Atopic dermatitis is a common inflammatory skin disease caused by interaction of genetic and environmental factors. On the basis of data from a genome-wide association study (GWAS) and a validation study comprising a total of 3,328 subjects with atopic dermatitis and 14,992 controls in the Japanese population, we report here 8 new susceptibility loci: IL1RL1-IL18R1-IL18RAP (P(combined) = 8.36 × 10(-18)), the major histocompatibility complex (MHC) region (P = 8.38 × 10(-20)), OR10A3-NLRP10 (P = 1.54 × 10(-22)), GLB1 (P = 2.77 × 10(-16)), CCDC80 (P = 1.56 × 10(-19)), CARD11 (P = 7.83 × 10(-9)), ZNF365 (P = 5.85 × 10(-20)) and CYP24A1-PFDN4 (P = 1.65 × 10(-8)). We also replicated the associations of the FLG, C11orf30, TMEM232-SLC25A46, TNFRSF6B-ZGPAT, OVOL1, ACTL9 and KIF3A-IL13 loci that were previously reported in GWAS of European and Chinese individuals and a meta-analysis of GWAS for atopic dermatitis. These findings advance the understanding of the genetic basis of atopic dermatitis.

A major problem with allergen-specific immunotherapy involving repeated injection of allergens is the risk of an anaphylactic reaction. We engineered the major house dust mite allergen, Der f 2, to reduce its capacity to induce skin test reactivity and histamine release from peripheral blood basophils in allergic patients. The engineered allergen, in which the disulfide bond that linked the N- and C-terminal sequences of Der f 2 was disrupted, retained T-cell epitopes essential for immunotherapy and ability to stimulate T-cell proliferation. Such engineered allergens are potentially useful for safer and more effective immunotherapy for allergies.

We found a novel polymorphism, −66T/C, in the promoter region of human FcεRIα, the specific component of the high affinity receptor for IgE (FcεRI), which is essential for the cell surface expression of FcεRI and the binding of IgE Ab. When the effect of the single nucleotide replacement on the promoter function was analyzed, the transcription activity of the T allele promoter was found to be higher than that of the C allele promoter, and was markedly up-regulated by the overexpression of GATA-1 when compared with the C allele promoter. This is probably because the promoter with T at −66 has an additional GATA-1-binding motif in the region, which may assure higher affinity of the transcription factor to the promoter. In accordance with this, EMSA actually indicated that GATA-1 bound to the T allele probe (−80/−59) with the affinity higher than that to the C allele probe. Statistical analysis suggested that a significant portion of nonallergic individuals has heterozygous −66T/C genotype, while most of allergic individuals have homozygous −66T/T genotype in Japanese population. Our findings for the first time demonstrate the presence of FcεRIα polymorphism related to the allergic diseases.

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Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six of the resulting N-acetyl-l-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward l-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM l-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM l-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.

Transcriptional regulation of the gene-encoding human FcεRI α-chain was analyzed in detail. EMSA revealed that either YY1 or PU.1 bound to the region close to that recognized by Elf-1. The α-chain promoter activity was up-regulated ∼2-fold by exogenously expressed YY1 or PU.1 and ∼7-fold by GATA-1, respectively, in KU812 cells. In contrast, coexpression of GATA-1 with either of PU.1 or YY1 dramatically activated the promoter ∼41- or ∼27-fold, respectively. Especially synergic activation by GATA-1 and PU.1 was surprising, because these transcription factors are known to inhibit the respective transactivating activities of each other. These up-regulating effects of PU.1 and YY1 with GATA-1 were inhibited by overexpression of Elf-1, indicating that Elf-1 serves as a repressor for the α-chain gene expression. Transcriptional regulation of the α-chain gene through four transcriptional factors is discussed.

House dust mites cause heavy atopic diseases such as asthma and dermatitis. Among allergens from Dermatophagoides farinae, Der f 2 shows the highest positive rate for atopic patients, but its biological function in mites has been perfectly unknown, as well as the functions of its homologs in human and other animals. We have determined the tertiary structure of Der f 2 by multidimensional nuclear magnetic resonance spectroscopy. Der f 2 was found to be a single-domain protein of immunoglobulin fold, and its structure was the most similar to those of the two regulatory domains of transglutaminase. This fact, binding to the bacterial surface, and other small pieces of information hinted that Der f 2 is related to the innate antibacterial defense system in mites. The immunoglobulin E epitopes are also discussed on the basis of the tertiary structure.

Cell-type-specific transcription of mouse high-affinity IgE receptor (Fc⑀RI) ␤-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse Fc⑀RI ␤-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the Fc⑀RI ␤-chain promoter. The expression levels of FOG-1, GATA-1, and ␤-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. IntroductionThe high-affinity IgE receptor Fc⑀RI is composed of an ␣-chain, a ␤-chain, and a ␥-chain. Allergen-IgE antibody complex-induced cross-linking of Fc⑀RI results in activation of mast cells, which subsequently secrete various chemical mediators that induce the symptoms of an allergic response.In humans, Fc⑀RI is expressed as a tetramer (␣␤␥ 2 ) on mast cells and basophils and as a trimer (␣␥ 2 ) on Langerhans cells, monocytes, and dendritic cells. [1][2][3][4][5] Thus, while ␤-chain may facilitate cell-surface expression of Fc⑀RI, 6 it is not necessarily required for cell-surface expression of human Fc⑀RI. By contrast, in mice, Fc⑀RI is expressed as a tetramer (␣␤␥ 2 ) only on mast cells and basophils, and the ␤-chain is necessary for cell-surface expression of mouse Fc⑀RI 7 and acts as an amplifier for Fc⑀RI signaling by increasing phosphorylation of the ␥-chain 8 and by enhancing signaling protein recruitment. 9 Therefore, characterization of the mechanisms of mouse ␤-chain expression is critical for the understanding of mast-cell-and basophil-specific transcriptional regulatory systems.We previously reported that the transcription factor GATA-1 positively regulated cell-type-specific ␤-chain expression via 4 GATA motifs in the promoter. 10 GATA-1 mediates the maturation of various cell lineages, including erythroid cells, megakaryocytes, eosinophils, basophils, and mast cells. [11][12][13] However, the expression of mouse ␤-chain is limited to mast cells but not observed in other GATA-1-positive cell lineages. Thus, other factors may regulate cell-type-specific transcription of the ␤-chain.A zinc finger cofactor, FOG-1, interacts with GATA-1 and can either enhance or repress GATA-1-dependent gene expression. 14-17 FOG-1 is abundantly expressed in erythroid cells and in megakaryocytes, where it regulates growth and differentiation. Erythroid and megakaryocyte lineage development is arrested at proerythroblast stage in Gata-1 Ϫ/Ϫ or Fog-1 Ϫ/Ϫ mice, 18,19 and FOG-1 mutants that lack GATA-binding activity result in abnormal differentiation of megakaryotic cells. 19,20 Recent studies have implicated abnormalities in GATA-1 and FOG-1 in various human diseases, including thrombocytopenia and idiopathic myelofibrosis (IM). [21][22][23] Thus, the goal of the present study was to characterize the role of GATA-associated cofactor FOG-1 in the regulation of the Fc⑀RI ␤-chain promoter. Materials and methods Cell culture...