternatively spliced transcripts between the high- and low-MWM-performance mice. Although the sets of DEGs and differentially alternatively spliced transcripts did not overlap, both were found to be enriched with genes related to the same type of biological processes: trans-synaptic signaling, cognition, and glutamatergic transmission. These findings were supported by the results of weighted-gene co-expression network analysis (WGCNA) revealing the enrichment of MWM-cognitive-performance-correlating gene modules with very similar Gene Ontology terms. High-MWM-performance mice manifested mostly higher expression of the genes associated with glutamatergic transmission and long-term potentiation implementation, which are processes necessary for memory acquisition and consolidation. In this set, there were genes participating in the regulation of trans-synaptic signaling, primarily AMPA receptor signaling (Nrn1, Nptx1, Homer3, Prkce, Napa, Camk2b, Syt7, and Nrgn) and calcium turnover (Hpca, Caln1, Orai2, Cpne4, and Cpne9). In high-MWM-performance mice, we also demonstrated significant upregulation of the "flip" splice variant of Gria1 and Gria2 transcripts encoding subunits of AMPA receptor. Altogether, our data helped to identify specific genes in the hippocampus that are associated with learning and long-term memory. We hypothesized that the differences in MWM cognitive performance between the mouse groups are linked with increased long-term potentiation, which is mainly mediated by increased glutamatergic transmission, primarily AMPA receptor signaling.

The Effects of Chronic Stress on Brain Myelination in Humans and in Various Rodent Models.

The myelination of axons, which is performed in brain tissues by specialized glial cells (oligodendrocytes) is crucial for correct formation of the complicated neural circuitry necessary for normal cognition, sensation, and motor function. Myelin-related anomalies are seen in many neurodegenerative diseases and in psychiatric disorders, including major depressive disorder and post-traumatic stress disorder. Chronic stress involving chronic stress early in life is believed to be a major etiological factor of neuropsychiatric disorders. Although molecular and cellular mechanisms underlying stress-induced psychopathologies are actively investigated, there is still little data about the role that is played in the development of these pathologies by myelin and oligodendrocyte impairments caused by chronic stress. In this article, after brief review of published data on myelin abnormalities in stress-related psychiatric disorders, we focus on recent cellular and molecular discoveries in various rodent models including models of chronic unpredictable stress, social isolation stress, chronic social defeat stress, and chronic immobilization stress. We also attempt to compile and analyze currently scarce data on myelin-related impairments resulting from early postnatal stress.

Potential regulatory SNPs in the ATXN7L3B and KRT15 genes are associated with gender-specific colorectal cancer risk.

Aim: According to the current data, a major factor for phenotypic variation of complex traits and disease susceptibility is the cis-acting effects of noncoding variants on gene expression. Our purpose was to evaluate the association between colorectal cancer (CRC) and six single nucleotide polymorphisms identified using our original bioinformatics approach as regulatory and putatively related to CRC. Materials: One hundred and sixty CRC patients and 185 healthy controls have been genotyped for rs590352, rs2072580, rs78317230, rs3829202, rs11542583 and rs4796672. Results: Genotypes and alleles distributions of rs590352 of ATXN7L3B gene were significantly different between the male CRC subjects and controls. Significant correlation of genotype with CRC is observable for women only for the rs4796672 of KRT15 gene. Analysis of haplotypes reveals that rs2072580 of the ISCU and SART3 genes can be also associated with CRC. Conclusion: We have identified three SNPs associated with CRC risk and demonstrated a gender specificity of rs590352 and rs4796672.

Regulatory SNPs and their widespread effects on the transcriptome.

Currently, it is generally accepted that the cis-acting effects of noncoding variants on gene expression are a major factor for phenotypic variation in complex traits and disease susceptibility. Meanwhile, the protein products of many target genes for the identified cis-regulatory variants (rSNPs) are regulatory molecules themselves (transcription factors, effectors, components of signal transduction pathways, etc.), which implies dramatic downstream effects of these variations on complex gene networks. Here, we brief the results of recent most comprehensive studies on the role of rSNPs in transcriptional regulation across the genome.

Novel functional variants at the GWAS-implicated loci might confer risk to major depressive disorder, bipolar affective disorder and schizophrenia.

BACKGROUND: A challenge of understanding the mechanisms underlying cognition including neurodevelopmental and neuropsychiatric disorders is mainly given by the potential severity of cognitive disorders for the quality of life and their prevalence. However, the field has been focused predominantly on protein coding variation until recently. Given the importance of tightly controlled gene expression for normal brain function, the goal of the study was to assess the functional variation including non-coding variation in human genome that is likely to play an important role in cognitive functions. To this end, we organized and utilized available genome-wide datasets from genomic, transcriptomic and association studies into a comprehensive data corpus. We focused on genomic regions that are enriched in regulatory activity-overlapping transcriptional factor binding regions and repurpose our data collection especially for identification of the regulatory SNPs (rSNPs) that showed associations both with allele-specific binding and allele-specific expression. We matched these rSNPs to the nearby and distant targeted genes and then selected the variants that could implicate the etiology of cognitive disorders according to Genome-Wide Association Studies (GWAS). Next, we use DeSeq 2.0 package to test the differences in the expression of the certain targeted genes between the controls and the patients that were diagnosed bipolar affective disorder and schizophrenia. Finally, we assess the potential biological role for identified drivers of cognition using DAVID and GeneMANIA. RESULTS: As a result, we selected fourteen regulatory SNPs locating within the loci, implicated from GWAS for cognitive disorders with six of the variants unreported previously. Grouping of the targeted genes according to biological functions revealed the involvement of processes such as 'posttranscriptional regulation of gene expression', 'neuron differentiation', 'neuron projection development', 'regulation of cell cycle process' and 'protein catabolic processes'. We identified four rSNP-targeted genes that showed differential expression between patient and control groups depending on brain region: NRAS-in schizophrenia cohort, CDC25B, DDX21 and NUCKS1-in bipolar disorder cohort. CONCLUSIONS: Overall, our findings are likely to provide the keys for unraveling the mechanisms that underlie cognitive functions including major depressive disorder, bipolar disorder and schizophrenia etiopathogenesis.

Consequences of early life stress on genomic landscape of H3K4me3 in prefrontal cortex of adult mice.

BACKGROUND: Maternal separation models in rodents are widely used to establish molecular mechanisms underlying prolonged effects of early life adversity on neurobiological and behavioral outcomes in adulthood. However, global epigenetic signatures following early life stress in these models remain unclear. RESULTS: In this study, we carried out a ChIP-seq analysis of H3K4 trimethylation profile in the prefrontal cortex of adult male mice with a history of early life stress. Two types of stress were used: prolonged separation of pups from their mothers (for 3 h once a day, maternal separation, MS) and brief separation (for 15 min once a day, handling, HD). Adult offspring in the MS group demonstrated reduced locomotor activity in the open field test accompanied by reduced exploratory activity, while the HD group showed decreased anxiety-like behavior only. In a group of maternal separation, we have found a small number (45) of slightly up-regulated peaks, corresponding to promoters of 70 genes, while no changes were observed in a group of handling. Among the genes whose promoters have differential enrichment of H3K4me3, the most relevant ones participate in gene expression regulation, modulation of chromatin structure and mRNA processing. For two genes, Ddias and Pip4k2a, increased H3K4me3 levels were associated with the increased mRNA expression in MS group. CONCLUSION: The distribution of H3K4me3 in prefrontal cortex showed relatively low variability across all individuals, and only some subtle changes were revealed in mice with a history of early life stress. It is possible that the observed long-lasting behavioral alterations induced by maternal separation are mediated by other epigenetic mechanisms, or other brain structures are responsible for these effects.

Molecular Adaptations to Social Defeat Stress and Induced Depression in Mice.

Chronic stress is a risk factor for major depression. Social defeat stress is a well-validated murine model of depression. However, little is known about the gene activity dynamics during the development of a depression-like state. We analyzed the effects of social defeat stress of varying duration (10 and 30 days) on the behavioral patterns and prefrontal-cortex transcriptome of C57BL/6 mice. The 10-day exposure to social defeat stress resulted in a high level of social avoidance with no signs of depression-associated behavior. Most animals exposed to 30 days of social defeat stress demonstrated clear hallmarks of depression, including a higher level of social avoidance, increased immobility in the forced swimming test, and anhedonic behavior. The monitoring of transcriptome changes revealed widespread alterations in gene expression on the 10th day. Surprisingly, the expression of only a few genes were affected by the 30th day of stress, apparently due to a reversal of the majority of the early stress-induced changes to the original basal state. Moreover, we have found that glucocorticoid-sensitive genes are clearly stimulated targets on the 10th day of stress, but these genes stop responding to the elevated corticosterone level by the 30th day of stress. The majority of genes altered by the 30-day stress were downregulated, with the most relevant ones participating in chromatin modifications and neuroplasticity (e.g., guanine nucleotide exchange factors of the Rho-family of GTPases). Very different molecular responses occur during short-term and long-term social stress in mice. The early-stress response is associated with social avoidance and with upregulation and downregulation of many genes, including those related to signal transduction and cell adhesion pathways. Downregulation of a few genes, in particular, genes for histone-modifying methyltransferases, is a signature response to prolonged stress that induces symptoms of depression. Altogether, our data show that the development of depression under social stress conditions is correlated with suppression of the overactive molecular response to induced stress, involving gene regulatory resistance to glucocorticoid molecules, potentially via a chromatin remodeling mechanism.

Brain-derived neurotrophic factor and early-life stress: Multifaceted interplay.

The brain-derived neurotrophic factor (BDNF) is a key regulator of neural development and plasticity. Longterm changes in the BDNF pathway are associated with childhood adversity and adult depression symptoms. Initially, stress-induced decreases in the BDNF pathway were found in some studies, but subsequent reports indicated the relationship between stress and BDNF to be much more complex, and the concept was significantly revised. In the present mini-review, we focus on the structure and regulation of the Bbnf gene as well as on the stress-BDNF interactions under early-life adverse conditions.

Regulatory single nucleotide polymorphisms (rSNPs) at the promoters 1A and 1B of the human APC gene.

BACKGROUND: Germline mutations in the coding sequence of the tumour suppressor APC gene give rise to familial adenomatous polyposis (which leads to colorectal cancer) and are associated with many other oncopathologies. The loss of APC function because of deletion of putative promoter 1A or 1B also results in the development of colorectal cancer. Since the regions of promoters 1A and 1B contain many single nucleotide polymorphisms (SNPs), the aim of this study was to perform functional analysis of some of these SNPs by means of an electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay. RESULTS: First, it was shown that both putative promoters of APC (1A and 1B) drive transcription in an in vitro reporter experiment. From eleven randomly selected SNPs of promoter 1A and four SNPs of promoter 1B, nine and two respectively showed differential patterns of binding of nuclear proteins to oligonucleotide probes corresponding to alternative alleles. The luciferase reporter assay showed that among the six SNPs tested, the rs75612255 C allele and rs113017087 C allele in promoter 1A as well as the rs138386816 T allele and rs115658307 T allele in promoter 1B significantly increased luciferase activity in the human erythromyeloblastoid leukaemia cell line K562. In human colorectal cancer HCT-116 cells, none of the substitutions under study had any effect, with the exception of minor allele G of rs79896135 in promoter 1B. This allele significantly decreased the luciferase reporter's activity CONCLUSION: Our results indicate that many SNPs in APC promoters 1A and 1B are functionally relevant and that allele G of rs79896135 may be associated with the predisposition to colorectal cancer.

Regulatory single nucleotide polymorphisms at the beginning of intron 2 of the human KRAS gene.

There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse K-ras gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T greater than A, rs12226937: G greater than A, and rs61761074: T greater than G) located in the same region of human KRAS. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G greater than A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T greater than G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.

Cytochrome P450 in fluke Opisthorchis felineus: identification and characterization.

Infection with the human liver fluke Opisthorchis felineus is a serious public health problem in Russia and other Eastern Europe countries. The aim of this work was to identify and sequence cytochrome P450 mRNA from O. felineus and to analyze its expression at different developmental stages. We found only one cytochrome P450 in O. felineus. It contains a conserved Pfam00067 domain which was typical of the CYP450 II eukaryotic microsomal type, and a putative transmembrane domain. Additionally, we identified a high degree of homology between a 3D model of O. felineus CYP450 and mammalian CYP2 structures. The level of O. felineus CYP mRNA expression in maritae (adult stage in definitive mammal host) is significantly higher than in metacercaria. This fact indicates an important role of this biotransformation enzyme in the biochemistry of the parasite at the maritae stage.

The increased CAR-dependent metabolism of thyroid hormones in mice with high cancer susceptibility.

AIM: our aim was to compare activation of the constitutive androstane receptor (CAR), hepatic expression of its target genes, and the serum thyroid hormone levels in C3H/He, C57BL/6J, and CC57BR/Mv mice following phenobarbital treatment. These differences, if present, could help to explain the different susceptibility to phenobarbital-induced liver tumor promotion among these strains of mice. MAIN METHODS: CAR DNA-binding activity and CAR content in nuclear protein extracts from mouse livers were assessed using the electrophoretic mobility shift assay and immunoblotting. Serum thyroid hormone concentrations were determined by radioimmunoassay. Real-time PCR was used to measure the hepatic expression level of CAR target genes. KEY FINDINGS: we found a 2.3-fold increase of CAR DNA-binding activity in response to phenobarbital in the sensitive C3H/He mice, but no change in the relatively resistant C57BL/6J and CC57BR/Mv mice. Phenobarbital treatment caused a significant decrease in triiodothyronine and free thyroxine concentrations (17% and 40%, respectively) in the sensitive C3H/He mice by the end of 60-day treatment, while in the resistant mice, these changes were not observed. In the sensitive C3H/He mice only, the expression of a CAR target gene encoding sulfotransferase Sult2a1, the thyroid hormone inactivation enzyme, increased by 260-fold after phenobarbital administration. The expression of another CAR target gene, Mdm2, was also increased by phenobarbital treatment in C3H/He mice. SIGNIFICANCE: we have shown that phenobarbital activates CAR and increases the expression of its target genes thereby accelerating the metabolism of thyroid hormones only in mice susceptible to liver tumor promotion by phenobarbital, but not in relatively resistant animals.

Recognition of interferon-inducible sites, promoters, and enhancers.

BACKGROUND: Computational analysis of gene regulatory regions is important for prediction of functions of many uncharacterized genes. With this in mind, search of the target genes for interferon (IFN) induction appears of interest. IFNs are multi-functional cytokines. Their effects are immunomodulatory, antiviral, antibacterial, and antitumor. The interaction of the IFNs with their cell surface receptors produces an activation of several transcription factors. Four regulatory factors, ISGF3, STAT1, IRF1, and NF-kappaB, are essential for the function of the IFN system. The aim of this work is the development of computational approaches for the recognition of DNA binding sites for these factors and computer programs for the prediction of the IFN-inducible regions. RESULTS: We developed computational approaches to the recognition of the binding sites for ISGF3, STAT1, IRF1, and NF-kappaB. Analysis of the distribution of these binding sites demonstrated that the regions -500 upstream of the transcription start site in IFN-inducible genes are enriched in putative binding sites for these transcription factors. Based on selected combinations of the sites whose frequencies were significantly higher than in the other functional gene groups, we developed methods for the prediction of the IFN-inducible promoters and enhancers. We analyzed 1004 sequences of the IFN-inducible genes compiled using microarray data analyses and also about 10,000 human gene sequences from the EPD and RefSeq databases; 74 of 1,664 human genes annotated in EPD were significantly IFN-inducible. CONCLUSION: Analyses of several control datasets demonstrated that the developed methods have a high accuracy of prediction of the IFN-inducible genes. Application of these methods to several datasets suggested that the number of the IFN-inducible genes is approximately 1500-2000 in the human genome.