CAGE: cap analysis of gene expression

Kodzius, Rimantas; Kojima, Miki; Nishiyori, Hiromi; Nakamura, Mari; Fukuda, Shiro; Tagami, Michihira; Sasaki, Daisuke; Imamura, Kengo; Cui, Kai; Harbers, Matthias; Hayashizaki, Yoshihide; Carninci, Piero

doi:10.1038/nmeth0306-211

Cited by 393 publications

(306 citation statements)

References 16 publications

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“…CAGE libraries were prepared as previously described [21,65] using RNAs isolated from the nuclear fraction of the untreated/control (at 0 hour), 500 nM JQ1- and DMSO-treated H23 cells (at 3, 6, 12, and 24 hours). All of these treatment conditions have three biological replicates.…”

Section: Methodsmentioning

confidence: 99%

“…Since stability of RNA sample affects the promoter hit rate and transcript complexity of a CAGE library, the RNA integrity of each sample was measured using an Agilent Bioanalyzer and all samples used in this study had RIN above 9.0. At least 3 μg RNA was used for CAGE library preparation [65]. Each CAGE library was sequenced at least 15 million reads.…”

Section: Methodsmentioning

confidence: 99%

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JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Handoko¹,

Kaczkowski²,

Hon³

et al. 2018

Epigenetics

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The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Handoko¹,

Kaczkowski²,

Hon³

et al. 2018

Epigenetics

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show abstract

“…Introns were also assessed in terms of intronic transcription, using the available data from the CAGE experiments within the ENCODE project 14,29,30 (files analysed are described in Supplementary Table 10). We focused on CAGE data collected using the 'normal' lymphoblastoid cell line GM12878: long poly-Anegative RNA from both genomic strands and from the nucleic and cytosolic fractions.…”

Section: Intron Length Repetitive Elements and Cage Data Analysismentioning

confidence: 99%

“…To assess cadherin intronic transcription, we mined the data from the pilot stage of the ENCODE project, 14 in particular data obtained by 5¢ cap analysis gene expression (CAGE) performed by Carninci and group 29 at the RIKEN Institute. This technique allowed for the detection of new TSS, and we focused on the data obtained using RNA extracted from cytosolic and nucleic fractions of the lymphoblastic human normal cell line GM12878 to assess transcription.…”

Section: Human Cadherin Introns Exhibit Significantly Increased Mir Andmentioning

confidence: 99%

Characterization of the intronic portion of cadherin superfamily members, common cancer orchestrators

et al. 2012

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Cadherins are cell-cell adhesion proteins essential for the maintenance of tissue architecture and integrity, and their impairment is often associated with human cancer. Knowledge regarding regulatory mechanisms associated with cadherin misexpression in cancer is scarce. Specific features of the intronic-structure and intronic-based regulatory mechanisms in the cadherin superfamily are unidentified. This study aims at systematically characterizing the intronic portion of cadherin superfamily members and the identification of intronic regions constituting putative targets/triggers of regulation, using a bioinformatic approach and biological data mining. Our study demonstrates that the cadherin superfamily genes harbour specific characteristics in comparison to all non-cadherin genes, both from the genomic and transcriptional standpoints. Cadherin superfamily genes display higher average total intron number and significantly longer introns than other genes and across the entire vertebrate lineage. Moreover, in the human genome, we observed an uncommon high frequency of MIR (mammalian-wide interspersed repeats) and MaLR (mammalian-wide interspersed repeats, a subtype of LTR) regulatory-associated repetitive elements at 5¢-located introns, concomitantly with increased de novo intronic transcription. Using this approach, we identified cadherin intronic-specific sites that may constitute novel targets/triggers of cadherin superfamily expression regulation. These findings pinpoint the need to identify mechanisms affecting particularly MIR and MaLR elements located in introns 2 and 3 of human cadherin genes, possibly important in the expression modulation of this superfamily in homeostasis and cancer.

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“…These modifications have also been successfully adopted in a human transcriptome analysis project in which over 30 million tags are sequenced from over 250 tissues 14 . Recently, the SAGE method was also modified to recover transcript information at 5¢ ends of mRNA [15][16][17] . MPSS was also developed for in-depth transcriptome analysis using a novel hybridization-based sequencing method 17 .…”

Section: Introductionmentioning

confidence: 99%

Robust analysis of 5′-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites

Gowda

Wang

2007

Nat Protoc

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The structure and diversity at the 3¢ ends of mRNA transcripts have been extensively characterized using several tag-based techniques in eukaryotes. However, the 5¢ ends of mRNA transcripts are not well understood, owing to a lack of efficient experimental approaches. We developed a new gene expression profiling method, called robust analysis of 5¢-transcript ends (5¢ RATE), to rapidly isolate the 5¢ ends of mRNA transcripts. After ligating RNA oligo linkers to the 5¢ regions of decapped mRNA, cDNA is synthesized and digested with the restriction enzyme NlaIII. Ditags are formed by ligating two individual NlaIII tags, and are then PCR-amplified, purified and sequenced using a pyrosequencing approach. The 5¢-RATE procedure is simple, fast and cost-effective because the complicated steps in comparative methods such as serial analysis of gene expression (including the formation of concatemers and their subsequent cloning and sequencing) have been eliminated. The longer 5¢-RATE tags (480 bp) provide more accurate matching to reference sequences for gene annotation and allow in-depth analysis of sequence diversity at the 5¢ regions of mRNA transcripts. Using our procedure, a 5¢-RATE library with about 180,000 end sequences can be generated within a week. We have successfully applied the 5¢-RATE method to characterize the transcriptome of various plant species including maize, rice and soybean. This method can be easily adapted to other eukaryotic organisms using the detailed procedures described in this protocol.

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CAGE: cap analysis of gene expression

Cited by 393 publications

References 16 publications

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Characterization of the intronic portion of cadherin superfamily members, common cancer orchestrators

Robust analysis of 5′-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites

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