Identification of Novel NRF2-Regulated Genes by ChIPSeq
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE37589 | GEO | 2012/06/18
SECONDARY ACCESSION(S): PRJNA161889
REPOSITORIES: GEO
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Project description: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
2012-06-18 | E-GEOD-37589 | biostudies-arrayexpress
Project description: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 human BEAS-2B cell line treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates.
2018-06-19 | GSE75812 | GEO
Project description:Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study transcriptomic and proteomic changes in the estrogen receptor negative, non tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R=0.679, P<0.05) and KEAP1 knockdown (R=0.853, P<0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and proteomic level. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast. MCF10A cells were treated with SFN or had KEAP1 knocked down by siRNA.
2011-04-22 | E-GEOD-28813 | biostudies-arrayexpress
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.
2022-01-22 | GSE181477 | GEO
Project description:We identified a SNP rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple ChIP-seq experiments, and its strong-binding allele increased transactivation, and accorded higher mRNA levels in cell lines and human brain. To confirm the allele-specific binding, we conducted ChIP tagmentaion sequencing experiment in human lymphoblastoid cell line GM12763 which is heterozygous for rs242561. NRF2 ChIP DNA was isolated from GM12763 cells treated with Sulforaphane (SFN) in triplicates. The region containing rs242561 was amplified using primers, Fwd 5â??-AGCCTTCCCTGTCCTTGATT-3â??, Rev 5â??-GGACCGAGCTTCCAGTCTAA-3â??, and tagmentated using Tn5-based transposition for library construction. Libraries were sequenced on Illumina MiSeq. NRF2 ChIP tagmentation sequencing of a 241bp intron region in MAPT gene using GM12763 treated with sulforaphane
2016-04-14 | E-GEOD-79569 | biostudies-arrayexpress
Project description:Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study transcriptomic and proteomic changes in the estrogen receptor negative, non tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R=0.679, P<0.05) and KEAP1 knockdown (R=0.853, P<0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and proteomic level. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.
2011-04-22 | GSE28813 | GEO
Project description:Sulforaphane (SFN), an isothiocyanate, is part of an important group of naturally occurring small molecules with antiinflammatory properties. Even though the published reports are vague, most are best conceivable with an inhibition of T cell functions. We therefore analyzed the effect of SFN on T cell-mediated autoimmune disease. Feeding mice with SFN protected from severe experimental autoimmune encephalomyelitis (EAE). Disease amelioration was associated with reduced interleukin (IL)-17 and IFN-gamma expression in draining lymph nodes. In vitro, SFN treatment of T cells did not directly alter T cell cytokine secretion. In contrast, SFN treatment of dendritic cells (DC) inhibited TLR4-induced IL-12 and IL-23 production and the cytokine profile of T cells stimulated by SFN-treated DC. SFN suppressed TLR4-induced nuclear factor kappa B (NFκB) activity, without affecting the degradation of its inhibitor (IκB). Instead, SFN treatment of DC resulted in strong expression of the stress response protein heme oxygenase-1 (HO-1), which interacts with NFκB p65 and inhibits its activity. Consistent with these findings, HO-1 bound to p65 and subsequently inhibited the p65 promoter activity within the IL23a and IL12b promoter region. Importantly, SFN suppressed Il23a and Il12b expression in vivo and silenced Th17/Th1 responses within the CNS . Our data show that SFN improves Th17/Th1-mediated autoimmune disease by inducing HO-1 and inhibiting p65-regulated IL-23 and IL-12 expression. Treatment-control experiment with two replicates per condition
2014-02-13 | E-GEOD-54980 | biostudies-arrayexpress
Project description:Sulforaphane (SFN), an isothiocyanate, is part of an important group of naturally occurring small molecules with antiinflammatory properties. Even though the published reports are vague, most are best conceivable with an inhibition of T cell functions. We therefore analyzed the effect of SFN on T cell-mediated autoimmune disease. Feeding mice with SFN protected from severe experimental autoimmune encephalomyelitis (EAE). Disease amelioration was associated with reduced interleukin (IL)-17 and IFN-gamma expression in draining lymph nodes. In vitro, SFN treatment of T cells did not directly alter T cell cytokine secretion. In contrast, SFN treatment of dendritic cells (DC) inhibited TLR4-induced IL-12 and IL-23 production and the cytokine profile of T cells stimulated by SFN-treated DC. SFN suppressed TLR4-induced nuclear factor kappa B (NFκB) activity, without affecting the degradation of its inhibitor (IκB). Instead, SFN treatment of DC resulted in strong expression of the stress response protein heme oxygenase-1 (HO-1), which interacts with NFκB p65 and inhibits its activity. Consistent with these findings, HO-1 bound to p65 and subsequently inhibited the p65 promoter activity within the IL23a and IL12b promoter region. Importantly, SFN suppressed Il23a and Il12b expression in vivo and silenced Th17/Th1 responses within the CNS . Our data show that SFN improves Th17/Th1-mediated autoimmune disease by inducing HO-1 and inhibiting p65-regulated IL-23 and IL-12 expression.
2014-02-13 | GSE54980 | GEO
Project description:Protective roles of Nrf2, a key transcription factor for antioxidant and defense genes, have been determined in oxidative lung injury, and health benefits of Nrf2 agonists including sulforaphane have been demonstrated. The current study was designed to investigate the effect of sulforaphane on model acute lung injury and sulforaphane-mediated transcriptome changes in mouse lungs. Adult mice genetically deficient in Nrf2 (Nrf2-/-) and wild-type controls (Nrf2+/+, ICR) received oral sulforaphane (9 mmol/daily) or vehicle before (-5, -3, -1 days) hyperoxia or air exposure (3 days), and lung injury and gene expression changes were assessed. Sulforaphane significantly reduced hyperoxia-induced airway injury, inflammation, and mucus hypersecretion in Nrf2+/+ mice while relatively marginal treatment effect was found in Nrf2-/- mice. Sulforaphane significantly altered expression of lung genes associated with oxidative phosphorylation and mitochondrial dysfunction (Atp2a2, Cox7a1, Ndufa1) basally and cell function/cycle and protein metabolism (Actr1a, Wasf2, Ccne1, Gtpbp4) after hyperoxia in Nrf2+/+ mice. Nrf2-dependently modulated lung genes by sulforaphane and hyperoxia were associated with tissue development and hereditary disorders (Slc25a3, Pccb, Psmc3ip). Results demonstrate preventive roles of sulforaphane against oxidant lung injury in mice, and reveal potential downstream mechanisms. Our observations also suggest Nrf2-independent mechanisms of sulforaphane in prevention of acute lung injury.
2015-06-01 | GSE58654 | GEO
Project description:H460 cells treated with vehicle or sulforaphane (SFN) for 48 hours were used to acquire expression profiles of a total of 1891 unique miRNAs. We aimed at identifying the differentially expressed miRNAs between cells treated or untreated with SFN.
2018-05-05 | GSE81143 | GEO