Massive transcriptional start site analysis of human genes in hypoxia cells.

Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible 'hot regions' in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.

Evaluation and application of RNA-Seq by MinION.

The current RNA-Seq method analyses fragments of mRNAs, from which it is occasionally difficult to reconstruct the entire transcript structure. Here, we performed and evaluated the recent procedure for full-length cDNA sequencing using the Nanopore sequencer MinION. We applied MinION RNA-Seq for various applications, which would not always be easy using the usual RNA-Seq by Illumina. First, we examined and found that even though the sequencing accuracy was still limited to 92.3%, practically useful RNA-Seq analysis is possible. Particularly, taking advantage of the long-read nature of MinION, we demonstrate the identification of splicing patterns and their combinations as a form of full-length cDNAs without losing precise information concerning their expression levels. Transcripts of fusion genes in cancer cells can also be identified and characterized. Furthermore, the full-length cDNA information can be used for phasing of the SNPs detected by WES on the transcripts, providing essential information to identify allele-specific transcriptional events. We constructed a catalogue of full-length cDNAs in seven major organs for two particular individuals and identified allele-specific transcription and splicing. Finally, we demonstrate that single-cell sequencing is also possible. RNA-Seq on the MinION platform should provide a novel approach that is complementary to the current RNA-Seq.

Intrinsic promoter activities of primary DNA sequences in the human genome.

In order to understand an overview of promoter activities intrinsic to primary DNA sequences in the human genome within a particular cell type, we carried out systematic quantitative luciferase assays of DNA fragments corresponding to putative promoters for 472 human genes which are expressed in HEK (human embryonic kidney epithelial) 293 cells. We observed the promoter activities of them were distributed in a bimodal manner; putative promoters belonging to the first group (with strong promoter activities) were designated as P1 and the latter (with weak promoter activities) as P2. The frequencies of the TATA-boxes, the CpG islands, and the overall G + C-contents were significantly different between these two populations, indicating there are two separate groups of promoters. Interestingly, similar analysis using 251 randomly isolated genomic DNA fragments showed that P2-type promoter occasionally occurs within the human genome. Furthermore, 35 DNA fragments corresponding to putative promoters of non-protein-coding transcripts (ncRNAs) shared similar features with the P2 in both promoter activities and sequence compositions. At least, a part of ncRNAs, which have been massively identified by full-length cDNA projects with no functional relevance inferred, may have originated from those sporadic promoter activities of primary DNA sequences inherent to the human genome.

Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer.

Here, we employed cDNA amplicon sequencing using a long-read portable sequencer, MinION, to characterize various types of mutations in cancer-related genes, namely, EGFR, KRAS, NRAS and NF1. For homozygous SNVs, the precision and recall rates were 87.5% and 91.3%, respectively. For previously reported hotspot mutations, the precision and recall rates reached 100%. The precise junctions of EML4-ALK, CCDC6-RET and five other gene fusions were also detected. Taking advantages of long-read sequencing, we conducted phasing of EGFR mutations and elucidated the mutational allelic backgrounds of anti-tumor drug-sensitive and resistant mutations, which could provide useful information for selecting therapeutic approaches. In the H1975 cells, 72% of the reads harbored both L858R and T790M mutations, and 22% of the reads harbored neither mutation. To ensure that the clinical requirements can be met in potentially low cancer cell populations, we further conducted a serial dilution analysis of the template for EGFR mutations. Several percent of the mutant alleles could be detected depending on the yield and quality of the sequencing data. Finally, we characterized the mutation genotypes in eight clinical samples. This method could be a convenient long-read sequencing-based analytical approach and thus may change the current approaches used for cancer genome sequencing.

The 5' terminal oligopyrimidine tract of human elongation factor 1A-1 gene functions as a transcriptional initiator and produces a variable number of Us at the transcriptional level.

The human elongation factor 1A-1 (eEF1A-1) gene is a member of the 5' terminal oligopyrimidine tract (5' TOP) gene family, and the number of thymidines (Ts) at the 5' TOP of cDNAs corresponding to this gene is known to show variation. Here we determined the 5'-end sequences of 125 eEF1A-1 clones and the complete sequences of 19 eEF1A-1 clones from an oligo-capped cDNA library and showed that variation in the number of Ts is generated by an in vivo process, not by an in vitro artifact during the construction of the cDNA library. Moreover, using green fluorescent protein transgenic mice, we demonstrated that the variation in T number is probably generated during or after transcription. We also introduced various mutations in the mRNA start site of this gene, particularly in the T stretch at the 5' TOP, and examined the effects on the promoter activity. The results showed that at least three Ts must exist at the 5' TOP for the high transcriptional activity of the eEF1A-1 gene promoter. Many other housekeeping genes, including ribosomal protein genes, are also members of the 5' TOP gene family, and the 5' TOP sequence may be an important core-promoter element of these genes.

Characterization of STAT6 target genes in human B cells and lung epithelial cells.

Using ChIP Seq, we identified 556 and 467 putative STAT6 target sites in the Burkitt's lymphoma cell line Ramos and in the normal lung epithelial cell line BEAS2B, respectively. We also examined the positions and expression of transcriptional start sites (TSSs) in these cells using our TSS Seq method. We observed that 44 and 132 genes in Ramos and BEAS2B, respectively, had STAT6 binding sites in proximal regions of their previously reported TSSs that were up-regulated at the transcriptional level. In addition, 406 and 109 of the STAT6 target sites in Ramos and BEAS2B, respectively, were located in proximal regions of previously uncharacterized TSSs. The target genes identified in Ramos and BEAS2B cells in this study and in Th2 cells in previous studies rarely overlapped and differed in their identity. Interestingly, ChIP Seq analyses of histone modifications and RNA polymerase II revealed that chromatin formed an active structure in regions surrounding the STAT6 binding sites; this event also frequently occurred in different cell types, although neither STAT6 binding nor TSS induction was observed. The rough landscape of STAT6-responsive sites was found to be shaped by chromatin structure, but distinct cellular responses were mainly mediated by distinct sets of transcription factors.

Distinct class of putative "non-conserved" promoters in humans: comparative studies of alternative promoters of human and mouse genes.

Although recent studies have revealed that the majority of human genes are subject to regulation of alternative promoters, the biological relevance of this phenomenon remains unclear. We have also demonstrated that roughly half of the human RefSeq genes examined contain putative alternative promoters (PAPs). Here we report large-scale comparative studies of PAPs between human and mouse counterpart genes. Detailed sequence comparison of the 17,245 putative promoter regions (PPRs) in 5463 PAP-containing human genes revealed that PPRs in only a minor fraction of genes (807 genes) showed clear evolutionary conservation as one or more pairs. Also, we found that there were substantial qualitative differences between conserved and non-conserved PPRs, with the latter class being AT-rich PPRs of relative minor usage, enriched in repetitive elements and sometimes producing transcripts that encode small or no proteins. Systematic luciferase assays of these PPRs revealed that both classes of PPRs did have promoter activity, but that their strength ranges were significantly different. Furthermore, we demonstrate that these characteristic features of the non-conserved PPRs are shared with the PPRs of previously discovered putative non-protein coding transcripts. Taken together, our data suggest that there are two distinct classes of promoters in humans, with the latter class of promoters emerging frequently during evolution.

Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

Large-scale collection and characterization of promoters of human and mouse genes.

We report the generation and initial characterization of a large-scale collection of sequences of putative promoter regions (PPRs) of human and mouse genes. Based on our unique collection of 400,225 and 580,209 human and mouse full-length cDNAs, we determined exact transcriptional start sites (TSSs). Using positional information of the TSSs, we could retrieve adjacent sequences as PPRs for 8,793 and 6,875 human and mouse genes, respectively. The positions of the PPRs were 4 kb upstream to previously reported 5'-ends of cDNAs on average, demonstrating that full-length cDNA information is indispensable for this purpose. Among those PPRs supported by experimentally validated TSSs, 3,324 could be paired as mutually homologous genes between human and mouse and were used for the comprehensive comparative studies. The sequence identities in the proximal regions of the TSSs were 45% on average, and 22,794 putative transcription factor binding sites that are conserved between human and mouse were identified. The data resource created in the present work and the results of the sequences' initial characterization should lay the firm foundation for deciphering the transcriptional modulations of human genes. All the data were deposited and made available through a database for comparative studies, DBTSS.