Transcription Factors MAF Research Articles

Single-cell RNA-seq mapping of chicken peripheral blood leukocytes

BackgroundSingle-cell transcriptomics provides means to study cell populations at the level of individual cells. In leukocyte biology this approach could potentially aid the identification of subpopulations and functions without the need to develop species-specific reagents. The present study aimed to evaluate single-cell RNA-seq as a tool for identification of chicken peripheral blood leukocytes. For this purpose, purified and thrombocyte depleted leukocytes from 4 clinically healthy hens were subjected to single-cell 3′ RNA-seq. Bioinformatic analysis of data comprised unsupervised clustering of the cells, and annotation of clusters based on expression profiles. Immunofluorescence phenotyping of the cell preparations used was also performed.ResultsComputational analysis identified 31 initial cell clusters and based on expression of defined marker genes 28 cluster were identified as comprising mainly B-cells, T-cells, monocytes, thrombocytes and red blood cells. Of the remaining clusters, two were putatively identified as basophils and eosinophils, and one as proliferating cells of mixed origin. In depth analysis on gene expression profiles within and between the initial cell clusters allowed further identification of cell identity and possible functions for some of them. For example, analysis of the group of monocyte clusters revealed subclusters comprising heterophils, as well as putative monocyte subtypes. Also, novel aspects of TCRγ/δ + T-cell subpopulations could be inferred such as evidence of at least two subtypes based on e.g., different expression of transcription factors MAF, SOX13 and GATA3. Moreover, a novel subpopulation of chicken peripheral B-cells with high SOX5 expression was identified. An overall good correlation between mRNA and cell surface phenotypic cell identification was shown.ConclusionsTaken together, we were able to identify and infer functional aspects of both previously well known as well as novel chicken leukocyte populations although some cell types. e.g., T-cell subtypes, proved more challenging to decipher. Although this methodology to some extent is limited by incomplete annotation of the chicken genome, it definitively has benefits in chicken immunology by expanding the options to distinguish identity and functions of immune cells also without access to species specific reagents.

Lnc-HC ameliorates steatosis by promoting miR-130b-3p biogenesis and the assembly of an RNA-induced silencing complex

Hepatic lipid deposition is the main cause of non-alcoholic fatty liver disease (NAFLD). Our previous study identified that lnc-HC prevents NAFLD by increasing the expression of miR-130b-3p. In the present study, we show that lnc-HC, an lncRNA derived from hepatocytes, positively controls miR-130b-3p maturation at multiple levels and contributes to its action by enhancing the assembly of an RNA-induced silencing complex (RISC). lnc-HC negatively regulates the downstream target genes of miR-130b-3p, including peroxisome proliferator-activated receptor gamma (PPARγ) and acyl-CoA synthetase long-chain family member 1 and 4 (Acsl1 and Acsl4, respectively), thus suppressing hepatic lipid droplet accumulation. Mechanistically, lnc-HC enhanced the promoter activity of miR-130b-3p by positively regulating the expression of transcription factors MAF bZIP transcription factor B (Mafb) and Jun proto-oncogene (Jun). Then, lnc-HC contributed the processing step of primary (pri-) miR-130b and strengthened the interaction between Drosha enzyme and the 5′-flanking sequence of pri-miR-130b to produce more precursor transcripts. Through direct binding with the chaperone heat shock protein 90 alpha family class A member 1 (HSP90AA1), lnc-HC contributed to RISC assembly, which was composed of HSP90AA1, argonaute RISC catalytic component 2 (AGO2) and miR-130b-3p. In a high-fat, high-cholesterol-induced hepatic lipid disorder E3 model, we confirmed that the hepatic expression of lnc-HC/miR-130b-3p negatively correlated with that of the target genes and was closely associated with liver triglycerides concentration. These findings provide a deeper understanding of the regulatory roles of lnc-HC in hepatic lipid metabolism and NAFLD development.

Transcription factors CncC and Maf connect the molecular network between pesticide resistance and resurgence of pest mites.

Pesticide resistance and resurgence are serious problems often occurring simultaneously in the field. In our long-term study of a fenpropathrin-resistant strain of Tetranychus cinnabaribus, enhancement of detoxification and modified fecundity mechanisms were both observed. Here we investigate the network across these two mechanisms and find a key node between resistance and resurgence. We show that the ecdysone pathway is involved in regulating the fecundity of T. cinnabaribus. The concentration change of ecdysone is consistent with the fecundity curve; the concentration of ecdysone is higher in the fenpropathrin-resistant strain which has stronger fecundity. The enhancement of ecdysone is due to overexpression of two P450 genes (CYP314A1 and CYP315A1) in the ecdysone synthesis pathway. Silencing expression of these CYP genes resulted in lower concentration of ecdysone, reduced expression of vitellogenin, and reduced fecundity of T. cinnabaribus. The expression of CYP315A1 is regulated by transcription factors Cap-n-collar isoform C (CncC) and Musculoaponeurotic fibrosarcoma protein (Maf), which are involved in regulating other P450 genes functioning in detoxification of fenpropathrin in T. cinnabaribus. A similar regulation is established in citrus pest mite Panonychus citri showing that the CncC pathway regulates expression of PcCYP315A1, which affects mite fecundity. Transcription factors are activated to upregulate detoxification genes facilitating pesticide resistance, while the "one to multiple" regulation mode of transcription factors simultaneously increases expression of metabolic enzyme genes in hormone pathways and alters the physiology of pests. This is an important response of arthropods to pesticides which leads to resistance and population resurgence.

The aging mouse lens transcriptome

Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.

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.

Human Tregs at the materno-fetal interface show site-specific adaptation reminiscent of tumor Tregs.

Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ.

Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua

Insect cytochrome P450 s (P450 s) are associated with the metabolic detoxification of toxic xenobiotics and their constitutive upregulation is often associated with resistance to natural and synthetic toxicants. The P450 s CYP321A16 and CYP332A1 are constitutively overexpressed in an insecticide-resistant strain of beet armyworm, Spodoptera exigua. However, the function and upstream regulation of these two P450 s remain unknown. Here, we investigated the function of CYP321A16 and CYP332A1 in resistance to the organophosphate insecticide, chlorpyrifos and their regulation by the transcription factors CncC and Maf. Transgenic strains of Drosophila melanogaster expressing CYP321A16 or CYP332A1 showed higher levels of tolerance to chlorpyrifos than the control flies with the same genetic background. Furthermore, recombinant CYP321A16 and CYP332A1 proteins metabolized chlorpyrifos. Analysis of the putative promoter sequences of the genes coding for CYP321A16 and CYP332A1 revealed conserved CncC/Maf binding sites. Transfection of luciferase reporter plasmids containing the promoter of CYP450 gene together with CncC and Maf expression plasmids significantly enhanced the activity of the reporter. Promoter truncation identified a site in the promoter of CYP321A16 that is critical for the CncC/Maf binding. These data demonstrate that resistance to chlorpyrifos in S. exigua is conferred by the combined action of CYP321A16 and CYP332A1 and uncovered their regulation by the transcription factors CncC and Maf.

Transcription factor cap n collar C regulates multiple cytochrome P450 genes conferring adaptation to potato plant allelochemicals and resistance to imidacloprid in Leptinotarsa decemlineata (Say)

Colorado potato beetle (CPB), Leptinotarsa decemlineata is a notorious pest of potato. Co-evolution with Solanaceae plants containing high levels of toxins (glycoalkaloids) helped this insect to develop an efficient detoxification system and resist almost every chemical insecticide introduced for its control. Even though the cross-resistance between plant allelochemicals and insecticides is well acknowledged, the underlying molecular mechanisms are not understood. Here, we investigated the molecular mechanisms involved in detoxification of potato plant allelochemicals and imidacloprid resistance in the field-collected CPB. Our results showed that the imidacloprid-resistant beetles employ metabolic detoxification of both potato plant allelochemicals and imidacloprid by upregulation of common cytochrome P450 genes. RNAi aided knockdown identified four cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25, and CYP9Z29) that are required for defense against both natural and synthetic chemicals. These P450 genes are regulated by the xenobiotic transcription factors Cap n Collar C, CncC and muscle aponeurosis fibromatosis, Maf. Studies on the CYP9Z25 promoter using the luciferase reporter assay identified two binding sites (i.e. GCAGAAT and GTACTGA) for CncC and Maf. Overall, these data showed that CPB employs the metabolic resistance mediated through xenobiotic transcription factors CncC and Maf to regulate multiple P450 genes and detoxify both imidacloprid and potato plant allelochemicals.

A Subset of Interleukin-21 + Chemokine Receptor CCR9 + T Helper Cells Target Accessory Organs of the Digestive System in Autoimmunity

This study describes a CD4+ T helper (Th) cell subset marked by coexpression of the cytokine interleukin 21 (IL-21) and the gut-homing chemokine receptor CCR9. Although CCR9+ Th cells were observed in healthy mice and humans, they were enriched in the inflamed pancreas and salivary glands of NOD mice and in the circulation of Sjögren's syndrome patients. CCR9+ Th cells expressed large amounts of IL-21, inducible T cell costimulator (ICOS), and the transcription factors Bcl6 and Maf, and also supported antibody production from B cells, thereby resembling T follicular B helper (Tfh) cells. However, in contrast to Tfh cells, CCR9+ Th cells displayed limited expression of CXCR5 and the targets of CCR9+ Th cells were CD8+ T cells whose responsiveness to IL-21 was necessary for the development of diabetes. Thus, CCR9+ Th cells are a subset of IL-21-producing T helper cells that influence regional specification of autoimmune diseases that affect accessory organs of the digestive system.

Comprehensive Mutational Screening in a Cohort of Danish Families with Hereditary Congenital Cataract

Identification of the causal mutations in 28 unrelated families and individuals with hereditary congenital cataract identified from a national Danish register of hereditary eye diseases. Seven families have been published previously, and the data of the remaining 21 families are presented together with an overview of the results in all families. A combined screening approach of linkage analysis and sequencing of 17 cataract genes were applied to mutation analyses of total 28 families. The study revealed a disease locus in seven of eight families that were amenable to linkage analysis. All loci represented known genes, and subsequent sequencing identified the mutations. Mutations were found in eight genes, among them crystallins (36%), connexins (22%), and the transcription factors HSF4 and MAF (15%). One family carried a complex CRYBB2 allele of three DNA variants, and a gene conversion is the most likely mutational event causing this variant. Ten families had microcornea cataract, and a mutation was identified in eight of those. Most families displayed mixed phenotypes with nuclear, lamellar, and polar opacities and no apparent genotype-phenotype correlation emerged. In total, 28 families were analyzed, and mutations were identified in 20 (71%) of them. Despite considerable locus heterogeneity, a high mutation identification rate was achieved by sequencing a limited number of major cataract genes. Provided these results are representative of Western European populations, the applied sequencing strategy seems to be suitable for the exploration of the large group of isolated cataracts with unknown etiology.

ERK1/2-dependent Activation of Transcription Factors Required for Acute and Chronic Effects of Glucose on the Insulin Gene Promoter

The insulin promoter is both positively and negatively regulated in response to conditions to which pancreatic beta-cells are exposed. Exposure of intact rat islets and INS-1 pancreatic beta-cells to 11 mm glucose for minutes to hours results in an enhancement in the rate of insulin gene transcription assessed with a reporter linked to the insulin gene promoter. In contrast, chronic exposure of rat islets or beta-cells to 11 mm glucose results in loss of the glucose responsiveness of the insulin gene promoter. By 48 h, glucose inhibits insulin gene promoter activity. Here we show that not only the acute effect of elevated glucose to stimulate the insulin gene promoter but also the chronic effect of elevated glucose to inhibit the insulin gene promoter depend on ERK1/2 mitogen-activated protein kinase activity. In examining the underlying mechanism, we found that acute exposure to 11 mm glucose resulted in the binding of the transcription factors NFAT and Maf to the glucose-responsive A2C1 element of the insulin gene promoter. An NFAT and C/EBP-beta complex was observed in cells chronically exposed to 11 mm glucose. Formation of NFAT-Maf and NFAT-C/EBP-beta complexes was sensitive to inhibitors of ERK1/2 and calcineurin, consistent with our previous finding that activation of ERK1/2 by glucose required calcineurin activity and the well documented regulation of NFAT by calcineurin. These results indicate that the ERK1/2 pathway modulates partners of NFAT, which may either stimulate or repress insulin gene transcription during stimulatory and chronic exposure to elevated glucose.

Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice.

Mouse aphakia (ak) is a recessive phenotype that spontaneously occurs in the 129/Sv-SlJ strain and is characterized by small eyes that lack a lens. We have recently identified a homeobox-containing gene, Pitx3, and have shown that it is expressed in the developing lens and maps to chromosome 19 close to ak in mouse. Human PITX3 gene was found to underlie anterior segment dysgenesis and cataracts. We have now obtained the entire sequence of the mouse Pitx3 gene including 10 kb of the 5' region and 5 kb of the 3' region. Of several microsatellite repeat regions identified within the Pitx3 sequence, one was informative for linkage analysis. No recombination was observed between ak and the Pitx3 marker, indicating that these two loci are closely linked (0.2 +/- 0.2 cM). Additionally, Pitx3 transcripts were not detected in the ak/ak mice either in the lens placode or at later developmental stages of the lens by in situ hybridization. Since no differences were previously found between ak/ak and wild-type sequences in the Pitx3 coding region, we hypothesized that an etiologic mutation is located in the promoter or other regulatory regions. To test this hypothesis we studied the 5' flanking region of the Pitx3 gene. This analysis revealed a deletion of 652 bp located 2.5 kb upstream from the start point of the Pitx3 5' UTR sequence in ak/ak mice. The deletion co-segregated with the ak mutation and was not detected in 16 samples from 10 different mouse strains including the founder strains. Analysis of the 652 bp region identified sequences similar to consensus binding sites for transcription factors AP-2 and Maf that were shown to play a critical role in lens determination. These lines of evidence suggest that the abnormal ocular development in the aphakia mouse is due to the deletion upstream of the Pitx3 gene.