Tissue-Specific Effects of Genetic and Epigenetic Variation on Gene Regulation and Splicing

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans’ lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.     

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.     

Human cardiac inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR) 8-dependent

The group B coxsackieviruses are single-stranded RNA viruses that have been implicated in viral myocarditis. Viral infection of the myocardium, as well as the associated inflammatory response are important determinants of the virus-associated myocardial damage. Although these viruses are known as cytopathic viruses that cause death of the host cell, their viral RNA has been shown to persist in cardiac muscle contributing to a chronic inflammatory cardiomyopathy. Thus, it is essential that we understand the mechanism by which Coxasckie B viruses (CBVs) trigger this inflammatory response. In this study we investigated the involvement of Toll-like receptors (TLRs) in the recognition of CBV virions as well as CBV single-stranded RNA. Here we report that the CBV-induced inflammatory response is mediated through TLR8 and to a lesser extent through TLR7.     

Laboratory Investigation and Phylogenetic Analysis of an Imported Middle East Respiratory Syndrome Coronavirus Case in Greece

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1^st and 2^nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.     

Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae

Chemical or biological synthesis of plant secondary metabolites has attracted increasing interest due to their proven or assumed beneficial properties and health promoting effects. Resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and the flavonols kaempferol and quercetin have been shown to possess high nutritional and agricultural value. Four metabolically engineered yeast strains harboring plasmids with heterologous genes for enzymes involved in the biosynthesis of these compounds from phenylalanine have been constructed. Time course analyses of precursor utilization and end-product accumulation were carried out establishing the production of 0.29–0.31 mg/L of trans-resveratrol, 8.9–15.6 mg/L of naringenin, 0.1–7.7 mg/L of genistein, 0.9–4.6 mg/L of kaempferol and 0.26–0.38 mg/L of quercetin in defined media under optimal growth conditions. The recombinant yeast strains can be used further for the construction of improved flavonoid- and stilbenoid-overproducers.