Carcinogenic roles of MAFG-AS1 in human cancers

The MAF bZIP transcription factor G-antisense RNA 1 (MAFG-AS1) is located on chromosome 17. MAFG-AS1 was upregulated in 15 human cancers. MAFG-AS1 not only suppresses 16 miRNAs but also directly impacts 22 protein-coding genes' expression. Notably, abnormal MAFG-AS1 expression is connected to clinicopathological characteristics and a worse prognosis in a variety of cancers. Moreover, MAFG-AS1 takes its part in the tumorigenesis and progression of various human malignancies by suppressing apoptosis and promoting proliferation, migration, invasion, aerobic glycolysis, ferroptosis, angiogenesis, EMT, and metastasis. Besides, it can predict treatment effectiveness in ER + breast cancer, urothelial bladder carcinoma, and liver cancer by functioning as a trigger of resistance to tamoxifen, sorafenib, and cisplatin. This study systematically presents the functions of MAFG-AS1 in various cancers, as well as the findings of bioinformatics analyses of the MAFG-AS1, which should give clear advice for future research (AU)

Carcinogenic roles of MAFG-AS1 in human cancers.

The MAF bZIP transcription factor G-antisense RNA 1 (MAFG-AS1) is located on chromosome 17. MAFG-AS1 was upregulated in 15 human cancers. MAFG-AS1 not only suppresses 16 miRNAs but also directly impacts 22 protein-coding genes' expression. Notably, abnormal MAFG-AS1 expression is connected to clinicopathological characteristics and a worse prognosis in a variety of cancers. Moreover, MAFG-AS1 takes its part in the tumorigenesis and progression of various human malignancies by suppressing apoptosis and promoting proliferation, migration, invasion, aerobic glycolysis, ferroptosis, angiogenesis, EMT, and metastasis. Besides, it can predict treatment effectiveness in ER + breast cancer, urothelial bladder carcinoma, and liver cancer by functioning as a trigger of resistance to tamoxifen, sorafenib, and cisplatin. This study systematically presents the functions of MAFG-AS1 in various cancers, as well as the findings of bioinformatics analyses of the MAFG-AS1, which should give clear advice for future research.

LncRNA MAFG-AS1 is involved in human cancer progression.

Long noncoding RNAs (lncRNAs) refer to a type of non-protein-coding transcript of more than 200 nucleotides. LncRNAs play fundamental roles in disease development and progression, and lncRNAs are dysregulated in many pathophysiological processes. Thus, lncRNAs may have potential value in clinical applications. The lncRNA, MAF BZIP Transcription Factor G (MAFG)-AS1, is dysregulated in several cancer, including breast cancer, lung cancer, liver cancer, bladder cancer, colorectal cancer, gastric cancer, esophagus cancer, prostate cancer, pancreatic cancer, ovarian cancer, and glioma. Altered MAFG-AS1 levels are also associated with diverse clinical characteristics and patient outcomes. Mechanistically, MAFG-AS1 mediates a variety of cellular processes via the regulation of target gene expression. Therefore, the diagnostic, prognostic, and therapeutic aspects of MAFG-AS1 have been widely explored. In this review, we discuss the expression, major roles, and molecular mechanisms of MAFG-AS1, the relationship between MAFG-AS1 and clinical features of diseases, and the clinical applications of MAFG-AS1.

Transcription factor ETV1-induced lncRNA MAFG-AS1 promotes migration, invasion, and epithelial-mesenchymal transition of pancreatic cancer cells by recruiting IGF2BP2 to stabilize ETV1 expression.

We investigated the mechanism of ETS-translocation variant 1 (ETV1)/lncRNA-MAFG-AS1 in pancreatic cancer (PC). MAFG-AS1 and ETV1 levels in PC cell lines and HPNE cells were determined using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB). After transfection with sh-MAFG-AS1, PC cell invasion, migration, proliferation, and epithelial-mesenchymal transition (EMT)-related proteins were measured by 5-ethynyl-2'-deoxyuridine (EdU), Transwell assay, and WB. The binding between ETV1 and MAFG-AS1 was studied using dual-luciferase assay and chromatin immunoprecipitation. The interactions between MAFG-AS1, IGF2BP2, and ETV1 were tested. Combined experiments were further performed using sh-MAFG-AS1 and pcDNA-ETV1 simultaneously. ETV1/MAFG-AS1 was highly expressed in PC cells. Blocking MAFG-AS1 inhibited the malignant behaviors of PC cells. ETV1 induced MAFG-AS1 transcription in PC cells. MAFG-AS1 stabilized ETV1 mRNA by recruiting IGF2BP2. ETV1 overexpression partially antagonized the suppression of silencing MAFG-AS1 on PC cells. ETV1-induced MAFG-AS1 stabilized the ETV1 expression by recruiting IGF2BP2 and promoted PC cell migration, invasion, proliferation, and EMT.

Stapled Peptides as Direct Inhibitors of Nrf2-sMAF Transcription Factors.

Nuclear factor erythroid-related 2-factor 2 (Nrf2) is a transcription factor traditionally thought of as a cellular protector. However, in many cancers, Nrf2 is constitutively activated and correlated with therapeutic resistance. Nrf2 heterodimerizes with small musculoaponeurotic fibrosarcoma Maf (sMAF) transcription factors, allowing binding to the antioxidant responsive element (ARE) and induction of transcription of Nrf2 target genes. While transcription factors are historically challenging to target, stapled peptides have shown great promise for inhibiting these protein-protein interactions. Herein, we describe the first direct cell-permeable inhibitor of Nrf2/sMAF heterodimerization. N1S is a stapled peptide designed based on AlphaFold predictions of the interactions between Nrf2 and sMAF MafG. A cell-based reporter assay combined with in vitro biophysical assays demonstrates that N1S directly inhibits Nrf2/MafG heterodimerization. N1S treatment decreases the transcription of Nrf2-dependent genes and sensitizes Nrf2-dependent cancer cells to cisplatin. Overall, N1S is a promising lead for the sensitization of Nrf2-addicted cancers.

ARE-PROTACs Enable Co-degradation of an Nrf2-MafG Heterodimer.

Proteolysis-targeting chimera (PROTAC) technology has emerged as a potential strategy to degrade "undruggable" proteins in recent years. Nrf2, an aberrantly activated transcription factor in cancer, is generally considered undruggable as lacking active sites or allosteric pockets. Here, we constructed the chimeric molecule C2, which consists of an Nrf2-binding element and a CRBN ligand, as a first-in-class Nrf2 degrader. Surprisingly, C2 was found to selectively degrade an Nrf2-MafG heterodimer simultaneously via the ubiquitin-proteasome system. C2 impeded Nrf2-ARE transcriptional activity significantly and improved the sensitivity of NSCLC cells to ferroptosis and therapeutic drugs. The degradation character of ARE-PROTACs suggests that the PROTAC hijacking the transcription element of TFs could achieve co-degradation of the transcription complex.

A review on the roles and molecular mechanisms of MAFG-AS1 in oncogenesis.

Long non-coding RNAs (lncRNAs) have more than 200 nucleotides and do not encode proteins. At the same time, they can regulate various biological functions and therefore play an essential role as oncogenes or tumor suppressors in human cancers. MAFG-AS1 is an antisense RNA of MAF BZIP Transcription Factor G (MAFG) located at chromosome 17q25.3 head-to-head with the MAFG encoding gene containing a transcript size of 1895 bp. Accumulating evidence shows that MAFG-AS1 is overexpressed in many cancers, functions as an oncogene, and is significantly associated with poor clinical characteristics and prognosis. In this review, we first discuss the recent literature regarding the role of MAFG-AS1 in different cancers as well as its diagnostic and prognostic values. Then we will provide insights into its biological functions, such as its role in cancer progression, competing endogenous RNA (ceRNA) activity, regulation of EMT, glycolysis, energy metabolism, transcription factors, proteasomal degradation, and signaling pathways.

lncRNA MAFG­AS1 enhances radioresistance of glioblastoma cells via miR­642a­5p/Notch1 axis.

This study was designed to explore the function of lncRNA MAFG­AS1/miR­642a­5p/Notch1 in glioblastoma (GBM) cells under radiation. GBM cells (M059K and M059J) were transfected or/and irradiated. Western blotting was used to detect Notch1 protein and its downstream Hes1 protein. Cell Counting Kit-8 assay and flow cytometry were applied for viability and apoptosis tests, respectively. Luciferase reporter plasmids and Ago2 antibody were used to verify the predicted binding of miR­642a­5p to MAFG­AS1 or Notch1 mRNA. Notch1 and MAFG­AS1 were highly expressed and miR­642a­5p was lowly expressed in radioresistant M059K cells. Knockdown of Notch1 inhibited radioresistance and promoted apoptosis in M059K cells. MAFG­AS1 competed with Notch1 mRNA for binding of miR­642a­5p and therefore promoted the expression of Notch1. Overexpression of miR­642a­5p or silencing of MAFG­AS1 inhibited the radioresistance of M059K cells. Knockdown of Notch1 or overexpression of miR­642a­5p reduced viability and increased apoptosis in irradiated M059J cells overexpressing MAFG­AS1. MAFG­AS1 reduces the radiosensitivity of GBM cells via miR­642a­5p/Notch1 axis.

AKR1C3 regulated by NRF2/MAFG complex promotes proliferation via stabilizing PARP1 in hepatocellular carcinoma.

Aldo-keto reductase family 1 member C3 (AKR1C3) serves as a contributor to numerous kinds of tumors, and its expression is elevated in patients with hepatocellular carcinoma (HCC). However, the biological function of AKR1C3 in HCC remains unclear. Here we investigated the role of AKR1C3 in liver carcinogenesis using in vitro and in vivo models. We determined that AKR1C3 is frequently increased in HCC tissues with poor prognosis. Genetically manipulated cells with AKR1C3 construction were examined to highlight the pro-tumoral growth of both wild-type AKR1C3 and mutant in vitro and in vivo. We observed promising treatment effects of AKR1C3 shRNA by intratumoral injection in mice. Mechanically, we demonstrated that the transcription factor heterodimer NRF2/MAFG was able to bind directly to AKR1C3 promoter to activate its transcription. Further, AKR1C3 stabilized PARP1 by decreasing its ubiquitination, which resulted in HCC cell proliferation and low sensitivity of Cisplatin. Moreover, we discovered that the tumorigenic role of AKR1C3 was non-catalytic dependent and the NRF2/MAFG-AKR1C3-PARP1 axis might be one of the important proliferation pathways in HCC. In conclusion, blockage of AKR1C3 expression provides potential therapeutic benefits against HCC.

LncRNA MAFG-AS1 Promotes Lung Adenocarcinoma Cell Migration and Invasion by Targeting miR-3196 and Regulating SOX12 Expression.

Lung adenocarcinoma (LUAD) patients exhibit poor prognosis, primarily due to metastasis. Emerging studies have demonstrated that long noncoding RNAs (lncRNAs) play critical roles in cancer progression and metastasis besides their physiological function. Here, we investigated the potential role of lncRNA MAF BZIP Transcription Factor G Antisense RNA 1 (MAFG-AS1) in LUAD metastasis by analyzing its expression in The Cancer Genome Atlas (TCGA) LUAD database, and its function in LUAD using in vitro and in vivo experiments. We performed bioinformatics analysis, western blotting, dual-luciferase reporter gene assay, RNA immunoprecipitation (RIP), and rescue assays to reveal the molecular mechanisms underlying MAFG-AS1 function. We observed augmented expression of MAFG-AS1 in LUAD tissues compared with normal adjacent tissues, and its association with poor prognosis. Furthermore, MAFG-AS1 overexpression promoted LUAD cell migration, proliferation, invasion, and epithelial mesenchymal transition (EMT). Besides, MAFG-AS1 also targeted miR-3196 directly by acting as an endogenous sponge, thereby rescuing the inhibition of SOX12, a target of miR-3196. Thus, the rescue assays demonstrated that MAFG-AS1 promotes cell migration, invasion, and EMT by modulating the miR-3196/SOX12 pathway. In conclusion, our findings suggest that MAFG-AS1/miR-3196/SOX12 axis regulates LUAD progression and is a potential therapeutic target for LUAD.

LncRNA MAFG-AS1 promotes the malignant phenotype of ovarian cancer by upregulating NFKB1-dependent IGF1.

Long non-coding RNAs (lncRNAs) were recently recognized to vitally function in a variety of cancer cellular events, including epithelial-mesenchymal transition (EMT), invasion, and migration, particularly in ovarian cancer (OC). Herein, we sought to investigate the potential role of MAFG-AS1 in the malignant behaviors of OC cells. The binding affinity between MAFG-AS1, miR-339-5p, NFKB1 or IGF1 was characterized so as to identify the underlying mechanism of corresponding their interactions. We conducted MAFG-AS1 overexpression or knockdown along with NFKB1 and IGF1 silencing to examine their effects on the EMT, migration, and invasion of OC cells. Tumors were xenografted in nude mice to validate the in vitro findings. Our data showed significantly high expression pattern of MAFG-AS1 in the OC tissues and cells. Further mechanistic investigations revealed that MAFG-AS1 upregulated the IGF1 expression pattern through recruitment of NFKB1, whereas MAFG-AS1 upregulated the NFKB1 expression pattern through binding to miR-339-5p. Thus, MAFG-AS1 overexpression accelerated the EMT, invasion, and migration of OC cells, which could be annulled by silencing of IGF1 or NFKB1. Besides, our in vitro findings were successfully recapitulated in the xenograft mice. These results determined that MAFG-AS1 stimulated the OC malignant progression by upregulating the NFKB1-mediated IGF1 via miR-339-5p, thus highlighting a novel potential therapeutic target against OC.

LncRNA MAFG-AS1 Upregulates Polo-Like Kinase-1 by Sponging miR-505 to Promote Gastric Adenocarcinoma Cell Proliferation.

Gastric cancer is a commonly diagnosed, often fatal malignancy and requires novel anticancer therapies and preventative approaches. This study described the involvement of MAFG-AS1, a lncRNA with important functions in cancer biology, in gastric adenocarcinoma (GA). Thirty-six male and forty-two female GA patients with an average age of 51.9 ± 5.7 years in the range of 35 to 68 years were enrolled. Paired gastric cancer (GC) and non-tumor tissues were collected from each patient. MAFG-AS1 expression was determined. RNA interaction prediction, dual luciferase reporter assay, RT-qPCR assay, Western blot, and CCK-8 assay were conducted. The results indicated that MAFG-AS1 was highly expressed in GA and closely correlated with poor survival. MAFG-AS1 interacted with miR-505, but MAFG-AS1 and miR-505 overexpression showed no significant effects on each other's expression. In addition, MAFG-AS1 increased the expression of PLK1, a miR-505 target. MAFG-AS1 and PLK1 overexpression increased GC cell proliferation rate. MiR-505 overexpression reduced the effects of MAFG-AS1 and PLK1 overexpression on cell proliferation. Therefore, MAFG-AS1 might upregulate PLK1 by sponging miR-505 to promote GA cell proliferation.

Proteome-scale profiling reveals MAFF and MAFG as two novel key transcription factors involved in palmitic acid-induced umbilical vein endothelial cell apoptosis.

BACKGROUND: Vascular endothelial cell apoptosis is the leading risk factor of atherosclerosis (AS). The purpose of our study was to use a new generation high-throughput transcription factor (TF) detection method to identify novel key TFs in vascular endothelial cell apoptosis induced by palmitic acid (PA). METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with 0, 300, or 500 µM PA. Candidate TFs in the three groups were identified by differential expression, pathway enrichment, Western Blot (WB), and RT-qPCR analyses. Apoptosis was assessed by fluorescence-activated cell sorting (FACS) using FITC-annexin V and propidium iodide staining. RESULTS: We established a HUVEC apoptosis model to simulate the process of atherosclerosis onset and identified 51 significant TFs. of the 51 TFs, v-maf musculoaponeurotic fibrosarcoma oncogene family protein G (MAFG) and v-maf musculoaponeurotic fibrosarcoma oncogene family protein F (MAFF), were matched to known AS signalling pathways and were validated by WB and RT-qPCR analyses in our study. Overexpression of MAFG or MAFF in HUVECs significantly inhibited PA-induced early apoptosis. CONCLUSIONS: We identified MAFF and MAFG as novel key TFs in vascular endothelial cell apoptosis.

Discovery and characterization of novel peptide inhibitors of the NRF2/MAFG/DNA ternary complex for the treatment of cancer.

Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI-H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).

The small protein MafG plays a critical role in MC3T3-E1 cell apoptosis induced by simulated microgravity and radiation.

Microgravity and radiation exposure-induced bone damage is one of the most significant alterations in astronauts after long-term spaceflight. However, the underlying mechanism is still largely unknown. Recent ground-based simulation studies have suggested that this impairment is likely mediated by increased production of reactive oxygen species (ROS) during spaceflight. The small Maf protein MafG is a basic-region leucine zipper-type transcription factor, and it globally contributes to regulation of antioxidant and metabolic networks. Our research investigated the role of MafG in the process of apoptosis induced by simulated microgravity and radiation in MC3T3-E1 cells. We found that simulated microgravity or radiation alone decreased MafG expression and elevated apoptosis in MC3T3-E1 cells, and combined simulated microgravity and radiation treatment aggravated apoptosis. Meanwhile, under normal conditions, increased ROS levels facilitated apoptosis and downregulated the expression of MafG in MC3T3-E1 cells. Overexpression of MafG decreased apoptosis induced by simulated microgravity and radiation. These findings provide new insight into the mechanism of bone damage induced by microgravity and radiation during space flight.

HBx-upregulated MAFG-AS1 promotes cell proliferation and migration of hepatoma cells by enhancing MAFG expression and stabilizing nonmuscle myosin IIA.

To identify hepatitis B virus (HBV)-related lncRNA(s), we previously examined the transcription profiles of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells by RNA deep sequencing and identified 38 upregulated long noncoding RNAs (lncRNAs). In the present study, the lncRNA MAFG-AS1 is investigated in detail because its gene is located adjacent to the MAFG gene, which is an important transcription factor involved in cell proliferation. The level of MAFG-AS1 is significantly higher in HCC tissue than in nontumor tissues. TCGA data show that the expression level of MAFG-AS1 is negatively correlated with survival of HCC patients. GEO cohort data show that compared with healthy tissues, the expression level of MAFG-AS1 is significantly higher in HBV-infected liver tissues. Real-time PCR and luciferase reporter assay data show that HBx can enhance the transcription of MAFG-AS1. Gain-of-function and loss-of-function experiments indicate that MAFG-AS1 promotes proliferation, migration, and invasion of HCC cells. Tumor formation assay results demonstrate that knockdown of MAFG-AS1 significantly inhibits cell proliferation in nude mice. Furthermore, MAFG-AS1 enhances the transcription of adjacent MAFG via E2F1. Additionally, MAFG-AS1 interacts with three subunits (MYH9, MYL12B, and MYL6) of nonmuscle myosin IIA (NM IIA). Knockdown of MAFG-AS1 inhibits ATPase activity of MYH9, interaction of NM IIA subunits, and cell cycle progression. Thus, the lncRNA MAFG-AS1 is upregulated by HBV and promotes proliferation and migration of HCC cells. Our findings suggest that MAFG-AS1 is a potential oncogene that may contribute to HBV-related HCC development.

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

Development of INSOL-tag for proteome-wide protein handling and its application in protein array analysis.

Proteomic analysis requires protein tags that enable high-throughput handling; however, versatile tags that can be used in in vitro expression systems are currently lacking. In this study, we developed an insoluble protein tag, INSOL-tag, derived from human transcription factor MafG. The INSOL-tagged target protein is expressed in a eukaryotic in vitro expression system and recovered as a pellet following centrifugation at 19,000 × g for 20 min. Comparisons of the target protein recovery rates of GST-tag and INSOL-tag using 111 cytoplasmic proteins revealed a fourfold increase in the yield of INSOL-tagged proteins. Using 267 cancer antigens purified with INSOL-tag, we subsequently developed an INSOL-CTA array method, for profiling autoantibodies in sera of cancer patients. The detection limit of the array was approximately 11.1 pg IgG, and the correlation with ELISA was high (R2  = .993, .955). Moreover, when autoantibody profiling of digestive cancer patient sera was performed, antigen spreading was observed. These data suggest that INSOL-tag is a versatile tag that can insolubilize a wide range of target proteins. It is therefore expected to become a powerful tool in comprehensive protein preparation for protein arrays, antibody production, and mass spectrometry.

Free heme regulates placenta growth factor through NRF2-antioxidant response signaling.

Free heme activates erythroblasts to express and secrete Placenta Growth Factor (PlGF), an angiogenic peptide of the VEGF family. High circulating levels of PlGF have been associated in experimental animals and in patients with sickle cell disease with echocardiographic markers of pulmonary hypertension, a life-limiting complication associated with more intense hemolysis. We now show that the mechanism of heme regulation of PlGF requires the contribution of the key antioxidant response regulator NRF2. Mimicking the effect of heme, the NRF2 agonist sulforaphane stimulates the PlGF transcript level nearly 30-fold in cultured human erythroblastoid cells. Heme and sulforaphane also induce transcripts for NRF2 itself, its partners MAFF and MAFG, and its competitor BACH1. Furthermore, heme induction of the PlGF transcript is significantly diminished by the NRF2 inhibitor brusatol and by siRNA knockdown of the NRF2 and/or MAFG transcription factors. Chromatin immunoprecipitation experiments show that heme induces NRF2 to bind directly to the PlGF promoter region. In complementary in vivo experiments, mice injected with heme show a significant increase in their plasma PlGF protein as early as 3 h after treatment. Our results reveal an important mechanism of PlGF regulation, adding to the growing literature that supports the pivotal importance of the NRF2 axis in the pathobiology of sickle cell disease.

Direct and Specific Functional Evaluation of the Nrf2 and MafG Heterodimer by Introducing a Tethered Dimer into Small Maf-Deficient Cells.

A group of cytoprotective genes is regulated by heterodimers composed of the cap'n'collar (CNC) family member Nrf2 and one of the small Maf (sMaf) proteins (MafF, MafG, or MafK) through the antioxidant response element (ARE, also referred to as the CNC-sMaf binding element [CsMBE]). Many lines of evidence support this model; however, a direct and specific evaluation of the Nrf2-sMaf heterodimer remains to be executed. To address this issue, we constructed a tethered Nrf2-MafG (T-N2G) heterodimer using a flexible linker peptide. We then introduced the T-N2G construct into cells lacking all three sMaf proteins to specifically evaluate the function of the tethered heterodimer without interference from other endogenous CNC-sMaf heterodimers or sMaf homodimers. In response to an Nrf2 activator, diethyl maleate, the T-N2G protein can widely activate the target genes of Nrf2 but not those of Nrf1, such as proteasome subunit genes. Genome-wide binding analysis showed that the T-N2G protein preferentially bound to the CsMBE motifs in the regulatory regions of the Nrf2 target genes. These results provide direct evidence that the Nrf2-MafG heterodimer acts as a transcriptional activator of Nrf2-dependent genes and show that this assay system will be a powerful tool to specifically examine the function of other CNC-sMaf heterodimers.