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

ABSTRACT: UNLABELLED:We identified 531 and 616 putative HIF-1? target sites by ChIP-Seq in the cancerous cell line DLD-1 and the non-cancerous cell line TIG-3, respectively. We also examined the positions and expression levels of transcriptional start sites (TSSs) in these cell lines using our TSS-Seq method. We observed that 121 and 48 genes in DLD-1 and TIG-3 cells, respectively, had HIF-1? binding sites in proximal regions of the previously reported TSSs that were up-regulated at the transcriptional level. In addition, 193 and 123 of the HIF-1? target sites, respectively, were located in proximal regions of previously uncharacterized TSSs, namely, TSSs of putative alternative promoters of protein-coding genes or promoters of putative non-protein-coding transcripts. The hypoxic response of DLD-1 cells was more significant than that of TIG-3 cells with respect to both the number of target sites and the degree of induced changes in transcript expression. The Nucleosome-Seq and ChIP-Seq analyses of histone modifications revealed that the chromatin formed an open structure in regions surrounding the HIF-1? binding sites, but this event occurred prior to the actual binding of HIF-1?. Different cellular histories may be encoded by chromatin structures and determine the activation of specific genes in response to hypoxic shock. ELECTRONIC SUPPLEMENTARY MATERIAL:The online version of this article (doi:10.1007/s11568-011-9150-9) contains supplementary material, which is available to authorized users.

SUBMITTER: Tanimoto K

PROVIDER: S-EPMC3051044 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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

Tanimoto Kousuke K Tsuchihara Katsuya K Kanai Akinori A Arauchi Takako T Esumi Hiroyasu H Suzuki Yutaka Y Sugano Sumio S

The HUGO journal 20101201 1-4

<h4>Unlabelled</h4>We identified 531 and 616 putative HIF-1α target sites by ChIP-Seq in the cancerous cell line DLD-1 and the non-cancerous cell line TIG-3, respectively. We also examined the positions and expression levels of transcriptional start sites (TSSs) in these cell lines using our TSS-Seq method. We observed that 121 and 48 genes in DLD-1 and TIG-3 cells, respectively, had HIF-1α binding sites in proximal regions of the previously reported TSSs that were up-regulated at the transcript ...[more]

PMID: 22132063

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Project description:The adaptive immune system generates a remarkable range of antigen-specific T-cell receptors (TCRs), allowing the recognition of a diverse set of antigens. Most of this diversity is encoded in the complementarity determining region 3 (CDR3) of the ? chain of the ?? TCR, which is generated by somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) gene segments. Deletion and non-templated insertion of nucleotides at the D-J and V-DJ junctions further increases diversity. Many of these gene segments are annotated as non-functional owing to defects in their primary sequence, the absence of motifs necessary for rearrangement, or chromosomal locations outside the TCR locus.We sought to utilize a novel method, based on high-throughput sequencing of rearranged TCR genes in a large cohort of individuals, to evaluate the use of functional and non-functional alleles. We amplified and sequenced genomic DNA from the peripheral blood of 587 healthy volunteers using a multiplexed polymerase chain reaction assay that targets the variable region of the rearranged TCR? locus, and we determined the presence and the proportion of productive rearrangements for each TCR? V gene segment in each individual. We then used this information to annotate the functional status of TCR? V gene segments in this cohort.For most TCR? V gene segments, our method agrees with previously reported functional annotations. However, we identified novel non-functional alleles for several gene segments, some of which were used exclusively in our cohort to the detriment of reported functional alleles. We also saw that some gene segments reported to have both functional and non-functional alleles consistently behaved in our cohort as either functional or non-functional, suggesting that some reported alleles were not present in the population studied.In this proof-of-principle study, we used high-throughput sequencing of the TCR? locus of a large cohort of healthy volunteers to evaluate the use of functional and non-functional alleles of individual TCR? V gene segments. With some modifications, our method has the potential to be extended to gene segments in the ?, ?, and ? TCR loci, as well as the genes encoding for B-cell receptor chains.

Project description:Hypoxia Inducible Factor (HIF) regulates a cascade of transcriptional events in response to decreased oxygenation, acting from the cellular to the physiological level. This response is evolutionarily conserved, allowing the use of zebrafish (Danio rerio) as a model for studying the hypoxic response. Activation of the hypoxic response can be achieved in zebrafish by homozygous null mutation of the von Hippel-Lindau (vhl) tumour suppressor gene. Previous work from our lab has focused on the phenotypic characterisation of this mutant, establishing the links between vhl mutation, the hypoxic response and cancer. To further develop fish as a model for studying hypoxic signalling, we examine the transcriptional profile of the vhl mutant with respect to Hif-1?. As our approach uses embryos consisting of many cell types, it has the potential to uncover additional HIF regulated genes that have escaped detection in analogous mammalian cell culture studies.We performed high-density oligonucleotide microarray analysis of the gene expression changes in von Hippel-Lindau mutant zebrafish, which identified up-regulation of well-known hypoxia response genes and down-regulation of genes primarily involved in lipid processing. To identify the dependency of these transcriptional changes on HIF, we undertook Chromatin Immunoprecipitation linked next generation sequencing (ChIP-seq) for the transcription factor Hypoxia Inducible Factor 1? (HIF-1?). We identified HIF-1? binding sites across the genome, with binding sites showing enrichment for an RCGTG motif, showing conservation with the mammalian hypoxia response element.Transcriptome analysis of vhl mutant embryos detected activation of key hypoxia response genes seen in human cell models of hypoxia, but also suppression of many genes primarily involved in lipid processing. ChIP-seq analysis of Hif-1? binding sites unveiled an unprecedented number of loci, with a high proportion containing a canonical hypoxia response element. Whether these sites are functional remains unknown, nevertheless their frequent location near transcriptional start sites suggests functionality, and will allow for investigation into the potential hypoxic regulation of genes in their vicinity. We expect that our data will be an excellent starting point for analysis of both fish and mammalian gene regulation by HIF.

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

Publications

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

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