TGF-ß specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf.

T follicular helper (TFH) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse TFH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-ß (TGF-ß) induces robust expression of TFH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4+ T cells in vitro. TGF-ß-induced mouse CXCR5+ TFH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated TFH cells and provide critical help to B cells. The study further reveals that TGF-ß-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-ß-containing T helper 17 (TH17)-inducing conditions also yield separate CXCR5+ and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of TFH and TH17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-ß-induced TFH cell program, that TFH and TH17 cells share a common developmental stage, and that c-Maf acts as a switch factor for TFH versus TH17 cell fates in TGF-ß-rich environments in vitro and in vivo.

Discovery of small molecule c-Maf inhibitors using molecular docking-based virtual screening, molecular dynamics simulation, and biological evaluation.

Multiple myeloma (MM) is a prevalent plasma cell malignancy in the blood system that remains incurable. Given the abnormally high expression of c-Maf in most MM patients, targeting c-Maf presents an attractive therapeutic approach for treating MM malignancies. In this study, we employed a combined strategy involving molecular docking-based virtual screening, molecular dynamics (MD) simulation, and molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculation on existing FDA-approved drugs. Six compounds were selected for further experimental assay: vemurafenib, sorafenib, sildenafil, fluvastatin, erlotinib, and glimepiride. Among these compounds, sorafenib and glimepiride exhibited significant inhibition of myeloma cell proliferation in the RPMI-8226 cell line. Moreover, both compounds simultaneously downregulated c-Maf protein expression to induce G1 phase arrest and apoptosis in myeloma cells. Collectively, sorafenib and glimepiride may be considered promising candidates for developing more potent c-Maf inhibitors in the future.

MAF amplification licenses ERα through epigenetic remodelling to drive breast cancer metastasis.

MAF amplification increases the risk of breast cancer (BCa) metastasis through mechanisms that are still poorly understood yet have important clinical implications. Oestrogen-receptor-positive (ER+) BCa requires oestrogen for both growth and metastasis, albeit by ill-known mechanisms. Here we integrate proteomics, transcriptomics, epigenomics, chromatin accessibility and functional assays from human and syngeneic mouse BCa models to show that MAF directly interacts with oestrogen receptor alpha (ERα), thereby promoting a unique chromatin landscape that favours metastatic spread. We identify metastasis-promoting genes that are de novo licensed following oestrogen exposure in a MAF-dependent manner. The histone demethylase KDM1A is key to the epigenomic remodelling that facilitates the expression of the pro-metastatic MAF/oestrogen-driven gene expression program, and loss of KDM1A activity prevents this metastasis. We have thus determined that the molecular basis underlying MAF/oestrogen-mediated metastasis requires genetic, epigenetic and hormone signals from the systemic environment, which influence the ability of BCa cells to metastasize.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

Identification of small compounds that inhibit multiple myeloma proliferation by targeting c-Maf transcriptional activity.

Multiple myeloma displays the clonal B cell expansion and the overproduction of monoclonal immunoglobulins. Genetic translocations at 14q32, particularly with partners like 16q23, lead to the dysregulation of oncogene expression, including the significant enhancement of c-Maf. This aberrant expression of c-Maf has prompted research into strategies for targeting this transcription factor as a potential therapeutic avenue for multiple myeloma treatment. In this study, we introduce a screening pipeline to test small compounds for their ability to inhibit c-Maf. Using a luciferase indicator driven by the Ccl8 gene promoter, we identified two small compounds that inhibit transcriptional activity of c-Maf. These molecules impede the proliferation of c-Maf-expressing myeloma cells, and repress the expression of c-Maf target genes such as ITGB7 and CCR1. Importantly, these molecules target c-Maf-expressing multiple myeloma cells, but not c-Maf-negative myeloma cells, showing potential for tailoring therapeutic intervention. In conclusion, our screening pipeline is effective to explore leads for a novel c-Maf inhibitor for multiple myeloma therapy.

RORγt+ c-Maf+ Vγ4+ γδ T cells are generated in the adult thymus but do not reach the periphery.

T cell receptor (TCR) Vγ4-expressing γδ T cells comprise interferon γ (IFNγ)- and interleukin-17 (IL-17)-producing effector subsets, with a preference for IL-17 effector fate decisions during early ontogeny. The existence of adult-thymus-derived IL-17+ T cells (γδ17) remains controversial. Here, we use a mouse model in which T cells are generated exclusively in the adult thymus and employ single-cell chromatin state analysis to study their development. We identify adult-thymus-derived Vγ4 T cells that have all the molecular programs to become IL-17 producers. However, they have reduced IL-17 production capabilities and rarely reach the periphery. Moreover, this study provides high-resolution profiles of Vγ4 T cells in the adult thymus and lymph nodes and identifies Zeb1 as a potential γδ17 cell regulator. Together, this study provides valuable insights into the developmental traits of Vγ4 T cells during adulthood and supports the idea of age-specific signals required for thymic export and/or peripheral maturation of γδ17 cells.

Large Maf transcription factor family is a major regulator of fast type IIb myofiber determination.

Myofibers are broadly characterized as fatigue-resistant slow-twitch (type I) fibers and rapidly fatiguing fast-twitch (type IIa/IIx/IIb) fibers. However, the molecular regulation of myofiber type is not entirely understood; particularly, information on regulators of fast-twitch muscle is scarce. Here, we demonstrate that the large Maf transcription factor family dictates fast type IIb myofiber specification in mice. Remarkably, the ablation of three large Mafs leads to the drastic loss of type IIb myofibers, resulting in enhanced endurance capacity and the reduction of muscle force. Conversely, the overexpression of each large Maf in the type I soleus muscle induces type IIb myofibers. Mechanistically, a large Maf directly binds to the Maf recognition element on the promoter of myosin heavy chain 4, which encodes the type IIb myosin heavy chain, driving its expression. This work identifies the large Maf transcription factor family as a major regulator for fast type IIb muscle determination.

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy.

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.

Dynamic behavior of P53-Mdm2-Wip1 gene regulatory network under the influence of time delay and noise.

The tumor suppressor protein P53 can regulate the cell cycle, thereby preventing cell abnormalities. In this paper, we study the dynamic characteristics of the P53 network under the influence of time delay and noise, including stability and bifurcation. In order to study the influence of several factors on the concentration of P53, bifurcation analysis on several important parameters is conducted; the results show that the important parameters could induce P53 oscillations within an appropriate range. Then we study the stability of the system and the existing conditions of Hopf bifurcation by using Hopf bifurcation theory with time delays as the bifurcation parameter. It is found that time delay plays a key role in inducing Hopf bifurcation and regulating the period and amplitude of system oscillation. Meanwhile, the combination of time delays can not only promote the oscillation of the system but it also provides good robustness. Changing the parameter values appropriately can change the bifurcation critical point and even the stable state of the system. In addition, due to the low copy number of the molecules and the environmental fluctuations, the influence of noise on the system is also considered. Through numerical simulation, it is found that noise not only promotes system oscillation but it also induces system state switching. The above results may help us to further understand the regulation mechanism of the P53-Mdm2-Wip1 network in the cell cycle.

Transcription factor c-Maf deletion improves streptozotocin-induced diabetic nephropathy by directly regulating Sglt2 and Glut2.

The transcription factor c-Maf has been widely studied and has been reported to play a critical role in embryonic kidney development; however, the postnatal functions of c-Maf in adult kidneys remain unknown as c-Maf-null C57BL/6J mice exhibit embryonic lethality. In this study, we investigated the role of c-Maf in adult mouse kidneys by comparing the phenotypes of tamoxifen-inducible (TAM-inducible) c-Maf-knockout mice (c-Maffl/fl; CAG-Cre-ERTM mice named "c-MafΔTAM") with those of c-Maffl/fl control mice, 10 days after TAM injection [TAM(10d)]. In addition, we examined the effects of c-Maf deletion on diabetic conditions by injecting the mice with streptozotocin, 4 weeks before TAM injection. c-MafΔTAM mice displayed primary glycosuria caused by sodium-glucose cotransporter 2 (Sglt2) and glucose transporter 2 (Glut2) downregulation in the kidneys without diabetes, as well as morphological changes and life-threatening injuries in the kidneys on TAM(10d). Under diabetic conditions, c-Maf deletion promoted recovery from hyperglycemia and suppressed albuminuria and diabetic nephropathy by causing similar effects as did Sglt2 knockout and SGLT2 inhibitors. In addition to demonstrating the potentially unique gene regulation of c-Maf, these findings highlight the renoprotective effects of c-Maf deficiency under diabetic conditions and suggest that c-Maf could be a novel therapeutic target gene for treating diabetic nephropathy.

Structural basis for the toxic activity of MafB2 from maf genomic island 2 (MGI-2) in N. meningitidis B16B6.

The Maf polymorphic toxin system is involved in conflict between strains found in pathogenic Neisseria species such as Neisseria meningitidis and Neisseria gonorrhoeae. The genes encoding the Maf polymorphic toxin system are found in specific genomic islands called maf genomic islands (MGIs). In the MGIs, the MafB and MafI encode toxin and immunity proteins, respectively. Although the C-terminal region of MafB (MafB-CT) is specific for toxic activity, the underlying enzymatic activity that renders MafB-CT toxic is unknown in many MafB proteins due to lack of homology with domain of known function. Here we present the crystal structure of the MafB2-CTMGI-2B16B6/MafI2MGI-2B16B6 complex from N. meningitidis B16B6. MafB2-CTMGI-2B16B6 displays an RNase A fold similar to mouse RNase 1, although the sequence identity is only ~ 14.0%. MafB2-CTMGI-2B16B6 forms a 1:1 complex with MafI2MGI-2B16B6 with a Kd value of ~ 40 nM. The complementary charge interaction of MafI2MGI-2B16B6 with the substrate binding surface of MafB2-CTMGI-2B16B6 suggests that MafI2MGI-2B16B6 inhibits MafB2-CTMGI-2B16B6 by blocking access of RNA to the catalytic site. An in vitro enzymatic assay showed that MafB2-CTMGI-2B16B6 has ribonuclease activity. Mutagenesis and cell toxicity assays demonstrated that His335, His402 and His409 are important for the toxic activity of MafB2-CTMGI-2B16B6, suggesting that these residues are critical for its ribonuclease activity. These data provide structural and biochemical evidence that the origin of the toxic activity of MafB2MGI-2B16B6 is the enzymatic activity degrading ribonucleotides.

Protein kinase CK2 modulates the activity of Maf-family bZIP transcription factor NRL in rod photoreceptors of mammalian retina.

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate interdependence of transcriptional and signaling pathways in maintaining rod homeostasis.

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.

Intestinal epithelial c-Maf expression determines enterocyte differentiation and nutrient uptake in mice.

The primary function of the small intestine (SI) is to absorb nutrients to maintain whole-body energy homeostasis. Enterocytes are the major epithelial cell type facilitating nutrient sensing and uptake. However, the molecular regulators governing enterocytes have remained undefined. Here, we identify c-Maf as an enterocyte-specific transcription factor within the SI epithelium. c-Maf expression was determined by opposing Noggin/BMP signals and overlapped with the zonated enrichment of nutrient transporters in the mid-villus region. Functionally, enterocytes required c-Maf to appropriately differentiate along the villus axis. Specifically, gene programs controlling carbohydrate and protein absorption were c-Maf-dependent. Consequently, epithelial cell-specific c-Maf deletion resulted in impaired enterocyte maturation and nutrient uptake, including defects in the adaptation to different nutrient availability. Concomitantly, intraepithelial lymphocytes were less abundant, while commensal epithelial cell-attaching SFB overgrew in a c-Maf-deficient environment, highlighting the close interdependence between the intestinal epithelium, immune system, and microbiota. Collectively, our data identified c-Maf as a key regulator of SI enterocyte differentiation and function, essential for nutrient, immune, and microbial homeostasis.

Broadening the genotypic and phenotypic spectrum of MAF in three Chinese Han congenital cataracts families.

Pathogenic variants in the v-maf avian musculoaponeurotic fibrosarcoma oncogene homologue (MAF) encoding a transcription factor (from a unique subclass of basic leucine zipper transcription factors) are associated with isolated congenital cataracts (CCs) and Aymé-Gripp syndrome (AYGRPS). We collected detailed disease histories from, and performed comprehensive ophthalmic and systemic examinations in 269 patients with CCs; we then performed whole-exome sequencing. Pathogenicity assessments were evaluated using multiple predictive tools. The clinical validities of the reported gene-disease relationships for MAF genes (MAF-CCs and MAF-AYGRPS) were assessed using the ClinGen gene curation framework. We identified two novel (c.173C>A, p.Thr58Asn and c.947T>C, p. Leu316Pro) variants and one known (c.173C>T, p.Thr58Ile) MAF missense variant in three patients. We described novel phenotypes including cleft palate, macular hypoplasia, and retinal neovascularization in the peripheral avascular area and analyzed the genotype-phenotype correlations. We demonstrated associations of variants in the MAF C-terminal DNA-binding domain with CCs and associations of variants in the N-terminal transactivation domain of MAF with AYGRPS. We thus expand the genotypic and phenotypic spectrum of the MAF gene. The ClinGen gene curation framework results suggested that variants in different domains of MAF are associated with different diseases.

Differential metabolic requirement governed by transcription factor c-Maf dictates innate γδT17 effector functionality in mice and humans.

Cellular metabolism has been proposed to govern distinct γδ T cell effector functions, but the underlying molecular mechanisms remain unclear. We show that interleukin-17 (IL-17)-producing γδ T (γδT17) and interferon-γ (IFN-γ)-producing γδ T (γδT1) cells have differential metabolic requirements and that the rate-limiting enzyme isocitrate dehydrogenase 2 (IDH2) acts as a metabolic checkpoint for their effector functions. Intriguingly, the transcription factor c-Maf regulates γδT17 effector function through direct regulation of IDH2 promoter activity. Moreover, mTORC2 affects the expression of c-Maf and IDH2 and subsequent IL-17 production in γδ T cells. Deletion of c-Maf in γδ T cells reduces metastatic lung cancer development, suggesting c-Maf as a potential target for cancer immune therapy. We show that c-Maf also controls IL-17 production in human γδ T cells from peripheral blood and in oral cancers. These results demonstrate a critical role of the transcription factor c-Maf in regulating γδT17 effector function through IDH2-mediated metabolic reprogramming.

c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 innate lymphoid cells.

Group-2 innate lymphoid cells (ILC2s), which are involved in type 2 inflammatory diseases such as allergy, can exhibit immunological memory, but the basis of this ILC2 "trained immunity" has remained unclear. Here, we found that stimulation with IL-33/IL-25 or exposure to the allergen papain induces the expression of the transcription factor c-Maf in mouse ILC2s. Chronic papain exposure results in high production of IL-5 and IL-13 cytokines and lung eosinophil recruitment, effects that are blocked by c-Maf deletion in ILCs. Transcriptomic analysis revealed that knockdown of c-Maf in ILC2s suppresses expression of type 2 cytokine genes, as well as of genes linked to a memory-like phenotype. Consistently, c-Maf was found highly expressed in human adult ILC2s but absent in cord blood and required for cytokine production in isolated human ILC2s. Furthermore, c-Maf-deficient mouse or human ILC2s failed to exhibit strengthened ("trained") responses upon repeated challenge. Thus, the expression of c-Maf is indispensable for optimal type 2 cytokine production and proper memory-like responses in group-2 innate lymphoid cells.

Molecular Mechanisms Driving IL-10- Producing B Cells Functions: STAT3 and c-MAF as Underestimated Central Key Regulators?

Regulatory B cells (Bregs) have been highlighted in very different pathology settings including autoimmune diseases, allergy, graft rejection, and cancer. Improving tools for the characterization of Bregs has become the main objective especially in humans. Transitional, mature B cells and plasma cells can differentiate into IL-10 producing Bregs in both mice and humans, suggesting that Bregs are not derived from unique precursors but may arise from different competent progenitors at unrestricted development stages. Moreover, in addition to IL-10 production, regulatory B cells used a broad range of suppressing mechanisms to modulate the immune response. Although Bregs have been consistently described in the literature, only a few reports described the molecular aspects that control the acquisition of the regulatory function. In this manuscript, we detailed the latest reports describing the control of IL-10, TGFß, and GZMB production in different Breg subsets at the molecular level. We focused on the understanding of the role of the transcription factors STAT3 and c-MAF in controlling IL-10 production in murine and human B cells and how these factors may represent an important crossroad of several key drivers of the Breg response. Finally, we provided original data supporting the evidence that MAF is expressed in human IL-10- producing plasmablast and could be induced in vitro following different stimulation cocktails. At steady state, we reported that MAF is expressed in specific human B-cell tonsillar subsets including the IgD+ CD27+ unswitched population, germinal center cells and plasmablast.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

c-Maf: The magic wand that turns on LSEC fate.

In this issue of Cell Stem Cell, Gómez-Salinero et al. (2022) identify c-Maf as a driver for murine liver sinusoidal endothelial cell (LSEC) fate and function during liver development, homeostasis, and repair. Similarly, c-Maf defines human LSECs, and its overexpression specializes generic HUVECs into functional induced-LSECs, potentiating regenerative therapeutics.