Masahide Seki

Low conservation and species-specific evolution of alternative splicing in humans and mice: comparative genomics analysis using well-annotated full-length cDNAs

Using full-length cDNA sequences, we compared alternative splicing (AS) in humans and mice. The alignment of the human and mouse genomes showed that 86% of 199 426 total exons in human AS variants were conserved in the mouse genome. Of the 20 392 total human AS variants, however, 59% consisted of all conserved exons. Comparing AS patterns between human and mouse transcripts revealed that only 431 transcripts from 189 loci were perfectly conserved AS variants. To exclude the possibility that the full-length human cDNAs used in the present study, especially those with retained introns, were cloning artefacts or prematurely spliced transcripts, we experimentally validated 34 such cases. Our results indicate that even retained-intron type transcripts are typically expressed in a highly controlled manner and interact with translating ribosomes. We found non-conserved AS exons to be predominantly outside the coding sequences (CDSs). This suggests that non-conserved exons in the CDSs of transcripts cause functional constraint. These findings should enhance our understanding of the relationship between AS and species specificity of human genes.

Long-term expansion of alveolar stem cells derived from human iPS cells in organoids

The stable expansion of tissue-specific stem cells in vitro has contributed to research on several organs. Alveolar epithelial type II (AT2) cells function as tissue stem cells in the lung, but robust models for studying human AT2 cells are lacking. Here we report a method for the efficient generation and long-term expansion of alveolar organoids (AOs) harboring SFTPC+ alveolar stem cells derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived SFTPC+ cells self-renewed, with transcriptomes and morphology consistent with those of AT2 cells, and were able to differentiate into alveolar epithelial type I (AT1)-like cells. Single-cell RNA-seq of SFTPC+ cells and their progenitors demonstrated that their differentiation process and cellular heterogeneity resembled those of developing AT2 cells in vivo. AOs were applicable to drug toxicology studies recapitulating AT2-cell-specific phenotypes. Our methods can help scientists overcome the limitations of current approaches to the modeling of human alveoli and should be useful for disease modeling and regenerative medicine.

A Comparison of the Rest Complex Binding Patterns in Embryonic Stem Cells and Epiblast Stem Cells

We detected and characterized the binding sites of the representative Rest complex components Rest, Sin3A, and Lsd1. We compared their binding patterns in mouse embryonic stem (ES) cells and epiblast stem (EpiS) cells. We found few Rest sites unique to the EpiS cells. The ES-unique site features were distinct from those of the common sites, namely, the signal intensities were weaker, and the characteristic gene function categories differed. Our analyses showed that the Rest binding sites do not always overlap with the Sin3A and Lsd1 binding sites. The Sin3A binding pattern differed remarkably between the ES and EpiS cells and was accompanied by significant changes in acetylated-histone patterns in the surrounding regions. A series of transcriptome analyses in the same cell types unexpectedly showed that the putative target gene transcript levels were not dramatically different despite dynamic changes in the Rest complex binding patterns and chromatin statuses, which suggests that Rest is not the sole determinant of repression at its targets. Nevertheless, we identified putative Rest targets with explicitly enhanced transcription upon Rest knock-down in 143 and 60 common and ES-unique Rest target genes, respectively. Among such sites, several genes are involved in ES cell proliferation. In addition, we also found that long, intergenic non-coding RNAs were apparent Rest targets and shared similar features with the protein-coding target genes. Interestingly, such non-coding target genes showed less conservation through evolution than protein-coding targets. As a result of differences in the components and targets of the Rest complex, its functional roles may differ in ES and EpiS cells.

Predicting promoter activities of primary human DNA sequences

We developed a computer program that can predict the intrinsic promoter activities of primary human DNA sequences. We observed promoter activity using a quantitative luciferase assay and generated a prediction model using multiple linear regression. Our program achieved a prediction accuracy correlation coefficient of 0.87 between the predicted and observed promoter activities. We evaluated the prediction accuracy of the program using massive sequencing analysis of transcriptional start sites in vivo. We found that it is still difficult to predict transcript levels in a strictly quantitative manner in vivo; however, it was possible to select active promoters in a given cell from the other silent promoters. Using this program, we analyzed the transcriptional landscape of the entire human genome. We demonstrate that many human genomic regions have potential promoter activity, and the expression of some previously uncharacterized putatively non-protein-coding transcripts can be explained by our prediction model. Furthermore, we found that nucleosomes occasionally formed open chromatin structures with RNA polymerase II recruitment where the program predicted significant promoter activities, although no transcripts were observed.

Repeated inversions within a pannier intron drive diversification of intraspecific colour patterns of ladybird beetles

How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis. Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata, suggesting that H. axyridis’ extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles.The harlequin ladybird beetle, Harmonia axyridis, has remarkable phenotypic diversity, with over 200 colour patterns. Here, Ando et al. show that this patterning is regulated by the transcription factor gene pannier and has diversified by repeated inversions and cis-regulatory modifications of pannier.

Fluorescence-Activated Cell Sorting and Gene Expression Profiling of GFP-Positive Cells from Transgenic Zebrafish Lines

Gene expression profiling is a useful approach for deeper understanding of the specificity of cells, tissues, and organs in the transcriptional level. Recent development of high-throughput next-generation sequence (NGS) allows the RNA-seq method for this profiling. This method provides precise information of transcripts about the quantitation and the structure such as the splicing variants. In this chapter, we describe a method for gene expression profiling of GFP-positive cells from transgenic zebrafish by RNA-seq. We labeled specific cells in the brain with GFP by crossing a Gal4 driver line with the UAS:GFP line, isolated those cells by fluorescence-activated cell sorting (FACS), and analyzed by RNA-seq.

Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines

The functional relevancy of mutations occurring in the regulatory regions in cancers remains mostly elusive. Here, we identified and analyzed regulatory mutations having transcriptional consequences in lung adenocarcinoma-derived cell lines. We phased the mutations in the regulatory regions to the downstream heterozygous SNPs in the coding regions and examined whether the ChIP-Seq variant tags of the regulatory SNVs and the RNA-Seq variant tags of their target transcripts showed biased frequency between the mutant and reference alleles. We identified 137 potential regulatory mutations affecting the transcriptional regulation of 146 RefSeq transcripts with at least 84 SNVs that create and/or disrupt potential transcription factor binding sites. For example, in the regulatory region of NFATC1 gene, a novel and active binding site for the ETS transcription factor family was created. Further examination revealed that 31 of these disruptions were presented in clinical lung adenocarcinoma samples and were associated with prognosis of patients.

Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish

We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm), were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells

The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding hXIST exon 4 was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers KLF4, KLF5, OCT3/4, SOX2, NANOG, UTF1, and GRB7 were lower, while that of hXIST exon 4, LEFTY1, and LEFTY2 was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation.