Massive transcriptional start site analysis of human genes in hypoxia cells

Abstract

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.

Genome-wide identification and annotation of HIF-1α binding sites in two cell lines using massively parallel sequencing

“…Putative transcription factor binding sites that were statistically significantly enriched (P \ 0.05) in the respective groups were selected. For details, see Supplementary Table 3. TSS-Seq analysis and RNA-Seq analysis of polysome fractions TSS-Seq tags were prepared and analyzed as described in the reference (Tsuchihara et al 2009). After clustering the TSS-tags using 500-bp bins, representative TSSs were selected as the position from which the largest number of TSS tags was mapped.…”

“…This dataset contains a total of 141,590,200 TSS tags, each of which corresponds to the cap site of a single mRNA (Tsuchihara et al 2009). We clustered the TSS tags into TSS clusters (TSCs) to separate individual putative promoter units (see the reference (Tsuchihara et al 2009) for details).…”

“…By utilizing Illumina GA and ChIP technologies (ChIP-Seq), the binding sites of several transcription factors have been reported (Valouev et al 2008;Robertson et al 2007;Kwon et al 2009;Welboren et al 2009;Park 2009). On the other hand, we also developed a method for sequencing the 5 0 -ends of mRNAs by combining our oligo-capping technique with the Illumina GA sequencer (Tsuchihara et al 2009). This method, which we named TSS-Seq, enables us to obtain precise information regarding the positions of the transcriptional start sites (TSSs) together with their expression levels in a high-throughput manner.…”

“…This method, which we named TSS-Seq, enables us to obtain precise information regarding the positions of the transcriptional start sites (TSSs) together with their expression levels in a high-throughput manner. In our previous report, we applied TSS-seq to observe gene expression changes in response to hypoxia in several cell lines (Tsuchihara et al 2009). …”

RNA-Seq and human complex diseases: recent accomplishments and future perspectives

“…However, it does not allow -unlike CAGE -to get the exact positions of all TSS for a given gene, even though an innovative approach based on a combination of NGS and Oligo-capping (TSS-tag sequencing) has been recently developed to overcome this limitation. 14 Nonetheless, RNA-Seq provides more information than SAGE and CAGE in terms of splicing, post-transcriptional RNA editing and SNPs expression across the entire length of (virtually) all expressed transcripts in a cell. Indeed, it allows to analyze at a single-nucleotide resolution, the allele-specific expression and the post-transcriptional RNA editing, to examine known splice junctions-or to discover novel splicing events and to detect fusion transcripts, crucial especially in cancer research.…”