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Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end.

ABSTRACT: Environmental factors stimulate alteration of DNA methylation level. Investigation of the genome-wide DNA methylation status is important for environmental health studies. We here designed a genomic DNA amplification and labeling protocol using a methylation-sensitive restriction enzyme HinP1 I. This method can specifically amplify genomic DNA fragments possessing methyl-CpG at the end. The fragments are a relatively short size and dominantly located on CpG-islands. By using the samples prepared by this method, a dioxin-induced change in the methylation level of the mouse Cyp1a1 promoter was successfully evaluated using oligonucleotide probes covalently bound onto a glass plate. The method developed in this paper would be useful for other genome-wide analysis platforms for the large scale epigenome-wide association studies (EWAS) including human epidemiological samples.

SUBMITTER: Kurita H

PROVIDER: S-EPMC5761092 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Detection of dioxin-induced demethylation of mouse <i>Cyp1a1</i> gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end.

Kurita Hisaka H Aiba Toshiki T Saito Toshiyuki T Ohsako Seiichiroh S

Genes and environment : the official journal of the Japanese Environmental Mutagen Society 20180110

Environmental factors stimulate alteration of DNA methylation level. Investigation of the genome-wide DNA methylation status is important for environmental health studies. We here designed a genomic DNA amplification and labeling protocol using a methylation-sensitive restriction enzyme <i>HinP1</i> I. This method can specifically amplify genomic DNA fragments possessing methyl-CpG at the end. The fragments are a relatively short size and dominantly located on CpG-islands. By using the samples p ...[more]

PMID: 29339976

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Project description:The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). When activated by dioxin, the cytosolic AhR protein complex translocates into the nucleus and dimerizes with the ARNT (Ah receptor nuclear translocator) protein. The heteromeric ligand:AhR/Arnt complex then recognizes and binds to its specific DNA recognition site, the dioxin response element (DRE). DREs are located upstream of cytochrome P4501A1 (CYP1A1) and other AhR-responsive genes, and binding of the AhR complex stimulates their transcription. Although CYP1A1 expression has been used as the model system to define the biochemical and molecular mechanism of AhR action, there is still limited knowledge about the roles of each of the seven DREs located in the CYP1A1 promoter. These seven DREs are conserved in mouse, human and rat. Deletion analysis showed that a single DRE at -488 was enough to activate the transcription. Truncation analysis demonstrated that the DRE at site -981 has the highest transcriptional efficiency in response to TCDD. This result was verified by mutation analysis, suggesting that the conserved DRE at site -981 could represent a significant and universal AhR regulatory element for CYP1A1. The reversed substituted intolerant core sequence (5'-GCGTG-3' or 5'-CACGC-3') of seven DREs reduced the transcriptional efficiency, which illustrated that the adjacent sequences of DRE played a vital role in activating transcription. The core DRE sequence (5'-TNGCGTG-3') tends to show a higher transcriptional level than that of the core DRE sequence (5'-CACGCNA-3') triggered by TCDD. Furthermore, in the core DRE (5'-TNGCGTG-3') sequence, when "N" is thymine or cytosine (T or C), the transcription efficiency was stronger compared with that of the other nucleotides. The effects of DRE orientation, DRE adjacent sequences and the nucleotide "N" in the core DRE (5'-TNGCGTG-3') sequence on the AhR-regulated CYP1A1 transcription in response to TCDD were studied systematically, and our study laid a good foundation for further investigation into the AhR-dependent transcriptional regulation triggered by dioxin and dioxin-like compounds.

Project description:BackgroundThe aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose.ResultsWe performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKI?). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKI?, a CaMKI isoform previously reported to interplay with the AhR pathway.ConclusionsThese data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

Dataset Information

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end.

Publications

Detection of dioxin-induced demethylation of mouse <i>Cyp1a1</i> gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end.

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