Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of Novel NRF2-Regulated Genes by ChIPSeq

ABSTRACT: Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to DNA-regulatory sequences near stress responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted ChIP-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high-confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRAhas implications for response to retinoid treatments and adipogenesis. In mouse 3T3-L1 cells SFN treatment affected Rxra expression early in adipogenesis and knockdown of Nrf2 delayed Rxra expression, both leading to impaired adipogenesis. ChIP-Seq analysis of NRF2 binding sites in human lymphoblastoid cells treated with sulforaphane or vehicle

ORGANISM(S): Homo sapiens

SUBMITTER: Xuting Wang

PROVIDER: E-GEOD-37589 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha.

Chorley Brian N BN Campbell Michelle R MR Wang Xuting X Karaca Mehmet M Sambandan Deepa D Bangura Fatu F Xue Peng P Pi Jingbo J Kleeberger Steven R SR Bell Douglas A DA

Nucleic acids research 20120511 15

Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanat ...[more]

PMID: 22581777

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Project description:Introduction: The ascending prevalence of obesity in recent decades is commonly associated with soaring rates of morbidity and mortality resulting in increased health care costs and decreased quality of life. A systemic state of stress characterized by low grade inflammation and pathologic formation of reactive oxygen species (ROS) usually manifest in obesity. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) is the master regulator of the redox homeostasis and plays a critical role in the resolution of inflammation. Our results indicate that the natural isothiocyanate and potent NRF2 activator sulforaphane reverses the diet-induced obesity in rodents and implicate involvement of leptin receptor singling in this effect. Purpose: Here, we investigate molecular pathways underlying the effects of sulforaphane in reversing diet-induced obesity and interrogate potential pathways that link NRF2 with leptin receptor signaling by conducting whole transcriptome analysis in 6 metabolically active tissues from the diet-induced obese mice that were treated with SFN. Methods: Male 12-week-old C57/BJ wild type mice were fed with high fat diet for 16-20 weeks. Around 28 weeks of age, mice were treated with daily ip injections of either SFN (5 mg/kg) or vehicle for one week. We probed the whole transcriptome in several metabolic tissues including the hypothalamus, liver, brown adipose tissue (BAT), epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and skeletal muscle following peripheral SFN administration. Total RNA samples from each tissue were prepared with Trizol. Whole transcriptome were conducted by using Novogene sequencing platform NovaSeq 6000 PE150. The following bioinformatics analysis have been conducted. 1. Data Quality Control: filtering reads containing adapter or with low quality. 2. Statistics Analysis of Data Production and Quality. 3. Mapping Reads to Reference Genome. 4. Gene Expression Quantification. 5. Correlation analysis (For biological replicates only). 6. Differential Expression Analysis (two or more groups of samples). 7. GO Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples). 8. KEGG Pathway Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples). 9. GSEA Enrichment Analysis of Expressed Genes (two or more groups of samples). 10. Protein Protein Interaction Analysis. 11. Reactome Pathway Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples and only for mouse samples). 12. Oncogene Functional Annotation analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples and only for mouse sample). Results: RNAseq results indicated a significant upregulation of the NRF2 target gene HMOX1 only in the skeletal muscle. We also conducted an enrichment analysis to identify potential transcription factors for the significantly upregulated genes. NRF2 was identified as the most significant transcription factor only for the skeletal muscle gene set, providing further support for the skeletal muscle being a primary target tissue of SFN action. We thus conducted a KEGG and GO pathway analysis, and identified ribosome biogenesis, proteasome pathway, and the RNA transport as the significantly upregulated pathways in the SFN-treated muscle samples. In addition to HMOX1, the RNA seq results indicate upregulation of other genes involved in oxidative stress response capacity in SFN treated mice in the skeletal muscle. GCLM (glutamate-cysteine ligase), the first rate-limiting enzyme of glutathione synthesis, SRXN1 (Sulfiredoxin), an oxidoreductase that reduces cysteine-sulfinate, and GSR (glutathione-disulfide reductase), a central antioxidant enzyme, which reduces oxidized glutathione disulfide (GSSG) to the sulfhydryl form glutathione (GSH), and TXNRD1 (thioredoxin reductase 1) which reduces thioredoxins are all upregulated in the skeletal muscle of the SFN-treated DIO animals. Conclusions: Here, we show that the natural isothiocyanate and potent NRF2 activator sulforaphane reverses diet-induced obesity through an NRF2-dependent mechanism that requires a functional leptin receptor signaling and hyperleptinemia. Transcriptional profiling of six major metabolically relevant tissues highlights that sulforaphane suppresses fatty acid synthesis while promoting ribosome biogenesis, reducing ROS accumulation and resolving inflammation, therefore representing a unique transcriptional program that leads to protection from obesity. Our findings argue for clinical evaluation of sulforaphane for weight loss and obesity-associated metabolic disorders.

Dataset Information

Identification of Novel NRF2-Regulated Genes by ChIPSeq

Publications

Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha.

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