Genomewide approaches for BACH1 target genes in mouse embryonic fibroblasts showed BACH1-Pparg pathway in adipogenesis.

The transcription repressor BTB and CNC homology 1 (BACH1) represses genes involved in heme metabolism and oxidative stress response. BACH1 also suppresses the p53-dependent cellar senescence in primary mouse embryonic fibroblasts (MEFs). To investigate the role of BACH1 in MEF other than its known functions, we carried out a genomewide mapping of binding site for BACH1 and its heterodimer partner MAFK in immortalized MEFs (iMEFs) using chromatin immunoprecipitation and next-generation sequencing technology (ChIP-sequence). The comparative analysis of the ChIP-sequence data and DNA microarray data from Bach1-deficient and wild-type (WT) iMEF showed 35 novel candidate target genes of BACH1. Among these genes, five genes (Pparg, Nfia, Ptplad2, Adcy1 and Ror1) were related with lipid metabolism. Bach1-deficient iMEFs showed increased expression of mRNA and protein of PPARγ, which is the key factor of adipogenesis. These cells also showed a concomitant increase in ligand-dependent activation of PPARγ target genes compared with wild-type iMEFs. Moreover, Bach1-deficient iMEFs efficiently differentiated to adipocyte compared with wild-type cells in the presence of PPARγ ligands. Our results suggest that BACH1 regulates expression of adipocyte-related genes including Pparg and potentiates adipocyte differentiation capacity.

Effects of mutant TDP-43 on the Nrf2/ARE pathway and protein expression of MafK and JDP2 in NSC-34 cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons and lacks an effective treatment. The disease pathogenesis has not been clarified at present. Pathological transactive response DNA-binding protein 43 (TDP-43) plays an important role in the pathogenesis of ALS. Nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is found in a mutant TDP-43 transgenic cell model, but its downstream antioxidant enzyme expression is decreased. To elucidate the specific mechanism of Nrf2/ARE (antioxidant responsive element) signaling dysfunction, we constructed an ALS cell model with human mutant TDP-43 using the NSC-34 cell line to evaluate the impact of the TDP-43 mutation on the Nrf2/ARE pathway. We found the nuclear translocation of Nrf2, but the expression of total Nrf2, cytoplasmic Nrf2, and downstream phase II detoxifying enzyme (NQO1) was decreased in NSC-34 cells transfected with the TDP-43-M337V plasmid. Besides, TDP-43-M337V plasmid-transfected NSC-34 cells were rounded with reduced neurites, shortened axons, increased levels of intracellular lipid peroxidation products, and decreased viability, which suggests that the TDP-43-M337V plasmid weakened the antioxidant capacity of NSC-34 cells and increased their susceptibility to oxidative damage. We further showed that expression of the MafK protein and the Jun dimerization protein 2 (JDP2) was reduced in TDP-43-M337V plasmid-transfected NSC-34 cells, which might cause accumulation of Nrf2 in nuclei but a decrease in NQO1 expression. Taken together, our results confirmed that TDP-43-M337V impaired the Nrf2/ARE pathway by reducing the expression of MafK and JDP2 proteins, and provided information for further research on the molecular mechanisms of TDP-43-M337V in ALS.

Comparison of Transcriptome Between Type 2 Diabetes Mellitus and Impaired Fasting Glucose.

BACKGROUND The aim of this study was to compare the transcriptome between impaired fasting glucose (IFG) and type 2 diabetes mellitus (T2DM), and further research their molecular mechanisms. MATERIAL AND METHODS The original microarray GSE21321, including miRNA and mRNA expression profiles, was downloaded from the GEO database. Data preprocessing was processed by limma package, and differentially expressed genes (DGs) and miRNA (DMs) were screened. Then, the regulatory relationships among miRNA, TF, and genes were screened and the regulatory network was constructed. Finally, DAVID was used for KEGG enrichment analysis. RESULTS There were 11 upregulated IFG-related DMs and five upregulated T2DM-related DMs. Three of the DMs overlapped. In addition, there were eight downregulated IFG-related DMs and two downregulated T2DM-related DMs. Only one downregulated DM overlapped. Similarly, there were 264 upregulated IFG-related DGs and 331 upregulated T2DM-related DGs; and 196 overlapping genes were obtained. In addition, there were 400 downregulated IFG-related DMs and 568 downregulated T2DM-related DMs. A total of 326 downregulated DMs were overlapped. The overlapped DGs were enriched in various pathways, including hematopoietic cell lineage, Fc gamma R-mediated phagocytosis, and MAPK signaling pathway. TAF1 (upregulated gene) and MAFK (downregulated gene) were hub nodes both in IFG- and T2DM-related miRNA-TF-gene regulatory network. In addition, miRNAs, including hsa-miR-29a, hsa-miR-192, and hsa-miR-144, were upregulated hub nodes in the two regulatory networks. CONCLUSIONS Genes including TAF1 and MAFK, and miRNAs including hsa-miR-29a, hsa-miR-192, and hsa-miR-144 might be potential target genes and important miRNAs for IFG and T2DM.

Oxidative stress regulates CFTR gene expression in human airway epithelial cells through a distal antioxidant response element.

Cystic fibrosis transmembrane conductance regulator gene (CFTR) expression in human airway epithelial cells involves the recruitment of distal cis-regulatory elements, which are associated with airway-selective DNase hypersensitive sites at -44 kb and -35 kb from the gene. The -35-kb site encompasses an enhancer that is regulated by the immune mediators interferon regulatory factor 1 and 2 and by nuclear factor Y. Here we investigate the -44-kb element, which also has enhancer activity in vitro in airway epithelial cells but is inactive in intestinal epithelial cells. This site contains an antioxidant response element (ARE) that plays a critical role in its function in airway cell lines and primary human bronchial epithelial cells. The natural antioxidant sulforaphane (SFN) induces nuclear translocation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcription factor that regulates genes with AREs in their promoters, many of which are involved in response to injury. Under normal conditions, the -44-kb ARE is occupied by the repressor BTB and CNC homology 1, basic leucine zipper transcription factor (Bach1), and v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) heterodimers. After 2 hours of SFN treatment, Nrf2 displaces these repressive factors and activates CFTR expression. Site-directed mutagenesis shows that both the ARE and an adjacent NF-κB binding site are required for activation of the -44-kb element in airway epithelial cells. Moreover, this element is functionally linked to the -35-kb enhancer in modulating CFTR expression in response to environmental stresses in the airway.

Compound mouse mutants of bZIP transcription factors Mafg and Mafk reveal a regulatory network of non-crystallin genes associated with cataract.

Although majority of the genes linked to early-onset cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. We report on small Maf transcription factors Mafg and Mafk as regulators of several non-crystallin human cataract-associated genes in fiber cells and establish their significance to this disease. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify Mafg and its co-regulators in the lens, and generated various null-allelic combinations of Mafg:Mafk mouse mutants for phenotypic and molecular analysis. By age 4 months, Mafg-/-:Mafk+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of Mafg-/-:Mafk+/- mouse lens reveals severely disorganized fiber cells, while microarray-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of Mafg-/-:Mafk+/- lens-DRGs with (1) binding motifs and genomic targets of small Mafs and their regulatory partners, (2) iSyTE lens expression data, and (3) interactions between DRGs in the String database, unravel a detailed small Maf regulatory network in the lens, several nodes of which are linked to cataract. This approach identifies 36 high-priority candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes oxidative stress and misregulation of sterol synthesis. These data identify Mafg and Mafk as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to human cataract.

Wnt1-induced MAFK expression promotes osteosarcoma cell proliferation.

Osteosarcoma is one of the most common primary bone tumors in children and young adults. In this study, we investigated the role of musculoaponeurotic fibrosarcoma oncogene homolog K (MAFK) in osteosarcoma cell proliferation in vitro and the possible pathways that contributed to MAFK-related osteosarcoma development. We first reported that MAFK was expressed at low levels in an osteosarcoma cell line. Furthermore, a significant correlation between MAFK and the Wnt signaling pathway was observed in osteosarcoma by using a gene microarray assay. We found that expression of MAFK could be induced by Wnt1 in a dose-dependent manner. Furthermore, Wnt1-induced MAFK expression caused a significant increase of cell viability, whereas a Wnt pathway inhibitor, IWR-1-endo, abolished Wnt1-induced effects on MAFK. Finally, cell cycle analysis showed that enhanced cell proliferation might be attributed to re-distribution of the cell cycle. Together, our results suggested that Wnt1-induced MAFK expression promoted cell proliferation in MG63 cells, and that the role of MAFK in osteosarcoma might be closely linked to the Wnt signaling pathway.

Transforming growth factor-ß induces transcription factors MafK and Bach1 to suppress expression of the heme oxygenase-1 gene.

Transforming growth factor-ß (TGF-ß) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-ß suppresses the transcriptional activation of the heme oxygenase-1 (HO-1) gene, which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor Nrf2. TGF-ß did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-ß induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-ß-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-ß suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-ß is activated after tissue injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.

Small MAF genes variants and chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is one of the most frequent hematological neoplasia worldwide. The abnormal accumulation of reactive oxygen species may be an important factor in CML development. The transcription factor NRF2 can regulate the transcription of a battery of antioxidant and detoxificant genes after heterodimerizing with small-Maf proteins. Although the participation of NRF2 in the development of chronic degenerative diseases has been thoroughly studied, the role of small-Maf genes has not been documented. We have identified polymorphisms in the three MAF genes (F, G and K) and assessed their association with CML. Over 266 subjects with CML and 399 unrelated healthy donors have been studied. After sequencing each MAF gene by Sanger technology, we found 17 variants in MAFF gene, eight in MAFG and seven in MAFK. In the case-control study, the homozygote genotype CC for the rs9610915 SNP of MAFF was significantly associated with CML. The frequency of the ACC haplotype from MAFK was significantly lower than controls. After stratification by gender, the ACC and GTG haplotypes were associated only with males with CML. These novel data suggest an association between MAFF and MAFG and the development of CML.

Metabolic aspects of epigenome: coupling of S-adenosylmethionine synthesis and gene regulation on chromatin by SAMIT module.

Histone and DNA methyltransferases utilize S-adenosyl-L-methionine (SAM), a key intermediate of sulfur amino acid metabolism, as a donor of methyl group. SAM is biosynthesized by methionine adenosyltransferase (MAT) using two substrates, methionine and ATP. Three distinct forms of MAT (MATI, MATII and MATIII), encoded by two distinct genes (MAT1A and MAT2A), have been identified in mammals. MATII consists of α2 catalytic subunit encoded by MAT2A and ß regulatory subunit encoded by MAT2B, but the physiological function of the ß subunit is not clear. MafK is a member of Maf oncoproteins and functions as both transcription activator and repressor by forming diverse heterodimers to bind to DNA elements termed Maf recognition elements. Proteomics analysis of MafK-interactome revealed its interaction with both MATIIα and MATIIß. They are recruited specifically to MafK target genes and are required for their repression by MafK and its partner Bach1. Because the catalytic activity of MATIIα is required for the MafK target gene repression, MATIIα is suggested to provide SAM locally on chromatin where it is recruited. One of the unexpected features of MATII is that MATIIα interacts with many chromatin-related proteins of diverse functions such as histone modification, chromatin remodeling, transcription regulation, and nucleo-cytoplasmic transport. MATIIα appears to generate multiple, heterogenous regulatory complexes where it provides SAM. Considering their function, the heterooligomer of MATIIα and ß is named SAMIT (SAM-integrating transcription) module within their interactome where it serves SAM for nuclear methyltransferases.

miR-503 targets MafK to inhibit subcutaneous preadipocyte adipogenesis causing a decrease of backfat thickness in Guanzhong Black pigs.

Reducing backfat thickness (BFT), determined by subcutaneous fat deposition, is vital in Chinese developed pig breeds. The level of miR-503 in the backfat of Guanzhong Black pigs was found to be lower than that in Large White pigs, implying that miR-503 may be related to BFT. However, the effect and mechanism of miR-503 on adipogenic differentiation in subcutaneous preadipocytes remain unknown. Compared with Large White pigs, the BFT and body fat content of Guanzhong Black pigs were greater, but the level of miR-503 was lower in subcutaneous adipose tissue (SAT) at 180 days of age. Furthermore, miR-503 promoted preadipocyte proliferation by increasing the proportion of S-phase and EdU-positive cells. However, miR-503 inhibited preadipocyte differentiation by downregulating adipogenic gene expression. Mechanistically, miR-503 directly targeted musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) in both proliferating and differentiating preadipocytes to repress adipogenesis. Our findings provide a novel miRNA biomarker for reducing pig BFT levels to improve carcass quality.

MafK accelerates Salmonella mucosal infection through caspase-3 activation.

Gastrointestinal homeostasis is critical for maintaining host health, and is affected by many factors. A recent report showed that Musculoaponeurotic fibrosarcoma K (MafK) expression is increased in patients that have ulcerative colitis (UC). Even so, MafK's significance in sustaining intestinal homeostasis has not been investigated. In this research, MafK overexpressing transgenic (MafK Tg) mice were found to be more susceptible to infection with Salmonella on the mucosa than the wild-type (WT) mice. Following Salmonella oral infection, MafK Tg mice suffered higher mortality and a lot more weight loss, damage to the intestines, and inflammation in the intestines than WT mice. MafK Tg mice were also unable to control Salmonella colonization and dissemination. In vivo data showed that increased MafK expression promoted epithelial cell apoptosis which was further confirmed by in vitro data. The rapid cleavage of caspase-3 in epithelial cells contributed to Salmonella dissemination and inflammation initiation. This study reveals that MafK participates in Salmonella pathogenesis acceleration by increasing caspase-3 activation.

Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of glutathione synthetic enzymes and contributes to cholestatic liver injury in mice.

UNLABELLED: We previously showed that hepatic expression of glutathione (GSH) synthetic enzymes and GSH levels fell 2 weeks after bile duct ligation (BDL) in mice. This correlated with a switch in nuclear anti-oxidant response element (ARE) binding activity from nuclear factor erythroid 2-related factor 2 (Nrf2) to c-avian musculoaponeurotic fibrosarcoma (c-Maf)/V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (MafG). Our current aims were to examine whether the switch in ARE binding activity from Nrf2 to Mafs is responsible for decreased expression of GSH synthetic enzymes and the outcome of blocking this switch. Huh7 cells treated with lithocholic acid (LCA) exhibited a similar pattern of change in GSH synthetic enzyme expression as BDL mice. Nuclear protein levels of Nrf2 fell at 20 hours after LCA treatment, whereas c-Maf and MafG remained persistently induced. These changes translated to ARE nuclear binding activity. Knockdown of c-Maf or MafG individually blunted the LCA-induced decrease in Nrf2 ARE binding and increased ARE-dependent promoter activity, whereas combined knockdown was more effective. Knockdown of c-Maf or MafG individually increased the expression of GSH synthetic enzymes and raised GSH levels, and combined knockdown exerted an additive effect. Ursodeoxycholic acid (UDCA) or S-adenosylmethionine (SAMe) prevented the LCA-induced decrease in expression of GSH synthetic enzymes and promoter activity and prevented the increase in MafG and c-Maf levels. In vivo knockdown of the Maf genes protected against the decrease in GSH enzyme expression, GSH level, and liver injury after BDL. CONCLUSION: Toxic bile acid induces a switch from Nrf2 to c-Maf/MafG ARE nuclear binding, which leads to decreased expression of GSH synthetic enzymes and GSH levels and contributes to liver injury during BDL. UDCA and SAMe treatment targets this switch.

MAFK Polymorphisms Located in 3'-UTR are Associated with Severity of Atrophy and CDKN2A Methylation Status in the Gastric Mucosa.

Objective: This study aimed to clarify the association of MAFK polymorphisms (rs4268033, rs3735656, and rs10226620) with the degree of gastric mucosal atrophy and CDKN2A CpG methylation status. Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Results: Either rs3735656 or rs10226620, located in the 3'-UTR of MAFK, was significantly associated with the severity of gastric mucosal atrophy using a dominant genetic model (odds ratio [OR], 2.10; p = 0.0012, and OR, 1.98; p = 0.0027, respectively). However, using a recessive genetic model, no significant association was found between three polymorphisms and gastric mucosal atrophy. The serum pepsinogen I/II ratio was significantly lower in subjects with minor alleles of rs3735656 and rs10226620 than in subjects with the wild homozygous allele (p = 0.018 and 0.013, respectively). In a subgroup including 400 of the 491 subjects, the CpG of p14ARF and p16 INK4a were methylated in 132 and 112 subjects, respectively. Fifty subjects had both CpG methylations and 206 subjects had neither methylation. When comparing the groups with both and neither methylations, there were no significant associations between three polymorphisms and CDKN2A methylation using a dominant genetic model. However, all polymorphisms investigated in this study (rs4268033, rs3735656, and rs10226620) were significantly associated with CDKN2A methylation in a recessive genetic model (OR, 3.58; p = 0.0071, OR, 4.49; p = 0.0004, and OR, 3.45; p = 0.0027, respectively). Conclusions: Our results indicate that carrying the minor allele of the MAFK polymorphisms, particularly when they are located in the 3'-UTR, has a high risk for the severity of gastric mucosal atrophy; furthermore, CDKN2A CpG methylation may develop in subjects with homozygous minor allele of these polymorphisms.

Optimization of Jiuzao protein hydrolysis conditions and antioxidant activity in vivo of Jiuzao tetrapeptide Asp-Arg-Glu-Leu by elevating the Nrf2/Keap1-p38/PI3K-MafK signaling pathway.

Tetrapeptide Asp-Arg-Glu-Leu (DREL) was isolated from Jiuzao protein hydrolysates (JPHs) by alkaline proteinase (AP) and exhibited antioxidant activity in the HepG2 cell model in the previous study. In this study, the hydrolysis method of Jiuzao protein (JP) was further optimized by using different proteinases under different conditions (i.e., temperature, time, ratio between proteinase and substrate, and pH). Considering the limitation of in vitro assays, the AAPH-induced oxidative stress Sprague-Dawley (SD) rat model was selected to measure the antioxidant capacity of DREL in vivo. Pepsin exhibited the highest hydrolysis degree under the optimum conditions after single factor analysis (SFA) among seven proteinases. The total yield of DREL in Jiuzao was 158.24 mg kg-1Jiuzao under the pepsin's optimum hydrolysis conditions determined by response surface methodology analysis (RSMA). In addition, DREL could activate the Nrf2/Keap1-p38/PI3K-MafK signaling pathway and downstream antioxidant enzymes to improve the antioxidant ability in vivo. DREL also preliminarily exhibited anti-inflammatory activity by inhibiting the secretion of inflammatory cytokines and the mediator through the activation of Nrf2. Of note, the addition of DREL in baijiu brought an inconspicuous change in the taste after sensory evaluation. This study finds out the better proteinase used for JP hydrolysis and verifies the antioxidant capacity of DREL in vivo, which provides a potential approach to apply functional components from Jiuzao for by-product utilization. Meanwhile the antioxidant function of baijiu or other foods can be increased through adding these functional peptides without affecting the original flavor and taste in the future.

Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function.

BACKGROUND: Akt regulates various cellular processes, including cell growth, survival, and metabolism. Recently, Akt's role in neurite outgrowth has also emerged. We thus aimed to identify neuronal function-related genes that are regulated by Akt. METHODS: We performed suppression subtractive hybridization on two previously established PC12 sublines, one of which overexpresses the wild-type (WT) form and the other, the dominant-negative (DN) form of Akt. These sublines respond differently to NGF's neuronal differentiation effect. RESULTS: A variety of genes was identified and could be classified into several functional groups, one of which was developmental processes. Two genes involved in neuronal differentiation and function were found in this group. v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) induces the neuronal differentiation of PC12 cells and immature telencephalon neurons, and synaptotagmin I (SytI) is essential for neurotransmitter release. Another gene, syntenin-1 (Syn-1) was also recognized in the same functional group into which MafK and SytI were classified. Syn-1 has been reported to promote the formation of membrane varicosities in neurons. Quantitative reverse transcription polymerase chain reaction analyses show that the transcript levels of these three genes were lower in PC12 (WT-Akt) cells than in parental PC12 and PC12 (DN-Akt) cells. Furthermore, treatment of PC12 (WT-Akt) cells with an Akt inhibitor resulted in the increase of the expression of these genes and the improvement of neurite outgrowth. These results indicate that dominant-negative or pharmacological inhibition of Akt increases the expression of MafK, SytI, and Syn-1 genes. Using lentiviral shRNA to knock down endogenous Syn-1 expression, we demonstrated that Syn-1 promotes an increase in the numbers of neurites and branches. CONCLUSIONS: Taken together, these results indicate that Akt negatively regulates the expression of MafK, SytI, and Syn-1 genes that all participate in regulating neuronal integrity in some way or another.

MafK Mediates Chromatin Remodeling to Silence IRF8 Expression in Non-immune Cells in a Cell Type-SpecificManner.

The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors, and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific transcription factor and its expression is augmented by interferon-γ. Therefore, it serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. Ahigh-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-Seq analysis revealed three MafK binding regions (-25 kb, -20 kb, and IRF8 6th intron) within the IRF8 locus. These MafK binding sites are sufficient to repress a reporter gene when cloned in genome-integrated lentiviral reporter constructs in only expression-restrictive cells. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding sites in mediating repressed chromatin assembly. Finally, a more thorough genomic analysis was performed, using CRISPR-Cas9 to delete MafK-int6 binding region in IRF8 expression-restrictive cells. Deleted clones exhibited an accessible chromatin conformation within the IRF8 locus that was accompanied by a significant increase in basal expression of IRF8 that was further induced by interferon-γ. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression in a celltype-specific manner.

NF-kappaB/p65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK.

Constitutively activated NF-kappaB occurs in many inflammatory and tumor tissues. Does it interfere with anti-inflammatory or anti-tumor signaling pathway? Here, we report that NF-kappaB p65 subunit repressed the Nrf2-antioxidant response element (ARE) pathway at transcriptional level. In the cells where NF-kappaB and Nrf2 were simultaneously activated, p65 unidirectionally antagonized the transcriptional activity of Nrf2. In the p65-overexpressing cells, the ARE-dependent expression of heme oxygenase-1 was strongly suppressed. However, p65 inhibited the ARE-driven gene transcription in a way that was independent of its own transcriptional activity. Two mechanisms were found to coordinate the p65-mediated repression of ARE: (1) p65 selectively deprives CREB binding protein (CBP) from Nrf2 by competitive interaction with the CH1-KIX domain of CBP, which results in inactivation of Nrf2. The inactivation depends on PKA catalytic subunit-mediated phosphorylation of p65 at S276. (2) p65 promotes recruitment of histone deacetylase 3 (HDAC3), the corepressor, to ARE by facilitating the interaction of HDAC3 with either CBP or MafK, leading to local histone hypoacetylation. This investigation revealed the participation of NF-kappaB p65 in the negative regulation of Nrf2-ARE signaling, and might provide a new insight into a possible role of NF-kappaB in suppressing the expression of anti-inflammatory or anti-tumor genes.

MafA-deficient and beta cell-specific MafK-overexpressing hybrid transgenic mice develop human-like severe diabetic nephropathy.

Transcription factor MafA is a key molecule in insulin secretion and the development of pancreatic islets. Previously, we demonstrated that some of the MafA-deficient mice develop overt diabetes mellitus, and the phenotype of these mice seems to be mild probably because of redundant functions of other Maf proteins. In this study, we generated hybrid transgenic mice that were MafA-deficient and also over-expressed MafK specifically in beta cells (MafA(-/-)MafK(+)). MafA(-/-)MafK(+) mice developed severe overt diabetes mellitus within 5weeks old, and showed higher levels of proteinuria and serum creatinine. Histological analysis revealed that embryonic development of beta cells in the MafA(-/-)MafK(+) mice was significantly suppressed and the reduced number of beta cells was responsible for the early onset of diabetes. Furthermore, after uninephrectomy, these mice demonstrated three characteristics of human diabetic nephropathy: diffuse, nodular, and exudative lesions. MafA(-/-)MafK(+) mice might be a useful model for the analysis of human diabetic nephropathy.

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

MafG sumoylation is required for active transcriptional repression.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.