Regulation of Cell Type-Specific Mouse FcεRI β-Chain Gene Expression by GATA-1 Via Four GATA Motifs in the Promoter

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The high-affinity IgE receptor, FcεRI, which is composed of α-, β-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of β mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIβ). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIβ, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIβ transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Critical Roles for PU.1, GATA1, and GATA2 in the Expression of Human FcεRI on Mast Cells: PU.1 and GATA1 Transactivate FCER1A, and GATA2 Transactivates FCER1A and MS4A2

Inage

Kasakura

Yashiro

et al. 2014

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“…We previously demonstrated that the transcription of genes encoding FcεRIa and FcεRIb is positively regulated by the transcription factors PU.1, GATA-1, and GATA-2 in mast cells (3,5,7,(38)(39)(40)(41). Additionally, GATA-2 also plays an important role in the transcription of the Kit gene (11).…”

Section: Tgf-b1 Suppresses Fc«ri and C-kit Expression In Mast Cellsmentioning

confidence: 99%

“…The nucleotide sequences of primer sets are listed in Supplemental Table I. The luciferase reporter plasmids pGL3-Basic (Promega) encoding Ms4a2 promoter region 2763 to +103 relative to the transcription start site (+1) (3,5) and Kit promoter region 2622 to +22 (11) were generated as in our previous studies. The expression plasmid pCR3.1 (Life Technologies) encoding FLAG-tagged mouse Ehf cDNA and its empty plasmid were used in this study.…”

Section: Luciferase Reporter Assaymentioning

confidence: 99%

The Transcription Factor Ehf Is Involved in TGF-β–Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells

Yamazaki

Nakano

Honjo

et al. 2015

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FcεRI, which is composed of α, β, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-β1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-β1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-β/Smad signaling in mouse bone marrow–derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIβ, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIβ, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-β1 was mimicked by forced expression of Ehf, suggesting that TGF-β1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.

“…In vitro transcription and translation were performed with a TNT T7 Quick coupled transcription/ translation system (Promega) using pCR-YY1 (16) as the template. The FITC-labeled probe was mixed with nuclear extract or the in vitro transcription/translation mixture and subjected to 4% PAGE as described in our previous report (14,20). Fluorescent images were analyzed on a FluorImager 595 (Molecular Dynamics, Sunnyvale, CA).…”

Section: Emsamentioning

confidence: 99%

Polymorphisms in the FcεRIβ Promoter Region Affecting Transcription Activity: A Possible Promoter-Dependent Mechanism for Association between FcεRIβ and Atopy

Nishiyama¹,

Akizawa²,

Nishiyama

et al. 2004

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The β subunit of the high-affinity IgE receptor (FcεRI) plays an important role in IgE-mediated allergic reactions as an amplifier for cell surface expression and signal transduction of FcεRI. FcεRIβ is presumed to be one of the genes linked with atopic diseases. However, the validity of the associations previously found between single nucleotide polymorphisms (SNPs) in FcεRIβ and atopic diseases is questionable. In the present study, we found correlation between the SNP of FcεRIβ at +6960A/G, resulting in a Glu237Gly amino acid substitution, and the cell surface expression level of FcεRI on blood basophils, although it has been shown that the Glu237Gly mutation itself does not affect the surface expression or function of FcεRI. We additionally found four SNPs in the promoter region of FcεRIβ, among which −426T/C and −654C/T were tightly linked with +6960A/G. Reporter plasmids carrying the −426C and −654T promoter displayed higher transcriptional activity than those carrying the −426T and −654C promoter. We found that transcription factor YY1 preferentially bound and transactivated the −654T promoter. Furthermore, expression of FcεRI β-chain mRNA in basophils from individuals who have the minor heterozygous genotype was significantly higher than that of the major homozygous genotype. These results suggest that the SNPs in the FcεRIβ promoter are causally linked with atopy via regulation of FcεRI expression.