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Topoisomerase (TOP) inhibitors were commonly used as chemotherapeutic agents in the treatment of cancers. In our present study, we found that etoposide (ETO), a topoisomerase 2 (TOP2) inhibitor, upregulated the production of Interleukin 10 (IL-10) in lipopolysaccharide (LPS)-stimulated macrophages. Besides, other TOP2 inhibitors including doxorubicin hydrochloride (DOX) and teniposide (TEN) were also able to augment IL-10 production. Meanwhile, the expression levels of pro-inflammatory factors, for example IL-6 and TNF-α, were also decreased accordingly by the treatment of the TOP2 inhibitors. Of note, ETO facilitated IL-10 secretion, which might be regulated by transcription factor Maf via PI3K/AKT pathway, as pharmaceutic blockage of kinase PI3K or AKT attenuated ETO-induced Maf and IL-10 expression. Further, in LPS-induced mice sepsis model, the enhanced generation of IL-10 was observed in ETO-treated mice, whereas pro-inflammatory cytokines were decreased, which significantly reduced the mortality of mice from LPS-induced lethal cytokine storm. Taken together, these results indicated that ETO may exhibit an anti-inflammatory role by upregulating the alteration of transcription factor Maf and promoting subsequential IL-10 secretion via PI3K/Akt pathway in LPS-induced macrophages. Therefore, ETO may serve as a potential anti-inflammatory agent and employed to severe pro-inflammatory diseases including COVID-19.

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis, correlating with a decrease of FOXP3+RORγt+ T cells proportion, dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly, FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon, despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1−/− mice transferred with naïve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus, MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions.

Background: Meta-analysis of clinical trials has shown that adjuvant bisphosphonates reduce bone metastases and improve survival in postmenopausal (PM) breast cancer and are now recommended for routine clinical use by international guidelines1. However, evaluation of menopause is imprecise and the biological rationale for lack of benefit in premenopausal women unclear. To address this, the biomarker, transcription factor MAF on 16q23 was tested retrospectively in the prospective randomized AZURE trial of standard adjuvant therapy +/- zoledronic acid (ZOL). Initial evaluation indicated that women with MAF negative tumors treated with ZOL had a lower rate of disease relapse irrespective of menopausal status.2 Here we present the long-term findings of this predictive biomarker on 10 year overall survival. Materials and methods: The biomarker analysis was completed on TMAs from primary tumors. Quadruplicate cores of breast tumor tissue were arrayed across replicate TMAs. MAF+ was detected using a validated (MAF/D16Z3) FISH test (Inbiomotion SL, Spain). A central laboratory (Targos, Germany) validated the assay for analytic and diagnostic performance, established acceptance criteria, included appropriate quality controls for each assay, and performed the analyses in a blinded fashion. A copy number cut-off ≥2.5 was preset for MAF+ for both prognostic and predictive testing. Interactions between MAF+ and effects of ZOL on Invasive disease free (IDFS), overall (OS) survival and time to bone metastases by menopausal status were evaluated using a Cox proportional hazards model. Results: 1769 of the 3360 AZURE pts donated primary tumor samples. Median follow-up was 117 (interquartile range 70.4-120) months. 865 pts (49%) had 2 FISH evaluable cores and were included in the analysis. These pts had similar disease and treatment characteristics to the overall study population as well as similar IDFS and OS at 10 years. 184 (21%) had MAF+ tumors and these tumors were more likely to be of higher grade, ER-ve and HER2+. In 680 pts with MAF- tumors, ZOL was associated with improved IDFS (HR=0.75; 95%CI:0.58-0.97, [P=0.02]), reduced relapse in bone (HR=0.65; 95%CI:0.45-0.94, [P=0.022] and, most importantly, better OS (HR=0.69; 95%CI:0.50-0.94, [P=0.019]). In the 185 patients with MAF+ tumors, there was a suggestion of worse outcome (IDFS HR=1.54; 95%CI:0.96-2.47 and OS HR 1.40; 95%CI:0.83-2.33), with a strong interaction between treatment effects and menopausal status. Outcomes in ZOL treated MAF+ pts who were non-PM appeared to be much worse (IDFS HR=2.31; 95%CI:1.18-4.42] and OS HR=2.28; 95%CI:1.07-4.82) due predominantly to an excess of extra-skeletal metastases in ZOL treated patients (HR=4.47; 95%CI:1.66-12.57). Conclusions: Adjuvant ZOL significantly improved disease outcomes in 79% of patients with MAF negative tumors, irrespective of menopausal status and other clinico-pathologic features. Conversely, more extra-skeletal metastases and breast cancer deaths were seen in women with MAF+ tumors who were not PM at the start of treatment. If validated in ongoing studies, the MAF FISH test could provide a clinically useful biomarker for selection of patients for adjuvant bisphosphonate treatment. 1EBCTCG, Lancet 2015; 2Coleman RE et al, Lancet Oncol 2017 Citation Format: Coleman R, Gregory W, Jean-Mairet J, Tercero JC, Torres-Martin J, Gomis R. Long term survival benefits of adjuvant zoledronic acid associated with maf status of primary tumor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-17-01.

MafF is a member of the basic leucine zipper (bZIP) transcription factor Maf family and is commonly downregulated in multiple cancers. But the expression and function of MafF in hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the relationship between endogenous MafF expression and HCC progression and explored the regulatory mechanism of MafF expression in HCC. We found that MafF decreased in HCC tissues and cells. Lentivirus-mediated MafF overexpression inhibited HCC cell proliferation and induced cell apoptosis. Bioinformatics analysis and luciferase assay identified MafF as a direct target of miR-224-5p. RNA pull-down assay demonstrated that circular RNA circ-ITCH could sponge miR-224-5p specifically in HCC. The rescue experiments further elucidated that the expression and antitumor effects of MafF could be regulated via the circ-ITCH/miR-224-5p axis. This study verified that MafF acted as a tumor suppressor in HCC and revealed the upstream regulation mechanism of MafF, which provided a new perspective for potential therapeutic targets of HCC.

High myopia is a leading cause of blindness worldwide. Myopia progression may lead to pathological changes of lens and affect the outcome of lens surgery, but the underlying mechanism remains unclear. Here, we find an increased lens size in highly myopic eyes associated with up-regulation of β/γ-crystallin expressions. Similar findings are replicated in two independent mouse models of high myopia. Mechanistic studies show that the transcription factor MAF plays an essential role in up-regulating β/γ-crystallins in high myopia, by direct activation of the crystallin gene promoters and by activation of TGF-β1-Smad signaling. Our results establish lens morphological and molecular changes as a characteristic feature of high myopia, and point to the dysregulation of the MAF-TGF-β1-crystallin axis as an underlying mechanism, providing an insight for therapeutic interventions., High myopia is associated with lens changes, but the underlying mechanisms are unclear. Here, the authors show increased equatorial diameter of the lens in subjects affected by high myopia, and find that these changes are associated with an increase in crystallin expression driven by the transcription factor MAF and TGF-β1 signaling.

The study investigated the effect of extremely low-frequency electromagnetic fields (ELF-EMFs) exposure at different magnetic flux densities on genes expression of transcription factor Maf (c-Maf), signal transducer and activator of transcription 6 (STAT6), and retinoid-related orphan receptor alpha (RORα) in the spleen and thymus of rats. Eighty adult male rats were separated into four ELF-EMFs exposed and were exposed to magnetic flux densities of 1, 100, 500, and 2000 µT at a frequency of 50 Hz for 2 h daily for up to 60 d. All rats were intraperitoneally immunized on d 31, 44, and 58 of exposure. The experimental results showed that the expression levels of c-Maf, STAT6, and RORα in the thymus were not significantly changed at different magnetic flux densities. The expression levels of RORα and c-Maf were significantly downregulated at the densities of 1 and 100 µT, while the expression of STAT6 was only significantly decreased at the density of 100 µT. In conclusion, low magnetic flux densities of ELF-EMFs may reduce the expression levels of c-Maf, STAT6, and RORα genes in the spleen.

Summary The generation of high-affinity neutralizing antibodies, the objective of most vaccine strategies, occurs in B cells within germinal centers (GCs) and requires rate-limiting “help” from follicular helper CD4+ T (Tfh) cells. Although Tfh differentiation is an attribute of MHC II-restricted CD4+ T cells, the transcription factors driving Tfh differentiation, notably Bcl6, are not restricted to CD4+ T cells. Here, we identified a requirement for the CD4+-specific transcription factor Thpok during Tfh cell differentiation, GC formation, and antibody maturation. Thpok promoted Bcl6 expression and bound to a Thpok-responsive region in the first intron of Bcl6. Thpok also promoted the expression of Bcl6-independent genes, including the transcription factor Maf, which cooperated with Bcl6 to mediate the effect of Thpok on Tfh cell differentiation. Our findings identify a transcriptional program that links the CD4+ lineage with Tfh differentiation, a limiting factor for efficient B cell responses, and suggest avenues to optimize vaccine generation.

This study was aimed at evaluation of pathogenic role of F охр 3, GATA-3, c-Maf, and T-bet transcription factors in asthma. Methods. 47 healthy individuals and 82 patients with bronchial asthma including 42 patients with allergic asthma (ABA) and 40 patients with non-allergic asthma (NABA) participated in the study. An expression of mRNA of Fохр3 (forkhead box P3), с-Maf (transcription factor Maf), GATA3 (GATA-binding protein 3), and T-bet (T-box protein, TBX21) transcription factors was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Serum concentrations of IgE and cytokines (IL-4, IL-13, IL-17, IL-6, IFN- γ ) were measured using ELISA method. Results. An imbalance in Th1/Th2/Foxp3+Treg and Th17/Foxp3+Treg systems was found in patients with asthma. Foxp3 mRNA expression was decreased in asthma patients compared to healthy individuals. This could probably reflect an impaired regulatory role of Treg in asthma. At the same time, asthma severity was related to concentrations of Foxp3 mRNA and IL-17 and IL-6. This inverse relationship could indicate an imbalance between Foxp3+Treg and Th17 towards the increase in Th17 activity and the reduction in Foxp3+Treg cell number in asthma. Blood mononuclear cells of ABA patients were characterized by significantly increased expression of mRNA of c-Maf and GATA3 Th2-specific transcription factors, associated cytokines (IL-4, IL-13) and IgE. Nevertheless, ABA patients had lower T-bet expression compared to healthy individuals and NABA patients. Conclusion . Asthma worsening in NABA patients was associated with reduced T-bet mRNA expression and increased GATA3 mRNA expression. This could be due to Th1/Th2-cell imbalance towards the increased Th2-response.

Oxidative stress (OS) is associated with many diseases ranging from cancer to neurodegenerative disorders. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is one of the most effective cytoprotective controller against OS. Modulation of Nrf2 pathway constitutes a remarkable strategy in the antineoplastic treatments. A big number of Nrf2-antioxidant response element activators have been screened for use as chemo-preventive drugs in OS associated diseases like cancer even though activation of Nrf2 happens in a variety of cancers. Research proved that hyperactivation of the Nrf2 pathway produces a situation that helps the survival of normal as well as malignant cells, protecting them against OS, anticancer drugs, and radiotherapy. In this review, the modulation of the Nrf2 pathway, anticancer activity and challenges associated with the development of an Nrf2-based anti-cancer treatment approaches are discussed.

Macrophages manifest distinct phenotype according to the organs in which they reside. In addition, they flexibly switch their character in adaptation to the changing environment. However, the molecular basis that explains the conversion of the macrophage phenotype has so far been unexplored. We find that CD169+ macrophages change their phenotype by regulating the level of a transcription factor Maf both in vitro and in vivo in C57BL/6J mice. When CD169+ macrophages were exposed to bacterial components, they expressed an array of acute inflammatory response genes in Maf-dependent manner and simultaneously start to downregulate Maf. This Maf suppression is dependent on accelerated degradation through proteasome pathway and microRNA-mediated silencing. The downregulation of Maf unlocks the NF-E2–related factor 2–dominant, cytoprotective/antioxidative program in the same macrophages. The present study provides new insights into the previously unanswered question of how macrophages initiate proinflammatory responses while retaining their capacity to repair injured tissues during inflammation.

Background Malaria control in Africa is dependent upon the use insecticides but intensive use of a limited number of chemicals has led to resistance in mosquito populations. Increased production of enzymes that detoxify insecticides is one of the most potent resistance mechanisms. Several metabolic enzymes have been implicated in insecticide resistance but the processes controlling their expression have remained largely elusive. Results Here, we show that the transcription factor Maf-S regulates expression of multiple detoxification genes, including the key insecticide metabolisers CYP6M2 and GSTD1 in the African malaria vector Anopheles gambiae. Attenuation of this transcription factor through RNAi induced knockdown reduced transcript levels of these effectors and significantly increased mortality after exposure to the pyrethroid insecticides and DDT (permethrin: 9.2% to 19.2% (p = 0.015), deltamethrin: 3.9% to 21.6% (p = 0.036) and DDT: 1% to 11.7% (p =

Background Cataract is a major cause of severe visual impairment in childhood. The purpose of this study was to determine the genetic cause of syndromic congenital cataract in an Australian mother and son. Method Fifty-one genes associated with congenital cataract were sequenced in the proband using a custom Ampliseq library on the Ion Torrent Personal Genome Machine (PGM). Reads were aligned against the human genome (hg19) and variants were annotated. Variants were prioritised for validation by Sanger sequencing if they were novel, rare or previously reported to be associated with paediatric cataract and were predicted to be protein changing. Variants were assessed for segregation with the phenotype in the affected mother. Result A novel likely pathogenic variant was identified in the transactivation domain of the MAF gene (c.176C > G, p.(Pro59Arg)) in the proband and his affected mother., but was absent in 326 unrelated controls and absent from public variant databases. Conclusion The MAF variant is the likely cause of the congenital cataract, Asperger syndrome, seizures, hearing loss and facial characteristics in the proband, providinga diagnosis of Aymé-Gripp syndrome for the family. Electronic supplementary material The online version of this article (doi:10.1186/s12881-017-0414-7) contains supplementary material, which is available to authorized users.

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Interferon (IFN)-γ can prime macrophages for inflammatory responses by several mechanisms, including enhancer establishment and gene activation. In this issue of Immunity, Kang et al. (2017) provide insight into the mechanisms of IFN-γ-mediated gene repression as they show that IFN-γ promotes the disassembly of select active enhancers by interfering with enhancer-binding transcription factor MAF.

The human gene MafF (hMafF) is a member of bZip transcription factor Maf family, but it alone cannot activate its target genes. In 2006, a novel hMafF interacting protein (MIP) was identified. Transient transfection assay in Hela cells suggested that co-expression of MIP and hMafF could activate US2-driven transcription. In this work, we constructed a series of plasmids and transformed YM4271 yeast strain to establish a recombinant yeast detection system. In this system, MIP's expression level could be regulated using glucose incubation or galactose-induced incubation. The expression level of reporter gene LacZ in obtained recombinant yeast strains was measured using quantitative liquid assay. By comparing and analyzing the beta-galactosidase activities of different yeast strains or the same yeast strain in different culture media, the effect of MIP on transactivation driven by nUS2-hMafF was finally determined. Only in the presence of both MIP and hMafF could the nUS2-pLacZi reporter in yeast genome be activated. More importantly, this work established a novel recombinant yeast detection system, which may serve as a powerful tool to study the regulatory mechanisms of transcription complex in the future.

Much has been learned in recent years about the central mechanisms controlling the initiation of mammalian puberty. It is now clear that this process requires the interactive participation of several genes. Using a combination of high throughput, molecular, and bioinformatics strategies, in combination with a system biology approach, we singled out from the hypothalamus of nonhuman primates and rats a group of related genes whose expression increases at the time of female puberty. Although these genes [henceforth termed tumor-related genes (TRGs)] have diverse cellular functions, they share the common feature of having been earlier identified as involved in tumor suppression/tumor formation. A prominent member of this group is KiSS1, a gene recently shown to be essential for the occurrence of puberty. Cis-regulatory analysis revealed the presence of a hierarchically arranged gene set containing five major hubs (CDP/CUTL1, MAF, p53, YY1, and USF2) controlling the network at the transcriptional level. In turn, these hubs are heavily connected to non-TRGs involved in the transcriptional regulation of the pubertal process. TRGs may be expressed in the mammalian hypothalamus as components of a regulatory gene network that facilitates and integrates cellular and cell-cell communication programs required for the acquisition of female reproductive competence.

The Maf family of proteins are a subgroup of basic region-leucine zipper (bZIP) transcription factors, which recognize a long palindromic DNA sequence [TGCTGAC(G)TCAGCA] known as the Maf recognition element (MARE). Interestingly, the functional target enhancer sequences present in the alphaA-crystallin gene contain a well-conserved half-site of MARE rather than the entire palindromic sequence. To resolve how Maf proteins bind to target sequences containing only MARE half-sites, we examined their binding activities using electrophoretic gel mobility shift assays as well as in vitro and in vivo reporter assays. Our results indicate that the 5'-flanking region of the MARE half-site is required for Maf proteins to bind both in vitro and in vivo. The critical 5'-flanking sequences for c-Maf were determined by a selection and amplification binding assay and show a preference for AT-rich nucleotides. Furthermore, sequence analysis of the regulatory regions of several target genes also suggests that AT-rich sequences are important. We conclude that Maf can bind to at least two types of target sequences, the classical MARE (palindrome type) and a 5'-AT-rich MARE half-site (half-site type). Our results provide important new insights into the DNA binding and site selection by bZIP transcription factors.

The murine autosomal dominant cataract mutants created in mutagenesis experiments have proven to be a powerful resource for modelling the biological processes involved in cataractogenesis. We report a mutant which in the heterozygous state exhibits mild pulverulent cataract named 'opaque flecks in lens', symbol Ofl. By molecular mapping, followed by a candidate gene approach, the mutant was shown to be allelic with a knockout of the bZIP transcription factor, Maf. Homozygotes for Ofl and for Maf null mutations are similar but a new effect, renal tubular nephritis, was found in Ofl homozygotes surviving beyond 4 weeks, which may contribute to early lethality. Sequencing identified the mutation as a G-->A change, leading to the amino-acid substitution mutation R291Q in the basic region of the DNA-binding domain. Since mice heterozygous for knockouts of Maf show no cataracts, this suggests that the Ofl R291Q mutant protein has a dominant effect. We have demonstrated that this mutation results in a selective alteration in DNA binding affinities to target oligonucleotides containing variations in the core CRE and TRE elements. This implies that arginine 291 is important for core element binding and suggests that the mutant protein may exert a differential downstream effect amongst its binding targets. The cataracts seen in Ofl heterozygotes and human MAF mutations are similar to one another, implying that Ofl may be a model of human pulverulent cortical cataract. Furthermore, when bred onto a different genetic background Ofl heterozygotes also show anterior segment abnormalities. The Ofl mutant therefore provides a valuable model system for the study of Maf, and its interacting factors, in normal and abnormal lens and anterior segment development.

Mechanisms by which interferon (IFN)-γ activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN-γ-mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription factor binding, and gene expression in IFN-γ-primed human macrophages. IFN-γ suppressed basal expression of genes corresponding to an ‘M2’-like homeostatic and reparative phenotype. IFN-γ repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN-γ disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage-determining transcription factors, and chromatin accessibility. Genes associated with MAF-binding enhancers were suppressed in macrophages isolated from rheumatoid arthritis patients, revealing a disease-associated signature of IFN-γ–mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN-γ-mediated gene repression, and reveal MAF as a regulator of the macrophage enhancer landscape that is suppressed by IFN-γ to augment macrophage activation.

Osteoporosis is a common, age-related bone disease that results from an imbalance between the processes of bone formation and bone resorption, resulting in reduced bone mass and increased risk of fracture. Mesenchymal stem cells have the capacity to differentiate into osteoblastic and adipogenic lineages; recent research suggests that the switch between these two fates may be key to the decreased bone density that occurs with aging. In this issue, Nishikawa et al. demonstrate that the basic leucine-zipper transcription factor Maf (also known as c-Maf) is central to osteoblast lineage commitment. In addition, they find that increased oxidative stress - as occurs with aging - decreases Maf expression. This work advances understanding of the transcriptional regulation of cell fate decisions and may help direct the development of new therapies to fight age-related bone loss.

The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation. We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated Myb:Maf status seen in differentiating cells can be recapitulated by overexpression of c-Maf in myeloid cell lines, we inducibly expressed the c-Maf cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of c-Maf protein led to marked increases in Myb:Maf complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that c-Maf likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of c-Myb regulated targets, such as Bcl-2 and CD13/APN, coupled with the activation of as yet undefined differentiation-promoting genes.

Primary Myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm characterized by the presence of hyperplastic/dysplastic megakaryocytes and the development of myelofibrosis and osteosclerosis. This disruption of the bone marrow (BM) structure results from a deregulated release of growth factors by malignant hematopoietic cells, particularly monocytes and megakaryocytes, that activate BM stromal cells (Desterke-C, Mediators Inflamm, 2015). Therefore, it remains of primary importance to identify novel therapeutic strategies to restore BM microenvironment homeostasis in PMF patients. In order to better characterize the molecular mechanisms underlying PMF pathogenesis, we have recently compared the gene expression profile (GEP) of CD34+ hematopoietic progenitor cells (HPCs) from PMF patients and healthy donors, and we observed the upregulation of the transcription factor MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) (Norfo-R, Blood, 2014). With the aim to investigate the role of MAF in PMF pathogenesis and to reproduce the condition observed in PMF CD34+ cells, we studied the effects of MAF overexpression on HPCs differentiation. Human cord blood-derived CD34+ cells were transduced with a retroviral vector carrying the coding sequences for the two MAF transcript variants (LMAFVAR1IDN and LMAFVAR2IDN). The megakaryocyte lineage-committed CD41+ population was increased in LMAFVAR1IDN (38.2±2.3%) and LMAFVAR2IDN (39.4±2.3%) samples compared with the empty vector LXIDN (16.1±2.7%) at day 3 after NGFR+ cells purification. Values are reported as mean ± SEM from 3 independent experiments, p Next, we analyzed the GEP of MAF-overexpressing CD34+ cells and observed the upregulation of genes involved in megakaryopoiesis, such as MERTK and RCAN1, and in monocyte/macrophage differentiation (e.g. EGR2 and KLF4). In particular, GEP revealed the upregulation of genes related to processes deregulated in PMF, such as fibrosis (i.e. ENPP2, ADAM9) and inflammation (i.e. TNF, CCL2 and CCL3). Noteworthy, several upregulated transcripts encoded for secreted proteins. Based on their role in inflammation and fibrosis, we selected a set of secreted molecules (IL8, MMP9, CCL2, SPP1, LGALS3 and PLAUR) and assessed them by quantitative enzyme-linked immunoassay (ELISA) in the culture supernatants from LMAFVAR1IDN-, LMAFVAR2IDN- and LXIDN-transduced cells. The levels in culture supernatants of all these molecules were increased in MAF-overexpressing samples compared to control. In order to correlate the expression of these molecules with MAF upregulation in PMF, we assessed them in plasma samples of PMF patients (n=30) and healthy donors (n=10). All the selected molecules displayed higher plasma levels in PMF patients than in healthy controls (Figure 1). Since the increase of SPP1 and LGALS3 has never been described before in PMF, we investigated which cell type could be responsible for SPP1 and LGALS3 production. Real Time qRTPCR performed on healthy donors peripheral blood-derived cells showed that the highest SPP1 expression levels were observed in CD34+ cells-derived megakaryocytes and granulocytes, whereas LGALS3 was expressed at higher levels in monocytes compared to the other cell types. Because the increased proliferation of fibroblasts is involved in BM fibrosis and is a PMF feature, we investigated the effects of LGALS3 and SPP1 on normal human primary dermal fibroblasts. The treatment with recombinant human LGALS3 (1000 ng/ml) or SPP1 (500 ng/ml) caused an increase in fibroblasts cell count of 20.9±2.7% (p Finally, we observed that increased SPP1 plasma levels in PMF patients correlate with a more severe fibrosis and lower overall survival (p In conclusion, our data unveil the involvement of MAF in PMF pathogenesis through the induction of a pro-fibrotic and pro-inflammatory phenotype in the malignant myeloid differentiated progeny. Disclosures Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

BackgroundAccuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures.MethodsA resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis.ResultsThe two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively). The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real-time qRT-PCR supported the meta-analysis results.ConclusionThe proposed meta-analysis approach has the ability to detect a set of differentially expressed genes with the least amount of within-group variability, thus providing highly stable gene lists for class prediction. Increased statistical power and stringent filtering criteria used in the present study also make identification of novel candidate genes possible and may provide further insight to improve our understanding of breast cancer development.

Background Inflammation is a key feature of asthma and allergic disease. The proinflammatory cytokines IL-4 , IL-5 , and IL-13 are clustered on chromosome 5q with GM-CSF in close proximity, and each of these cytokines has been implicated in the pathogenesis of inflammatory disease. Although the expression of IL-4, IL-5, and IL-13 is coordinately regulated, the T H 2-associated transcription factor c-Maf is thought to be involved only in the regulation of IL-4, the cytokine thought to be the main driver of T H 2 differentiation. Objective We sought to determine whether c-Maf influenced the expression of proinflammatory cytokines other than IL-4 in the Jurkat human T-cell line. Methods RT-PCR, ELISA, and promoter-driven CAT assays were used to determine the effect of c-Maf overexpression on cytokine genes. A biotinylated oligo pulldown assay was used to demonstrate recruitment of c-Maf to the GM-CSF promoter. Results We found that in addition to induction of IL-4, c-Maf could upregulate GM-CSF expression at both mRNA and protein levels, and that c-Maf could strongly activate the promoters of GM-CSF and IL-4 but not IL-5 . Recruitment of c-Maf to the -33 to -97 bp region of the GM-CSF promoter was demonstrated. Conclusion We propose a novel role for c-Maf in the transcriptional regulation of GM-CSF in human T cells. Clinical implications These data suggest that c-Maf may be a therapeutic target affecting both IL-4 and GM-CSF.

Aims: To report the detailed clinical findings in a three generation pedigree with autosomal dominant cataract, microcornea, and coloboma resulting from mutation of the lens development gene, MAF. Methods: Five members of a three generation pedigree with progressive cataracts underwent detailed ophthalmic examination to characterise associated ocular phenotypic features. Results: The cataracts present in all affected individuals were cortical, and/or nuclear, pulverulent opacities. Corneal diameters of 10–10.25 mm were present in two family members. Axial lengths were in the normal range. Bilateral iris coloboma in the 6 o'clock position was present in one patient. Uveal melanoma was present in one patient, with uveal naevi in this and one other patient. Conclusion: The bZIP transcription factor MAF is a key lens development gene that regulates the expression of the crystallins. Individuals with a mutation in MAF may have pulverulent cataract alone or cataract in association with microcornea or iris coloboma.

Human congenital cataract and ocular anterior segment dysgenesis both demonstrate extensive genetic and phenotypic heterogeneity. We identified a family where ocular developmental abnormalities (cataract, anterior segment dysgenesis and microphthalmia) co-segregated with a translocation, t(5;16)(p15.3;q23.2), in both balanced and unbalanced forms. We hypothesized that this altered the expression of a gene of developmental significance in the human lens and ocular anterior segment. Cloning the 16q23.2 breakpoint demonstrated that it transected the genomic-control domain of MAF, a basic region leucine zipper (bZIP) transcription factor, first identified as an oncogene, which is expressed in vertebrate lens development and regulates the expression of the eye lens crystallins. The homozygous null mutant Maf mouse embryo demonstrates defective lens formation and microphthalmia. Through mutation screening of a panel of patients with hereditary congenital cataract we identified a mutation in MAF in a three-generation family with cataract, microcornea and iris coloboma. The mutation results in the substitution of an evolutionarily highly conserved arginine with a proline at residue 288 (R288P) in the basic region of the DNA-binding domain of MAF. Our findings further implicate MAF/Maf in mammalian lens development and highlight the role of the lens in anterior segment development. The 16q23.2 breakpoint transects the common fragile site, FRA16D, providing a molecular demonstration of a germline break in a common fragile site.