Maf family transcription factors are required for nutrient uptake in the mouse neonatal gut.

There are fundamental differences in how neonatal and adult intestines absorb nutrients. In adults, macromolecules are broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely on internalization of whole macromolecules and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors MAFB and c-MAF as markers of terminally differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α and HNF4γ, master regulators of enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of macromolecular nutrient uptake. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number, in response to loss of Maf proteins. Finally, we show that loss of BLIMP1, a repressor of adult enterocyte genes, shows highly overlapping changes in gene expression and similar defects in macromolecular uptake. This work defines transcriptional regulators that are necessary for nutrient uptake in neonatal enterocytes.

Mafba and Mafbb regulate microglial colonization of zebrafish brain via controlling chemotaxis receptor expression.

Microglia are the central nervous system (CNS)-resident macrophages involved in neural inflammation, neurogenesis, and neural activity regulation. Previous studies have shown that naturally occurring neuronal apoptosis plays a critical role in regulating microglial colonization of the brain in zebrafish. However, the molecular signaling cascades underlying neuronal apoptosis-mediated microglial colonization and the regulation of these cascades remain undefined. Here, we show that basic leucine zipper (b-Zip) transcription factors, Mafba and Mafbb, two zebrafish orthologs of mammalian MAFB, are key regulators in neuronal apoptosis-mediated microglial colonization of the brain in zebrafish. We document that the loss of Mafba and Mafbb function perturbs microglial colonization of the brain. We further demonstrate that Mafba and Mafbb act cell-autonomously and cooperatively to orchestrate microglial colonization, at least in part, by regulating the expression of G protein-coupled receptor 34a (Gpr34a), which directs peripheral macrophage recruitment into the brain through sensing the lysophosphatidylserine (lysoPS) released by the apoptotic neurons. Our study reveals that Mafba and Mafbb regulate neuronal apoptosis-mediated microglial colonization of the brain in zebrafish via the lysoPS-Gpr34a pathway.

Zebrafish mafbb Mutants Display Osteoclast Over-Activation and Bone Deformity Resembling Osteolysis in MCTO Patients.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia with osteolysis at the carpal and tarsal bones. Heterozygous missense mutations in the transcription factor MAFB are found in patients with MCTO. MAFB is reported to negatively regulate osteoclastogenesis in vitro. However, the in vivo function of MAFB and its relation to MCTO remains unknown. In this study, we generated zebrafish MAFB homolog mafbb mutant utilizing CRISPR/Cas9 technology. Mafbb deficient zebrafish demonstrated enhanced osteoclast cell differentiation and abnormal cartilage and bone development resembling MCTO patients. It is known that osteoclasts are hematopoietic cells derived from macrophages. Loss of mafbb caused selective expansion of definitive macrophages and myeloid cells, supporting that mafbb restricts myeloid differentiation in vivo. We also demonstrate that MAFB MCTO mutations failed to rescue the defective osteoclastogenesis in mafbb-/- embryos, but did not affect osteoclast cells in wild type embryos. The mechanism of MCTO mutations is likely haploinsufficiency. Zebrafish mafbb mutant provides a useful model to study the function of MAFB in osteoclastogenesis and the related MCTO disease.

Aberrant TGF-ß1 signaling activation by MAF underlies pathological lens growth in high myopia.

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 ß/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 ß/γ-crystallins in high myopia, by direct activation of the crystallin gene promoters and by activation of TGF-ß1-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-ß1-crystallin axis as an underlying mechanism, providing an insight for therapeutic interventions.

MAFB and MAF Transcription Factors as Macrophage Checkpoints for COVID-19 Severity.

Defective IFN production and exacerbated inflammatory and pro-fibrotic responses are hallmarks of SARS-CoV-2 infection in severe COVID-19. Based on these hallmarks, and considering the pivotal role of macrophages in COVID-19 pathogenesis, we hypothesize that the transcription factors MAFB and MAF critically contribute to COVID-19 progression by shaping the response of macrophages to SARS-CoV-2. Our proposal stems from the recent identification of pathogenic lung macrophage subsets in severe COVID-19, and takes into consideration the previously reported ability of MAFB to dampen IFN type I production, as well as the critical role of MAFB and MAF in the acquisition and maintenance of the transcriptional signature of M-CSF-conditioned human macrophages. Solid evidences are presented that link overexpression of MAFB and silencing of MAF expression with clinical and biological features of severe COVID-19. As a whole, we propose that a high MAFB/MAF expression ratio in lung macrophages could serve as an accurate diagnostic tool for COVID-19 progression. Indeed, reversing the macrophage MAFB/MAF expression ratio might impair the exacerbated inflammatory and profibrotic responses, and restore the defective IFN type I production, thus becoming a potential strategy to limit severity of COVID-19.

Chicken lens development: complete signature of expression of galectins during embryogenesis and evidence for their complex formation with α-, ß-, δ-, and τ-crystallins, N-CAM, and N-cadherin obtained by affinity chromatography.

The emerging multifunctionality of galectins by specific protein-glycan/protein interactions explains the interest to determine their expression during embryogenesis. Complete network analysis of all seven chicken galectins (CGs) is presented in the course of differentiation of eye lens that originates from a single type of progenitor cell. It answers the questions on levels of expression and individual patterns of distribution. A qualitative difference occurs in the CG-1A/B paralogue pair, underscoring conspicuous divergence. Considering different cell phenotypes, lens fiber and also epithelial cells can both express the same CG, with developmental upregulation for CG-3 and CG-8. Except for expression of the lens-specific CG (C-GRIFIN), no other CG appeared to be controlled by the transcription factors L-Maf and Pax6. Studying presence and nature of binding partners for CGs, we tested labeled galectins in histochemistry and in ligand blotting. Mass spectrometric (glyco)protein identification after affinity chromatography prominently yielded four types of crystallins, N-CAM, and, in the cases of CG-3 and CG-8, N-cadherin. Should such pairing be functional in situ, it may be involved in tightly packing intracellular lens proteins and forming membrane contact as well as in gaining plasticity and stability of adhesion processes. The expression of CGs throughout embryogenesis is postulated to give meaning to spatiotemporal alterations in the local glycome.

Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection.

BACKGROUND: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. RESULTS: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. CONCLUSION: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.

IL-27 promotes NK cell effector functions via Maf-Nrf2 pathway during influenza infection.

Influenza virus targets epithelial cells in the upper respiratory tract. Natural Killer (NK) cell-mediated early innate defense responses to influenza infection include the killing of infected epithelial cells and generation of anti-viral cytokines including interferon gamma (IFN-γ). To date, it is unclear how the underlying cytokine milieu during infection regulates NK cell effector functions. Our data show during influenza infection myeloid cell-derived IL-27 regulates the early-phase effector functions of NK cells in the bronchioalveolar and lung tissue. Lack of IL-27R (Il27ra-/-) or IL-27 (Ebi3-/-) resulted in impaired NK cell effector functions including the generation of anti-viral IFN-γ responses. We identify CD27+CD11b+ NK cells as the primary subset that expresses IL-27R, which predominantly produces IFN-γ within the upper respiratory tract of the infected mice. IL-27 alone was incapable of altering the effector functions of NK cells. However, IL-27 sensitizes NK cells to augment both in vitro and in vivo responses mediated via the NKG2D receptor. This 'priming' function of IL-27 is mediated partly via transcriptional pathways regulated by Mafs and Nrf2 transcriptionally regulating TFAM and CPT1. Our data for the first time establishes a novel role for IL-27 in regulating early-phase effector functions of NK cells during influenza infection.

A dynamic and integrated epigenetic program at distal regions orchestrates transcriptional responses to VEGFA.

Cell behaviors are dictated by epigenetic and transcriptional programs. Little is known about how extracellular stimuli modulate these programs to reshape gene expression and control cell behavioral responses. Here, we interrogated the epigenetic and transcriptional response of endothelial cells to VEGFA treatment and found rapid chromatin changes that mediate broad transcriptomic alterations. VEGFA-responsive genes were associated with active promoters, but changes in promoter histone marks were not tightly linked to gene expression changes. VEGFA altered transcription factor occupancy and the distal epigenetic landscape, which profoundly contributed to VEGFA-dependent changes in gene expression. Integration of gene expression, dynamic enhancer, and transcription factor occupancy changes induced by VEGFA yielded a VEGFA-regulated transcriptional regulatory network, which revealed that the small MAF transcription factors are master regulators of the VEGFA transcriptional program and angiogenesis. Collectively these results revealed that extracellular stimuli rapidly reconfigure the chromatin landscape to coordinately regulate biological responses.

Chicken GRIFIN: binding partners, developmental course of localization and activation of its lens-specific gene expression by L-Maf/Pax6.

Tissue lectins appear to be involved in a broad range of physiological processes, as reflected for the members of the family of galectins by referring to them as adhesion/growth-regulatory effectors. In order to clarify the significance of galectin presence, key challenges are to define their binding partners and the profile of localization. Having identified the chicken galectin-related interfiber protein (C-GRIFIN) as lens-specific protein present in the main body of adult lens, we here report its interaction with lens proteins in ligand blotting. The assumption for pairing with α-, ß- and δ-crystallins was ascertained by mass spectrometric detection of their presence in eluted fractions obtained by affinity chromatography. Biochemical and immunohistochemical monitoring revealed protein presence from about 3-day-old embryos onwards, mostly in the cytoplasm of elongated posterior cells, later in secondary lens fiber cells. On the level of gene expression, its promoter was activated by transcription factor L-Maf alone and together with Pax6 like a crystallin gene, substantiating C-GRIFIN's status as lens-specific galectin. Using this combined strategy for counterreceptor and expression profiling by bio- and histochemical methods including light, electron and fluorescence microscopy, respective monitoring in lens development can now be taken to the level of the complete galectin family.

The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis.

The Kelch-like ECH-associated protein 1-NF-E2-related factor 2 (KEAP1-NRF2) system forms the major node of cellular and organismal defense against oxidative and electrophilic stresses of both exogenous and endogenous origins. KEAP1 acts as a cysteine thiol-rich sensor of redox insults, whereas NRF2 is a transcription factor that robustly transduces chemical signals to regulate a battery of cytoprotective genes. KEAP1 represses NRF2 activity under quiescent conditions, whereas NRF2 is liberated from KEAP1-mediated repression on exposure to stresses. The rapid inducibility of a response based on a derepression mechanism is an important feature of the KEAP1-NRF2 system. Recent studies have unveiled the complexities of the functional contributions of the KEAP1-NRF2 system and defined its broader involvement in biological processes, including cell proliferation and differentiation, as well as cytoprotection. In this review, we describe historical milestones in the initial characterization of the KEAP1-NRF2 system and provide a comprehensive overview of the molecular mechanisms governing the functions of KEAP1 and NRF2, as well as their roles in physiology and pathology. We also refer to the clinical significance of the KEAP1-NRF2 system as an important prophylactic and therapeutic target for various diseases, particularly aging-related disorders. We believe that controlled harnessing of the KEAP1-NRF2 system is a key to healthy aging and well-being in humans.

Nrf2-Keap1 signaling in oxidative and reductive stress.

Nrf2 and its endogenous inhibitor, Keap1, function as a ubiquitous, evolutionarily conserved intracellular defense mechanism to counteract oxidative stress. Sequestered by cytoplasmic Keap1 and targeted to proteasomal degradation in basal conditions, in case of oxidative stress Nrf2 detaches from Keap1 and translocates to the nucleus, where it heterodimerizes with one of the small Maf proteins. The heterodimers recognize the AREs, that are enhancer sequences present in the regulatory regions of Nrf2 target genes, essential for the recruitment of key factors for transcription. In the present review we briefly introduce the Nrf2-Keap1 system and describe Nrf2 functions, illustrate the Nrf2-NF-κB cross-talk, and highlight the effects of the Nrf2-Keap1 system in the physiology and pathophysiology of striated muscle tissue taking into account its role(s) in oxidative stress and reductive stress.

Regulation of tRNA synthesis by the general transcription factors of RNA polymerase III - TFIIIB and TFIIIC, and by the MAF1 protein.

The synthesis of transfer RNA (tRNA) is directed by RNA polymerase III (Pol III) specialized in high-level transcription of short DNA templates. Pol III recruitment to tRNA genes is controlled by two general initiation factors, TFIIIB and TFIIIC. They are multi-protein complexes regulated at the level of expression of individual subunits, as well as through phosphorylation and interaction with partner proteins. Here, we describe particular aspects of TFIIIB and TFIIIC control in yeast and human cells. Under stress conditions, tRNA synthesis is negatively regulated by the MAF1 protein, which interacts directly with Pol III. Sequence and function of MAF1 are conserved among eukaryotic organisms from yeast to humans. MAF1 is a phosphoprotein which mediates diverse regulatory signals to Pol III. Interestingly, there is a subset of housekeeping tRNA genes, both in the yeast and human genome, which are less sensitive to MAF1-dependent repression. The possible mechanisms responsible for this differential regulation of tRNA synthesis by MAF1 are discussed.

The transcription factor Maf-S regulates metabolic resistance to insecticides in the malaria vector Anopheles gambiae.

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 = <0.01), whilst dramatically decreasing mortality induced by the organophosphate malathion (79.6% to 8.0% (p = <0.01)). Additional genes regulated by Maf-S were also identified providing new insight into the role of this transcription factor in insects. CONCLUSION: Maf-S is a key regulator of detoxification genes in Anopheles mosquitoes. Disrupting this transcription factor has opposing effects on the mosquito's response to different insecticide classes providing a mechanistic explanation to the negative cross resistance that has been reported between pyrethroids and organophosphates.

Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome).

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.

Maf1 is a negative regulator of transcription in Trypanosoma brucei.

RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes.

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

MafA is critical for maintenance of the mature beta cell phenotype in mice.

AIMS/HYPOTHESIS: The plasticity of adult somatic cells allows for their dedifferentiation or conversion to different cell types, although the relevance of this to disease remains elusive. Perturbation of beta cell identity leading to dedifferentiation may be implicated in the compromised functions of beta cells in diabetes, which is a current topic of islet research. This study aims to investigate whether or not v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker, is involved in maintaining mature beta cell phenotypes. METHODS: The fate and gene expression of beta cells were analysed in Mafa knockout (KO) mice and mouse models of diabetes in which the expression of MafA was reduced in the majority of beta cells. RESULTS: Loss of MafA reduced the beta to alpha cell ratio in pancreatic islets without elevating blood glucose to diabetic levels. Lineage tracing analyses showed reduced/lost expression of insulin in most beta cells, with a minority of the former beta cells converted to glucagon-expressing cells in Mafa KO mice. The upregulation of genes that are normally repressed in mature beta cells or transcription factors that are transiently expressed in endocrine progenitors was identified in Mafa KO islets as a hallmark of dedifferentiation. The compromised beta cells in db/db and multiple low-dose streptozotocin mice underwent similar dedifferentiation with expression of Mafb, which is expressed in immature beta cells. CONCLUSIONS/INTERPRETATION: The maturation factor MafA is critical for the homeostasis of mature beta cells and regulates cell plasticity. The loss of MafA in beta cells leads to a deeper loss of cell identity, which is implicated in diabetes pathology.