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, 142 (4), 1173-1184

The Transcription Factors GATA2 and Microphthalmia-Associated Transcription Factor Regulate Hdc Gene Expression in Mast Cells and Are Required for IgE/mast Cell-Mediated Anaphylaxis

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The Transcription Factors GATA2 and Microphthalmia-Associated Transcription Factor Regulate Hdc Gene Expression in Mast Cells and Are Required for IgE/mast Cell-Mediated Anaphylaxis

Yapeng Li et al. J Allergy Clin Immunol.

Abstract

Background: Histamine is a critical mediator of IgE/mast cell-mediated anaphylaxis. Histamine is synthesized by decarboxylating the amino acid histidine, a reaction catalyzed by the histidine decarboxylase (Hdc) gene-encoded enzyme HDC. However, regulation of the Hdc gene in mast cells is poorly understood.

Objective: We sought to investigate the in vivo regulation of IgE/mast cell-mediated anaphylaxis by the transcription factors GATA2 and microphthalmia-associated transcription factor (MITF) and the mechanisms by which GATA2 and MITF regulate Hdc gene expression in mouse and human mast cells.

Methods: Mice deficient in the transcription factors Gata2, aryl hydrocarbon receptor (Ahr), aryl hydrocarbon receptor repressor (Ahrr), or basic helix-loop-helix family member E40 (Bhlhe40) were assessed for anaphylactic reactions. Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers. Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene. The short hairpin RNA knockdown approach was used to determine the role of MITF in regulating mouse and human HDC gene expression.

Results: Connective tissue mast cell-specific Gata2-deficient mice did not have IgE/mast cell-mediated anaphylaxis. GATA2 induced the expression of Mitf, Ahr, Ahrr, and Bhlhe40 in mast cells. MITF, but not AHR, AHRR, or BHLHE40, was required for anaphylaxis. MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. MITF overexpression largely restored Hdc gene expression in the Gata2-deficient mast cells. In the human mast cell line LAD2, MITF was required for the HDC gene expression and histamine synthesis.

Conclusion: The transcription factors GATA2 and MITF regulate Hdc gene expression in mast cells and are required for IgE/mast cell-mediated anaphylaxis.

Keywords: GATA2; Mast cell; anaphylaxis; connective tissue mast cell; enhancer; histamine synthesis; histidine decarboxylase; microphthalmia-associated transcription factor.

Figures

FIG E1.
FIG E1.
Neutrophils (Neu), Macrophages (MФ), T cells, B cells, Dendritic cells (DCs), and NK cells are not affected in Gata2f/f Mcpt5-Cre mice. A. Flow analysis of the indicated cell lineages in the bone marrow (Neutrophils and macrophages) and spleen (T cells, B cells, Dendritic cells and NK cells). B. Total number of cells (mean ± SEM, n=3 mice). FIG E1 related to FIG 1.
FIG E2.
FIG E2.
Plasma histamine and serum MMCP-1 levels are decreased in Gata2f/f Mcpt5-Cre mice. A. ELISA analysis of plasma histamine level (mean ± SEM, triplicates). The left, middle, and right panels show three independent experiments. B. ELISA analysis of serum MMCP-1 level (mean ± SEM, triplicates). The left, middle, and right panels show three independent experiments. FIG E2A related to FIG 1E; FIG E2B related to FIG 1F.
FIG E3.
FIG E3.
Transcription factors AHR, AHRR, BHLHE40 and MITF depend on GATA2 for their expression in mast cells. qPCR analysis of the mRNA expression in BMMCs (MC) versus basophils (Ba) (A), and Gata2+/+ BMMCs versus Gata2−/− BMMCs (B) (mean ± SEM, triplicates). The left, middle, and right panels each show three independent experiments. FIG E3A related to FIG 2B; FIG E3B related to FIG 2C.
FIG E4.
FIG E4.
The transfection factor MITF, but not AHR, AHRR or BHLHE40 regulates the Hdc gene expression. qPCR analysis of the Hdc mRNA expression and ELISA analysis of histamine content in BMMCs (A), PCMCs (C), and human mast cell line LAD2 (D) with knocked down Mitf mRNA (ShMitf) or with shRNA control (ShCTRL) (mean ± SEM, triplicates); B. qPCR analysis of the Hdc mRNA expression in BMMCs with knocked down Ahr mRNA (ShAhr), Ahrr mRNA (ShAhrr), and Bhlhe40 mRNA (ShBhlhe40) or with shRNA control (ShCTRL) (mean ± SEM, triplicates). The left, middle, and right panels each show three independent experiments. FIG E4A related to FIG 3A; FIG E4B related to FIG 3B; FIG E4C related to FIG 3D; FIG E4D related to FIG 3E.
FIG E5.
FIG E5.
Knockdown of Mitf mRNA expression does not lead to mast cell death or down regulation of FceR1a expression. A. Number of live ShCTRL or ShMitf PCMCs at different days after puromycin selection (mean ± SEM, n=3 samples). B. qPCR analysis of the Fcer1a mRNA expression in ShCTRL or ShMitf PCMCs (mean ± SEM, n=3 samples). FIG E5 related to FIG 3.
FIG E6.
FIG E6.
The transfection factors AHR, AHRR and BHLHE40 are not required for the Hdc gene expression in PCMCs. qPCR analysis of the Hdc mRNA expression in PCMCs of the indicated mice (mean ± SEM, triplicates). FIG E6 related to FIG 3.
FIG E7.
FIG E7.
Ahrf/fCpa3-Cre mice have normal numbers of mast cells and develop passive cutaneous anaphylaxis and passive systemic anaphylaxis normally. A. Flow analysis of peritoneal cavity mast cells and total number of peritoneal cavity mast cells (mean ± SEM, n=3 mice) (left panel). Toluidine blue staining of mast cells (magnification ×40; insert, ×100). Mast cells are indicated by arrows. Average number of mast cells in ten randomly selected fields (×40) (mean ± SEM, n=3 mice) (right panels). B. PCA analysis, mean ± SEM, n=3 mice. C. PSA analysis, mean ± SEM, n=3 mice. FIG E7 related to FIG 3.
FIG E8.
FIG E8.
Ahrr−/− mice have normal numbers of mast cells and develop passive cutaneous anaphylaxis and passive systemic anaphylaxis normally. A. Flow analysis of peritoneal cavity mast cells and total number of peritoneal cavity mast cells (mean ± SEM, n=3 mice) (left panel). Toluidine blue staining of mast cells (magnification ×40; insert, ×100). Mast cells are indicated by arrows. Average number of mast cells in ten randomly selected fields (×40) (mean ± SEM, n = 3 mice) (right panels). B. PCA analysis, mean ± SEM, n = 3 mice. C. PSA analysis, mean ± SEM, n=3 mice. FIG E8 related to FIG 3.
FIG E9.
FIG E9.
Bhlhe40−/− mice have normal numbers of mast cells and develop passive cutaneous anaphylaxis and passive systemic anaphylaxis normally. A. Flow analysis of peritoneal cavity mast cells and total number of peritoneal cavity mast cells (mean ± SEM, n=3 mice) (left panel). Toluidine blue staining of mast cells (281 magnification ×40; insert, ×100). Mast cells are indicated by arrows. Average number of mast cells in ten randomly selected fields (×40) (mean ± SEM, n=3 mice) (right panels). B. PCA analysis, mean ± SEM, n=3 mice. C. PSA analysis, mean ± SEM, n=3 mice. FIG E9 related to FIG 3.
FIG E10.
FIG E10.
Identification of putative Hdc enhancers. Luciferase reporter analysis of the Hdc enhancer activities in the CFTL-15 mast cell line. Error bars represent mean ± SEM, triplicates. The left, middle, and right panels each show three independent experiments. HdcMP: Hdc minimal promoter. FIG E10 related to FIG 4B.
FIG E11.
FIG E11.
MITF binds to the -8.8 enhancer. A. ChIP-qPCR analysis of MITF binding (mean ± SEM, triplicates). B, qPCR analysis of the Hdc gene expression in untreated or cholera toxin (CT) treated BMMCs, and PCMCs (mean ± SEM, triplicates). C. ELISA analysis of histamine content in untreated or cholera toxin (CT) treated BMMCs and PCMCs (mean ± SEM, triplicates). D. ChIP-qPCR analysis of MITF binding in untreated or cholera toxin (CT) treated BMMCs and PCMCs (mean ± SEM, triplicates). The left and right panels (A) or the left, middle, and right panel (B, C, and D) each show two or three independent experiments, respectively. FIG E11A related to FIG 5B; FIG E11B related to FIG 5C; FIG E11C related to FIG 5D; FIG E11D related to FIG 5E.
FIG E12.
FIG E12.
MITF drives the −8.8 enhancer activity. A, Luciferase reporter gene analysis of Hdc enhancer activities in CFTL-15 cells with knocked down Mitf mRNA or shRNA control (mean ± SEM, triplicates). B. Luciferase reporter gene analysis of the Hdc enhancer activities in HEK293 cells co-transfected with vector control or the MITF expression construct (mean ± SEM, triplicates). C. Luciferase reporter gene analysis of the activity of Hdc enhancers that have a mutated MITF binding site(s) (mean ± SEM, triplicates). The left and right panels (A, B, and C) or the upper and lower panel (D) each show two independent experiments. FIG E12A related to FIG 6A; FIG E12B related to FIG 6B; FIG E12C related to FIG 6C.
FIG E13.
FIG E13.
MITF partially restores Hdc gene expression in Gata2 deficient mast cells. qPCR analysis of the Hdc mRNA expression in Gata2−/− BMMCs overexpressing MITF, GATA2 or a control vector (CTRL) (mean ± SEM, triplicates). The left, middle, and right panels each show three independent experiments. FIG E13 related to FIG 7B.
FIG 1.
FIG 1.
GATA2 is critical for connective tissue mast cell differentiation and IgE/mast cell-mediated anaphylaxis. A, Flow analysis of peritoneal cavity mast cells (MC) and bone marrow basophils (Ba). Total number of cells (mean ± SEM, n=3 mice) (right panel). B, Toluidine blue staining of mast cells (magnification 3×40; insert, 3×100). Mast cells are indicated by arrows. Average number of mast cells in ten randomly selected fields (mean ± SEM, n=3 mice). C, PCA analysis, mean ± SEM, n = 3 mice; D, PSA analysis, mean ± SEM, n = 6 mice. E, ELISA analysis of histamine, mean ± SEM, n=3 samples from 3 individual mice. F, ELISA analysis of MMCP-1 (mean ± SEM, n=3 samples from 3 individual mice). G, PSA analysis of the Gata2f/fMcpt5-Cre mice reconstituted BMMCs (mean ± SEM, n=5 mice). Statistical differences were analyzed by student’s t test (A, B, C, E, F) or by ANOVA (D, G). *: p<0.05; **: p<0.01; ***: p<0.001.
FIG 2.
FIG 2.
Transcription factors AHR, AHRR, BHLHE40, and MITF depend on GATA2 for their expression in mast cells. A, Microarray analysis of transcription factor expression profiles in BMMCs versus basophils. B, qPCR analysis of mRNA expression in BMMCs versus basophils. The numbers indicate the fold of difference in mRNA expression between basophils and BMMCs (mean ± SEM, n=3 samples from 3 individual mice). C, qPCR analysis of mRNA expression in Gata2+/+ or Gata2−/− BMMCs (mean ± SEM, n=3 samples from 3 individual mice).
FIG 3.
FIG 3.
Transcription factors MITF, but not AHR, AHRR or BHLHE40, regulate Hdc gene expression and IgE/mast cell-mediated anaphylaxis. A, qPCR analysis of Hdc mRNA expression in BMMCs with knocked down Mitf mRNA (shMitf) or with shRNA control (shCTRL) (mean ± SEM, n=3 samples from 3 individual mice). ELISA analysis of the histamine contents in BMMCs with knocked down Mitf mRNA (shMitf) or with shRNA control (mean ± SEM, n=3 samples from 3 individual mice) (right panel). B, qPCR analysis of Hdc mRNA expression in BMMCs with knocked down Ahr mRNA (shAhr), Ahrr mRNA (shAhrr) and Bhlhe40 mRNA (shBhlhe40) or with shRNA control (shCTRL) (mean ± SEM, n=3 samples from 3 individual mice). C, PSA analysis of Gata2f/f Mcpt5-Cre mice reconstituted with BMMCs with knocked down Mitf mRNA (n=4 mice), Ahr mRNA (n=3 mice), Ahrr mRNA (n=3 mice), Bhlhe40 mRNA (n=3 mice) or with shRNA control (n=4 mice). Statistical differences in PSA analysis were analyzed by ANOVA. *: p<0.05; **: p<0.01. D, qPCR analysis of Hdc gene expression (left panel) and ELISA analysis of histamine content (right panel) in PCMCs with knocked down Mitf mRNA (shMitf) or with shRNA control (shCTRL) (mean ± SEM, n=3 samples from 3 individual mice). E, qPCR analysis HDC gene expression (left panel) and ELISA analysis of the histamine contents (right panel) in the human mast cell line LAD2 with knocked down Mitf mRNA (shMitf) or with shRNA control (shCTRL) (mean ± SEM, n=3 transduced samples).
FIG 4.
FIG 4.
Identification of putative Hdc enhancers. A, BMMCs were used for ChIP-seq (duplicate samples were used for H3K4me1 ChIP-seq and triplicate samples were used for H3K27ac ChIP-seq). H3K4me1 and H3K27ac ChIP-seq data were analyzed with the Integrative Genomics Viewer. Red bars indicate putative Hdc enhancers that show significant H3K4me1 and H3K27ac modifications. RPM: reads per million nucleotides; E: enhancer, +0.3 indicates the distance from the beginning of the downstream Hdc enhancer to the TSS of the Hdc gene, and −8.8 indicates the distance from the beginning of the upstream Hdc enhancer to the TSS of the Hdc gene. B, Scheme of the luciferase constructions (upper panel) and luciferase reporter analysis of the Hdc enhancer activities in the CFTL-15 mast cell line_(lower panel). Error bars represent mean ± SEM, n=3 transfectants. HdcMP: Hdc minimal promoter.
FIG 5.
FIG 5.
MITF binds to the −8.8 enhancer in immature and mature mast cells. A, Enlarged ChIP-seq peak plots of the Hdc E-8.8 region and Hdc E+0.3. The published MITF ChIP-seq data were aligned with our H3K4me1 and H3K27ac ChIP data using the Integrative Genomics Viewer. The black bar beneath the MITF ChIP-seq peak plot indicates the MITF-binding peak that is statistically significant. The red bars indicate the positions of consensus MITF-binding sites (BSs). B, ChIP-qPCR analysis of MITF binding (mean ± SEM, n=2 samples from 2 independent experiments). BMMCs, anti-MITF antibody or control IgG was used for immunoprecipitation. C, qPCR analysis of the Hdc mRNA expression (mean ± SEM, n=3 samples from 3 individual mice); D, ELISA analysis of histamine content (mean ± SEM, n=3 samples from 3 individual mice); and E, ChIP-qPCR analysis of MITF binding in untreated BMMCs, cholera toxin treated BMMCs, and PCMCs (mean ± SEM, n=3 samples from 3 independent experiments). CT: Cholera Toxin.
FIG 6.
FIG 6.
MITF drives the −8.8 Hdc enhancer activity. A, Luciferase reporter gene analysis of Hdc enhancer activities in CFTL-15 cells with knocked down Mitf mRNA or shRNA control (mean ± SEM, n=2 transfectants). B. Luciferase reporter gene analysis of the Hdc enhancer activities in HEK293 cells co-transfected with vector control or the MITF expression construct (mean ± SEM, n=2 transfectants). C. Luciferase reporter gene analysis of the activity of Hdc enhancers that have a mutated MITF binding site(s) (mean ± SEM, n=2 transfectants).
FIG 7.
FIG 7.
MITF is sufficient to fully restore cKIT and FcεRI expression but can only partially restore Hdc gene expression. A, Flow analysis of cKIT and FcεRI expression in Gata2−/− BMMCs overexpressing MITF, GATA2 or control vector (CTRL). YFP+ GFP+ cell populations are shown. Data represent three independent experiments with similar results. B, qPCR analysis of the Hdc mRNA expression in Gata2−/− BMMCs overexpressing MITF, GATA2 or a control vector (CTRL). YFP+ GFP+ BMMCs were FACS-sorted for qPCR analysis. Error bars represent mean ± SEM, n=3 samples from 3 independent experiments.

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