Monoallelic, antisense and total RNA transcription in an in vitro neural differentiation system based on F1 hybrid mice.

We developed an in vitro system to differentiate embryonic stem cells (ESCs) derived from reciprocally crossed F1 hybrid mice into neurons, and used it to investigate poly(A)+ and total RNA transcription at different stages of cell differentiation. By comparing expression profiles of transcripts assembled from 20 RNA sequencing datasets [2 alleles×(2 cell lines×4 time-points+2 mouse brains)], the relative influence of strain, cell and parent specificities to overall expression could be assessed. Divergent expression profiles of ESCs converged tightly at neural progenitor stage. Patterns of temporal variation of monoallelically expressed transcripts and antisense transcripts were quantified. Comparison of sense and antisense transcript pairs within the poly(A)+ sample, within the total RNA sample, and across poly(A)+ and total RNA samples revealed distinct rates of pairs showing anti-correlated expression variation. Unique patterns of sharing of poly(A)+ and poly(A)- transcription were identified in distinct RNA species. Regulation and functionality of monoallelic expression, antisense transcripts and poly(A)- transcription remain elusive. We demonstrated the effectiveness of our approach to capture these transcriptional activities, and provided new resources to elucidate the mammalian developmental transcriptome.

Occupation exposed to road-traffic emissions and respiratory health among Congolese transit workers, particularly bus conductors, in Kinshasa: a cross-sectional study.

OBJECTIVES: Road-traffic emissions (RTE) induce adverse health effects, notably respiratory symptoms and respiratory diseases, as a result of pollutants deposited into the respiratory tract. The aim of this study was to evaluate the association between occupation groups of Congolese transit workers exposed to RTE, particularly bus conductors and respiratory health, in Kinshasa. METHODS: A cross-sectional study was conducted from 2015 April 20th to May 14th, whose participants were bus conductors (n = 110), bus drivers (n = 107), taxi-motorcyclists (n = 102) and high school teachers (control group; n = 106). Subjects had completed the American Thoracic Society respiratory symptom questionnaire. Lung function test was performed by spirometry. Air pollutants levels of PM2.5, NO2 and SO2 were measured between 7:30 and 8:30 and 16:30-17:30 using a portable gas monitor. Multivariate analysis was performed to evaluate the association between occupation exposed to RTE and impaired pulmonary function, after adjustment by plausible confounders. RESULTS: The prevalence of mixed syndrome was 21.9% for bus conductors, 10.9% for bus drivers, 15.4% for taxi-motorcyclists and 7.1% for high school teachers with (p < 0.05). The risk of developing a mixed syndrome was seven times higher among bus conductors [OR = 7.64; 95% CI: 1.83-31.67; p < 0.05] than other groups. Additionally, the prevalence of respiratory syndromes increased with the duration of exposure. CONCLUSIONS: Occupation exposed to RTE is associated with impaired pulmonary function and the prevalence of respiratory symptoms among transit workers, especially bus conductors. Furthermore, this association increases with the duration of exposure suggesting the necessity to regulate these categories of occupations and to apply preventives measures.

Fibrotic gene expression coexists with alveolar proteinosis in early indium lung.

Occupational inhalation of indium compounds can cause the so-called "indium lung disease". Most affected individuals show pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease. In animal experiments, inhalation of indium tin oxide or indium oxide has been shown to cause lung damage. However, the mechanisms by which indium compounds lead to indium lung disease remain unknown. In this study, we constructed a mouse model of indium lung disease and analyzed gene expression in response to indium exposure. Indium oxide (In2O3, 10 mg/kg, primary particle size <100 nm) was administered intratracheally to C57BL/6 mice (male, 8 weeks of age) twice a week for 8 weeks. Four weeks after the final instillation, histopathological analysis exhibited periodic acid-Schiff positive material in the alveoli, characteristic of PAP. Comprehensive gene expression analysis by RNA-Seq, however, revealed expression of fibrosis-related genes, such as surfactant associated protein D, surfactant associated protein A1, mucin 1, and collagen type I and III, was significantly increased, indicating that fibrotic gene expression progresses in early phase of indium lung. These data supported the latest hypothesis that PAP occurs as an acute phase response and is replaced by fibrosis after long-term latency.

Secondary structure-based analysis of mouse brain small RNA sequences obtained by using next-generation sequencing.

In order to find novel structured small RNAs, next-generation sequencing was applied to small RNA fractions with lengths ranging from 40 to 140 nt and secondary structure-based clustering was performed. Sequences of structured RNAs were effectively clustered and analyzed by secondary structure. Although more than 99% of the obtained sequences were known RNAs, 16 candidate mouse structured small non-coding RNAs (MsncRs) were isolated. Based on these results, the merits of secondary structure-based analysis are discussed.

Regulation of Gene Expression by Sodium Valproate in Epithelial-to-Mesenchymal Transition.

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is an important mechanism in cancer metastasis and pulmonary fibrosis. Previous studies demonstrated effect of histone H3 and H4 acetylation in cancer and pulmonary fibrosis, so we hypothesized that histone modification might play a crucial role in gene regulation during EMT. In this study, we investigated the mechanism behind EMT by analyzing comprehensive gene expression and the effect of sodium valproate (VPA), a class I histone deacetylase inhibitory drug, on histone modification. METHODS: EMT was induced in human alveolar epithelial cells (A549) using 5 ng/mL of transforming growth factor (TGF)-ß1. Various concentrations of VPA were then administered, and Western blotting was used to analyze histone acetylation or methylation. Comprehensive gene expression analysis was carried out by RNA sequencing, and chromatin immunoprecipitation was performed with an anti-acetyl histone H3 lysine 27 antibody. RESULTS: TGF-ß1 stimulation led to a decrease in histone acetylation, especially that of histone H3K27, and H3K27ac localization was decreased at particular gene loci. This decrease was recovered by VPA treatment, which also up-regulated the mRNA expression of genes down-regulated by TGF-ß1, and correlated with the localization of H3K27ac. However, genes up-regulated by TGF-ß1 stimulation were not suppressed by VPA, with the exception of COL1A1. CONCLUSIONS: Histone acetylation was down-regulated by TGF-ß1 stimulation in A549 cells. VPA partially inhibited EMT and the decrease of histone acetylation, which plays an important role in the progression of EMT.

ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner.

Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.

Inter-subspecies mouse F1 hybrid embryonic stem cell lines newly established for studies of allelic imbalance in gene expression.

Allele-specific monoallelic gene expression is a unique phenomenon and a great resource for analyzing gene regulation. To study this phenomenon, we established new embryonic stem (ES) cell lines derived from F1 hybrid blastocysts from crosses between four mouse subspecies (Mus musculus domesticus, C57BL/6; M. musculus molossinus, MSM/Ms; M. musculus musculus, PWK; M. musculus castaneus, HMI/Ms) and analyzed the expression levels of undifferentiated pluripotent stem cell markers and karyotypes of each line. To demonstrate the utility of our cell lines, we analyzed the allele-specific expression pattern of the Inpp5d gene as an example. The allelic expression depended on the parental alleles; this dependence could be a consequence of differences in compatibility between cis- and trans-elements of the Inpp5d gene from different subspecies. The use of parental mice from four subspecies greatly enhanced genetic polymorphism. The F1 hybrid ES cells retained this polymorphism not only in the Inpp5d gene, but also at a genome-wide level. As we demonstrated for the Inpp5d gene, the established cell lines can contribute to the analysis of allelic expression imbalance based on the incompatibility between cis- and trans-elements and of phenotypes related to this incompatibility.

Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription.

We investigated the allele- and strand-specific transcriptional landscape of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A) by means of a highly parallel SNP genotyping platform. We have successfully identified maternal-specific expression of Ube3a and its antisense counterpart (Ube3a-ATS) in brain, but not in liver. Because of the use of inter-subspecies hybrid mice, this megabase-wide analysis provided high-resolution picture of the transcriptional patterns of this region. First, we showed that brain-specific maternal expression of Ube3a is restricted to the second half part of the locus, but is absent from the first half part. Balance of allelic expression is altered in the middle of the locus. Second, we showed that expression of the brain-specific Ube3a-ATS appeared to be terminated in the region upstream to the Ube3a transcription start site. The present study highlights the importance of locus-wide competition between sense and antisense transcripts.

Expression analysis of box C/D snoRNAs with SNPs between C57BL/6 and MSM/Ms strains in male mouse.

MSM/Ms mouse derived from the Japanese wild mouse has unique characteristics compared to the widely used C57BL/6 mouse. To examine the usefulness of the MSM/Ms mouse for the comparative genomic analysis, expression of small RNAs were analyzed by the large-scale sequence analysis for two strains of mouse, C57BL/6 and MSM/Ms. As a trial, expression of box C/D snoRNAs, which are the most abundant small RNAs in the cell, were analyzed. By the comparison of the read number for each fragment, 11 snoRNAs with single nucleotide polymorphisms (SNPs) were detected. One of the snoRNAs, SNORD53, shows the expression only for MSM/Ms and this snoRNA has a mutation in the box sequence in C57BL/6. Thus, it was demonstrated that the proposed experimental system using SNPs can give new insight for the gene expression regulation.

Reduction of ribosome biogenesis with activation of the mTOR pathway in denervated atrophic muscle.

Mammalian target of rapamycin (mTOR) pathway positively regulates the cell growth through ribosome biogenesis in many cell type. In general, myostatin is understood to repress skeletal muscle hypertrophy through inhibition of mTOR pathway and myogenesis. However, these relationships have not been clarified in skeletal muscle undergoing atrophy. Here, we observed a significant decrease of skeletal muscle mass at 2 weeks after denervation. Unexpectedly, however, mTOR pathway and the expression of genes related to myogenesis were markedly increased, and that of myostatin was decreased. However, de novo ribosomal RNA synthesis and the levels of ribosomal RNAs were dramatically decreased in denervated muscle. These results indicate that ribosome biogenesis is strongly controlled by factors other than the mTOR pathway in denervated atrophic muscle. Finally, we assessed rRNA transcription factors expression and observed that TAFIa was the only factor decreased. TAFIa might be a one of the limiting factor for rRNA synthesis in denervated muscle.

Fundamental study of detection of muscle hypertrophy-oriented gene doping by myostatin knock down using RNA interference.

To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. Key pointsUsing a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray.

Genome-wide analysis of expression modes and DNA methylation status at sense-antisense transcript loci in mouse.

The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite-strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.

Comparative expression analysis uncovers novel features of endogenous antisense transcription.

Increasing numbers of sense-antisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural sense-antisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-protein-coding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of sense-antisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (i) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of sense-antisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome.

Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes.

BACKGROUND: Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. RESULTS: First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions. CONCLUSION: Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.

Markers associated with neuron-specific Ube3a imprinting during neuronal differentiation of mouse embryonic stem cells.

Understanding gene expression in the brain requires allele-specific transcriptome analysis because of the presence of neuron-specific imprinted genes, which are expressed in a neuron-specific and parent-of-origin-specific manner. Ube3a is a neuron-specific imprinted gene with an expression pattern that changes from biallelic to maternal only (Ube3a imprinting) during differentiation. Because Ube3a imprinting occurs only in neurons, it has the potential to be a marker to assess the quality of neurons produced by in vitro neuronal differentiation of embryonic stem cells (ESCs). For the analysis of Ube3a imprinting, genetic polymorphisms between the two alleles are necessary to identify the parental origin of each. However, ESCs derived from commonly used inbred mouse strains have no genetic polymorphisms. To overcome this problem, we examined 10 markers of neurogenesis to determine whether they were associated with Ube3a imprinting. We measured the relative expression levels of these 10 gene markers and assessed the Ube3a imprinting status of 54 neuron samples differentiated under various in vitro conditions. Then we divided the samples into two groups depending on their Ube3a imprinting status and selected markers statistically associated with Ube3a imprinting. The identified markers included the antisense noncoding transcript of Ube3a and a mature neuron marker Mtap2, consistent with the markers we used empirically in our previous study to assess the quality of differentiated neurons. These findings provide new quality control criteria for differentiated neurons, and could also be applied to human ESCs and induced pluripotent stem cells.

Comparative analysis of cis-encoded antisense RNAs in eukaryotes.

Recent large-scale transcriptomic analyses have identified numerous endogenously encoded cis-antisense RNAs that are thought to play important roles in diverse cellular processes although comprehensive comparative studies among multiple species have yet to be performed. To investigate conserved genomic features across various species that may be related to sense-antisense regulation, we performed comparative analysis of approximately 1000-2000 cis-encoded antisense RNA pairs from five model eukaryotes (Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana, and Oryza sativa). Analysis of overlapping patterns relative to the exon-intron structure revealed that the number of pairs sharing the 3' part of the transcripts was larger than that of the 5'-sharing pairs except in rice. Moreover, most of the well-conserved sense-antisense pairs between human and mouse exhibited 3'-overlaps, suggesting that regulatory mechanisms involving these regions may be important in sense-antisense transcription. Functional classification using Gene Ontology revealed that genes related to catalytic activity, nucleotide binding, DNA metabolism, and mitochondria were preferentially distributed within the set of exon-overlapping sense-antisense genes compared to the non-exon-overlapping group in animals. Despite the numerous sense-antisense pairs identified in human and mouse individually, the number of conserved pairs was extremely small (6.6% of the entire set). Whereas both genes of most of the conserved sense-antisense pairs had protein-coding potential, nearly half of the non-conserved pairs included a non-coding RNA, suggesting that non-coding sense-antisense RNAs may function in species-specific regulatory pathways.

Comparison of two types of non-adherent plate for neuronal differentiation of mouse embryonic stem cells.

In vitro differentiation systems of mouse embryonic stem cells (ESCs) are widely used as tools for studies of cell differentiation, organogenesis, and regenerative medicine. We have studied the regulation of neuron-specific imprinting genes, Ube3a and its antisense transcripts (Ube3a ATS), using in vitro neuronal differentiation of F1 hybrid ESCs. Each different non-adherent plate used for embryoid body (EB) formation during differentiation is associated with different costs; notably, plates coated with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer are more expensive than untreated polystyrene plates. Here, we assessed whether the polymer-coated plates gave better results than the untreated plates. The first stage of differentation was performed in the MPC polymer-coated or untreated plates. The formed EBs were then passaged onto laminin-coated plates for further differentiation into neurons. Neither the neuron-specific imprinting status of Ube3a nor the expression levels of the neuron-specific markers Ube3a ATS and Mtap2 differed between neurons prepared on untreated plates and those prepared on MPC polymer-coated plates. These results suggest that the two non-adherent plates displayed almost the same characteristics for inducing neuronal differentiation of mouse ESCs and EB formation. Our study proved that untreated polystyrene plates are a cost-effective choice for EB formation in in vitro differentiation systems of mouse ESCs.

Sequence and structure analysis of a mirror tRNA located upstream of the cytochrome oxidase I mRNA in mouse mitochondria.

RNA fragments corresponding to the mirror tRNA that is located upstream of the cytochrome oxidase I (COXI) gene in the mouse mitochondrial genome were found in the sequences obtained from the mouse brain by the next generation sequencing. RNA fragments corresponding to the 5' terminal of COXI mRNA were also found and it was suggested that the precursor of the COXI mRNA is processed at three residues upstream of the first AUG codon. The mirror tRNA fragment has poly(A) in its 3' terminal and variable 5' terminal, suggesting that this RNA is produced during the 5' processing of COXI mRNA. Secondary structure prediction and NMR analysis indicated that the mirror tRNA is folded into a tRNA-like secondary structure, suggesting that the tRNA-like conformation of the 5' adjacent sequence of COXI mRNA is involved in the COXI mRNA maturation in the mouse mitochondria.

Genome-wide impact of endogenous antisense transcripts in eukaryotes.

Recent transcriptomic studies revealed that extensive proportions of genomes are transcribed, despite the limited fraction of protein-coding gene loci in the whole genome. Most transcripts are considered to be 'cryptic' output of the genome because of the lack of functional evidence; however, recent progress in molecular analyses has revealed that some of these transcripts at least have functional significance. This review article examines evidence of the functional significance of endogenous cis-antisense transcripts, which are the transcriptional output from the opposite strand of annotated genes. These transcripts are one of the most common types of transcripts that do not correspond to any protein-coding loci. Historical molecular studies revealed the existence of antisense transcripts associated with dozens of gene loci, whereas more recent genome-wide studies have shown that many genes have an antisense counterpart thus stimulating investigations into the functional significance of endogenous antisense transcripts. Here, we summarize the recent progress in the genome-wide characterization of the antisense transcriptome, and discuss the biological mechanisms that underlie the regulatory machinery of eukaryotic gene expression with respect to the potential roles of endogenous cis-antisense transcripts.