FOG-1 represses GATA-1-dependent FcϵRI β-chain transcription: transcriptional mechanism of mast-cell-specific gene expression in mice by Keiko Maeda, Chiharu Nishiyama, Tomoko Tokura, Hiroyasu Nakano, Shunsuke Kanada, Makoto Nishiyama, Ko Okumura, and Hideoki Ogawa Blood Volume 108(1):262-269 July 1, 2006 ©2006 by American Society of Hematology

The β-chain, GATA-1, and FOG-1 mRNA levels in Ba/F3 and PT18. The β-chain, GATA-1, and FOG-1 mRNA levels in Ba/F3 and PT18. (A) RT-PCR was performed to measure FcϵRI β-chain, FOG-1, GATA-1, and GAPDH mRNA levels, as visualized by ethidium bromide staining of agarose gels. (B) Western blotting analyses of FOG-1 and GATA-1. Whole-cell lysates (1 × 106 cells per lane) were applied into each well and were analyzed with anti-FOG-1 or anti-GATA-1 Ab as the primary Abs followed by peroxidase-conjugated anti-goat IgG donkey Ab or peroxidase-conjugated anti-rat IgG rabbit Ab as the secondary Abs, respectively. (C) Transcription activities of the β-chain and αIIb promoters. Five micrograms of each reporter plasmid was introduced into Ba/F3 or PT18. The relative luciferase activity driven by β-69/pGL3-Basic (β-69; ▪) or αIIb/pGL3-Basic (αIIb; ▦)is represented as the ratio to the activity driven by pGL3-Basic (Basic; □). Each experiment was conducted in duplicate for each sample, and the results are expressed as mean ± SD for more than 3 independent experiments in Figures 1, 2, 3, and 5. Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology

Transcriptional activity of β-chain promoter was downregulated by FOG-1 expression in mast cells. Transcriptional activity of β-chain promoter was downregulated by FOG-1 expression in mast cells. Five micrograms of each reporter plasmid was introduced into PT18 cells with or without 3 μg of expression plasmid. The ratio of luciferase activity of each construct in the absence of expression plasmid to that of pGL3-Basic was represented as relative luciferase activity. □ indicates without expression plasmid; ▪, with mock expression plasmid; and ▦, with FOG-1 expression plasmid. Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology

Direct interaction between FOG-1 and GATA-1 is required for suppression of the β-chain promoter. Direct interaction between FOG-1 and GATA-1 is required for suppression of the β-chain promoter. (A) CV-1 cells were transfected with 500 ng each of reporter plasmids (pGL3-Basic; β-69/pGL3-Basic; or β M1234/pGL3-Basic [β-chain mutant promoter lacking 4 GATA motifs by nucleotide replacement]) and 100 ng each of expression plasmids (pCR-GATA-1; pCR-GATA-1 V205G [GATA-1 mutant lacking FOG-1 binding activity]; and/or pCR-FOG-1). Total amount of expression plasmids was adjusted to 200 ng by addition of the empty plasmid pCR3.1. The ratio of luciferase activity of each expression plasmid in the presence of reporter plasmid to that of the empty expression plasmid was represented as fold activation. (B) Transcriptional level of endogenously produced GATA-1 and FOG-1 in CV-1 cells. RT-PCR was performed with total RNA prepared from each transfectant. The expression level of GATA-1 V205G was similar to that of wild-type GATA-1 (data not shown). (C) Dose dependency of the FOG-1 effect on β-chain promoter activity. CV-1 cells were transfected with 100 ng of reporter plasmid β-69/pGL3-Basic with or without 100 ng of pCR-3.1 (empty vector), pCR-GATA-1, or various amounts of pCR-FOG-1 (10, 30, 100, or 300 ng). The ratio of luciferase activity of each construct in the absence of expression plasmid to that of pGL3-Basic was represented as relative luciferase activity. Protein levels of exogenously expressed GATA-1 and FOG-1 and control YY1 are analyzed by Western blotting (bottom). Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology

Overexpression of FOG-1 induced the downregulation of β-chain transcription and surface expression of FcϵRI in PT18 cells. Overexpression of FOG-1 induced the downregulation of β-chain transcription and surface expression of FcϵRI in PT18 cells. (A) Western blot analysis of transfected cells. PT18 cells were transfected with pCR-FOG-1 or pCR3.1 by electroporation and cultured in the presence of G418 for longer than 2 weeks. Lysates (1 × 106 cells per lane) were analyzed using anti-FOG-1, anti-GATA-1, or anti-YY1 Abs. (B) Surface expression of FcϵRI on transfectants. PT18 cells transfected with pCR-FOG-1 (FOG-1, bold line) or empty vector (mock, solid line) were stained with FITC-conjugated mouse IgE antibody. FOG-1 transfectants without antibody are shown by the dotted line. (C) Quantitative real-time RT-PCR analysis of transfected cells. The mRNA levels of FOG-1, α-chain, and β-chain in FOG-overproducing cells were represented as the ratio to that of mock transfectants. □ indicates PT18 cells transfected with pCR3.1; and ▪, PT18 cells transfected with pCR-FOG-1. Data are represented as the average ± SD of triplicate samples. Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology

Inhibition of FOG-1 expression by siRNA resulted in upregulation of β-chain promoter activity. Inhibition of FOG-1 expression by siRNA resulted in upregulation of β-chain promoter activity. (A) Quantification of FOG-1 mRNA in siRNA transfectants using real-time RT-PCR. Ba/F3 cells (2 × 106) were transfected with 5 μL of 20 μM FOG-1 siRNA or shuffled control siRNA. After 20-hour culture, total RNA was extracted from each transfectant, and the amount of FOG-1 and GAPDH mRNAs was analyzed by ABI7500. FOG-1 mRNA levels are represented as the ratio to that of control (without siRNA). (B) Western blot analysis of transfectants. After 44 hours of culture, lysates (5 × 105 cells per lane) were analyzed using anti-FOG-1 or anti-YY1 Abs. (C) Transcription activity of the β-chain promoter. One microgram of reporter plasmid was introduced into Ba/F3 cells with or without 2.5 or 5 μL of 20 μM siRNA or shuffled control siRNA. The ratio of luciferase activity of each transfectant to that of control transfectant (without siRNA) was represented as fold activation. Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology

Binding of FOG-1 and GATA-1 to β-chain promoter in BMMCs at different developing stages. Binding of FOG-1 and GATA-1 to β-chain promoter in BMMCs at different developing stages. (A) Expression profile of c-kit and FcϵRI analyzed by flow cytometry. 0W indicates freshly prepared Lin- cells; 2W, immature BMMCs developed from Lin- cells after 2-week culture; and 6W, mature BMMCs developed from Lin- cells after 6-week culture. (B) Transcription levels of FOG-1 and β-chain during BMMC development. The mRNA levels of FOG-1 and β-chain in different developing stages of BMMCs and PT18 were measured by quantitative PCR and are represented as the ratio to that of Lin- (0W). Data are represented as the average ± SD of triplicate samples. (C) FOG-1 and GATA-1 in vivo binding to β-chain promoter. Binding between β-chain promoter (-197/+128) and FOG-1 or GATA-1 was analyzed by ChIP assay using anti-FOG-1, anti-GATA-1, or each isotype control Ab. The GAPDH gene served as control. (D) Quantitative analysis of FOG-1 and GATA-1 binding to β-chain gene by ChIP assay using real-time PCR. ▪ indicates anti-FOG-1 Ab; ▨, goat IgG (control of anti-FOG-1 Ab); ▦, anti-GATA-1 Ab; □, rat IgG (control of anti-GATA-1 Ab). The results are expressed as mean ± SD for 2 PCRs with duplicate samples on each 2 independent ChIPs. Relative input units are calculated from Ct values as described in “ChIP assay.” Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology

Effect of FOG-1 mutants lacking NuRD or CtBP binding on β-chain promoter. Effect of FOG-1 mutants lacking NuRD or CtBP binding on β-chain promoter. (A) Structure of FOG-1 mutants: ΔN-FOG-1, lacking NuRD-binding N-terminus27; ΔCtBP-FOG-1, with replacement of 2 amino acids essential for CtBP binding28; ΔN/ΔCtBP-FOG-1, carrying both mutations of ΔN and ΔCtBP. (B) Coexpression analysis using FOG-1 mutants. PT18 cells were transfected with 5 μg of reporter plasmid β-69/pGL3-Basic with or without 3 or 10 μg of pCR-3.1 (mock), pCR-FOG-1 (wild-type), pCR-ΔN-FOG-1 (ΔN), pCR-ΔCtBP-FOG-1 (ΔCtBP), or pCR-ΔN/ΔCtBP-FOG-1 (ΔN/ΔCtBP). The ratio of each luciferase activity to that without coexpression plasmid was represented as relative luciferase activity. Data represent the average ± SD of triplicate samples. Keiko Maeda et al. Blood 2006;108:262-269 ©2006 by American Society of Hematology