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Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

ABSTRACT: 8-Oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair (BER) is the primary pathway to remove the pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Recent studies documented 8-oxoG serves as an epigenetic-like mark and OGG1 modulates gene expression in oxidatively stressed cells. For this new role of OGG1, two distinct mechanisms have been proposed: one is coupled to base excision, while the other only requires substrate binding of OGG1--both resulting in conformational adjustment in the adjacent DNA sequences providing access for transcription factors to their cis-elements. The present study aimed to examine if BER activity of OGG1 is required for pro-inflammatory gene expression. To this end, Ogg1/OGG1 knockout/depleted cells were transfected with constructs expressing wild-type (wt) and repair-deficient mutants of OGG1. OGG1's promoter enrichment, oxidative state, and gene expression were examined. Results showed that TNF? exposure increased levels of oxidatively modified cysteine(s) of wt OGG1 without impairing its association with promoter and facilitated gene expression. The excision deficient K249Q mutant was even a more potent activator of gene expression; whereas, mutant OGG1 with impaired substrate recognition/binding was not. These data suggested the interaction of OGG1 with its substrate at regulatory regions followed by conformational adjustment in the adjacent DNA is the primary mode to modulate inflammatory gene expression.

SUBMITTER: Hao W

PROVIDER: S-EPMC7318607 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

Hao Wenjing W Wang Jing J Zhang Yuanhang Y Wang Chenxin C Xia Lan L Zhang Wenhe W Zafar Muhammad M Kang Ju-Yong JY Wang Ruoxi R Ali Bohio Ameer A Pan Lang L Zeng Xianlu X Wei Min M Boldogh Istvan I Ba Xueqing X

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20200506 6

8-Oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair (BER) is the primary pathway to remove the pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Recent studies documented 8-oxoG serves as an epigenetic-like mark and OGG1 modulates gene expression in oxidatively stressed cells. For this new role of OGG1, two distinct mechanisms have been proposed: one is coupled to base excision, while the other only requires substrate binding of OGG1--both resulting in conformational adju ...[more]

PMID: 32378256

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Project description:Purpose: The aim of this study is to evaluate the global gene expression induced by OGG1-BER product 8-oxoG in mouse airways. Methods: RNA extracted from individual mouse lungs (experimental group: n=5) were pooled and a total 1 M-NM-<g RNA was used for Next-Generation Sequencing (NGS) analyses on an Illumina HiSeq 1000 sequencing system. Sequence analysis were performed in duplicate. First- and second-strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq Sample Preparation Kit as recommended by the manufacturer (Illumina). Library quality was evaluated by using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Library DNA templates were quantitated by qPCR using known reference starndards. Cluster formation of the library of DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation. Paired-end, 50-base sequencing was performed with a TruSeq SBS kit v3 (Illumina) on the Illumina HiSeq 1000 by protocols defined by the manufacturer. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Sequence data were analyzed with the Bowtie2, Tophat2 and GFOLD programs. Processed data are presented as reads per kilobase transcript per million (RPKM), normalized to the experimental control (RNA from saline-challenged lungs) and reported as fold change (test/control). Results: We mapped an average of 24.76 million sequence reads per sample and identified 23,337 transcripts in total RNA extracted from lungs of Balb/cJ mice as described in Methods. Approximately 10% of the transcripts showed differential expression between the saline-challenged control and 8-oxoguanine-challeged mouse lungs, with a fold change M-bM-^IM-%3.0. We validated the expression changes of 7 selected pro-inflammatory cytokines and chemokines of interest for our studies by qRT-PCR. Hierarchical clustering followed by Protein ANalysis THrough Evolutionary Relationships database (PANTHER) analysis of differentially expressed genes. Results showed overrepresentation of various biological functions (GO terms) including immune system process (GO:0002376; p=5.24e-12) among others. Pathway analysis (PANTHER) indicated that the most overrepresented pathway was inflammation mediated by chemokine and cytokine (P00031, p=<0.01). In addition to gene expression analysis, we confirmed OGG1M-bM-^@M-"8-oxoG-dependent RAS activation in lungs by active RAS pull-down assays, airways neutrophil accumulation by bronchoalveolar lavage fluid (BALF) differential cell counts and airway inflammation by histological examination (H&E staining) of lung sections. Conclusions: This is the first study at the whole-transcriptome level to show induction of innate immune response gene expression in mouse lungs after exposure to OGG1-BER product 8-oxoG. Balb/cJ mice (5 per group) were intranasally challenged with 8-oxoguanine (1 M-BM-5M, 60 M-BM-5l) for 30, 60 and 120 min. Control group mice were intranasally challenged with saline (60 M-BM-5l). RNA from individual mice whithin the same group was pooled and subjected to deep-sequencing analysis in duplicate using NGS on an Illumina HiSeq 1000 sequencing system. After alignment and processing, the resulting RPKM from treatment groups (8-oxoG-challenged) were normalized to the control group (saline-challenged).

Project description:Purpose: The aim of this study is to evaluate the global gene expression induced by OGG1-BER product 8-oxoG in mouse airways. Methods: RNA extracted from individual mouse lungs (experimental group: n=5) were pooled and a total 1 μg RNA was used for Next-Generation Sequencing (NGS) analyses on an Illumina HiSeq 1000 sequencing system. Sequence analysis were performed in duplicate. First- and second-strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq Sample Preparation Kit as recommended by the manufacturer (Illumina). Library quality was evaluated by using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Library DNA templates were quantitated by qPCR using known reference starndards. Cluster formation of the library of DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation. Paired-end, 50-base sequencing was performed with a TruSeq SBS kit v3 (Illumina) on the Illumina HiSeq 1000 by protocols defined by the manufacturer. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Sequence data were analyzed with the Bowtie2, Tophat2 and GFOLD programs. Processed data are presented as reads per kilobase transcript per million (RPKM), normalized to the experimental control (RNA from saline-challenged lungs) and reported as fold change (test/control). Results: We mapped an average of 24.76 million sequence reads per sample and identified 23,337 transcripts in total RNA extracted from lungs of Balb/cJ mice as described in Methods. Approximately 10% of the transcripts showed differential expression between the saline-challenged control and 8-oxoguanine-challeged mouse lungs, with a fold change ≥3.0. We validated the expression changes of 7 selected pro-inflammatory cytokines and chemokines of interest for our studies by qRT-PCR. Hierarchical clustering followed by Protein ANalysis THrough Evolutionary Relationships database (PANTHER) analysis of differentially expressed genes. Results showed overrepresentation of various biological functions (GO terms) including immune system process (GO:0002376; p=5.24e-12) among others. Pathway analysis (PANTHER) indicated that the most overrepresented pathway was inflammation mediated by chemokine and cytokine (P00031, p=<0.01). In addition to gene expression analysis, we confirmed OGG1•8-oxoG-dependent RAS activation in lungs by active RAS pull-down assays, airways neutrophil accumulation by bronchoalveolar lavage fluid (BALF) differential cell counts and airway inflammation by histological examination (H&E staining) of lung sections. Conclusions: This is the first study at the whole-transcriptome level to show induction of innate immune response gene expression in mouse lungs after exposure to OGG1-BER product 8-oxoG.

Dataset Information

Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

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Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

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