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The Journal of Immunology, 2006, 177: 4605-4611.
Copyright © 2006 by The American Association of Immunologists, Inc.

Molecular Mechanisms for Transcriptional Regulation of Human High-Affinity IgE Receptor beta-Chain Gene Induced by GM-CSF1

Kyoko Takahashi*,{dagger}, Natsuko Hayashi*,{ddagger}, Shuichi Kaminogawa{dagger} and Chisei Ra2,*

* Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, Tokyo, Japan; {dagger} Nihon University College of Bioresource Sciences, Kanagawa, Japan; and {ddagger} Department of Applied Biological Chemistry, Tamagawa University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The beta-chain of the high-affinity receptor for IgE (Fc{epsilon}RI) plays an important role in regulating activation of Fc{epsilon}RI-expressing cells such as mast cells in allergic reactions. We already reported that the transcription factor myeloid zinc finger (MZF) 1 which formed a high m.w. complex including four and a half LIM-only protein (FHL)3 in the nucleus repressed human beta-chain gene expression through an element in the fourth intron. We also found that GM-CSF induced expression of MZF-1 and nuclear translocation of FHL3. We screened a human cDNA library and identified NFY which was reported to bind histone deacetylases (HDACs) as a constituent of the complex. The C-subunit of NFY was demonstrated to form a ternary complex with MZF-1/FHL3 and interact with a beta-chain gene region including the element in the fourth intron. HDAC1 and HDAC2 were also shown to interact with the fourth intron region of the beta-chain gene. In a human mast cell line HMC-1 cultured with GM-CSF, both beta-chain expression and acetylation of histones interacting with the fourth intron region of the beta-chain gene were decreased. Collectively, these results indicated that HDACs, which were recruited to the beta-chain gene through the element in the fourth intron by MZF-1/FHL3/NFY, repressed beta-chain gene transcription by deacetylation of histones in the presence of GM-CSF. These mechanisms will be involved in not only the cell type-specific repression of beta-chain gene expression in differentiating hemopoietic cells but also the repression of beta-chain gene expression in the peripheral cells under specific circumstances.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cells of limited types such as mast cells, basophils, eosinophils (1), monocytes (2), Langerhans cells (3, 4), platelets (5, 6), and neutrophils (7) express the high-affinity receptor for IgE (Fc{epsilon}RI). Fc{epsilon}RI plays an important role in triggering IgE-mediated allergic reaction. Cross-linking of Fc{epsilon}RI on effector cells, for example mast cells, by Ag (allergen)-IgE complexes triggers allergic reaction by activating intracellular signal cascades to induce not only the release of chemical mediators in the early-phase reaction but also cytokine gene expressions leading to the late-phase reaction. Moreover, Fc{epsilon}RI on epidermal Langerhans cells in the skin is known to be involved in the Ag presentation by facilitating uptake of IgE-associated allergens, suggesting its pivotal role in the pathophysiology of atopic dermatitis.

Fc{epsilon}RI is composed of three different subunits, {alpha}, beta, and {gamma}, of which the {alpha}-chain directly binds IgE through its extracellular domain, while the beta- and {gamma}-chains are responsible for mediating intracellular signals. Although a functional receptor is expressed both as tetramers ({alpha}beta{gamma}2) and trimers ({alpha}{gamma}2) in humans (8), intracellular signals (9, 10), in addition to cell surface expression of the receptor (11), were reported to be significantly amplified by beta-chain, indicating that beta-chain increases cell activation sensitivity to the stimulation by allergens. Recently, it was revealed that beta-chain amplified degranulation and leukotriene secretion but suppressed cytokine production (12), indicating that beta-chain regulates distinct intracellular signaling events in both positive and negative manners by the same molecule of itself. Furthermore, an alternative splice variant which encoded only an N-terminal portion of beta-chain was found to be expressed in human mast cells (13). This truncation variant competed with full-length beta and prevented Fc{epsilon}RI surface expression by inhibiting {alpha}-chain maturation (13). Because all of these findings indicate that beta-chain is a fine regulator of Fc{epsilon}RI-mediated cell activation to precisely control the allergic reaction, elucidation of regulatory mechanisms of beta-chain expression will make a meaningful contribution to medical intervention for allergy. In addition, because beta-chain has only been reported to associate with Fc{gamma}RIIIA except for Fc{epsilon}RI (14) and its expression is limited in the specific types of cells such as mast cells and basophils, applications which are targeted for beta-chain are expected to exert cell type-specific effects.

The genomic structure of the human Fc{epsilon}RI beta-chain gene was already determined (15), however, only a few analyses were performed on regulatory mechanisms of beta-chain gene transcription. Two Oct-1-binding sites in the 5' untranslated region were essential for activation of beta-chain gene promoter (16). We revealed that the transcription factor myeloid zinc finger (MZF)3 1 repressed beta-chain gene expression through an element in the fourth intron (17). It was suggested that this transcriptional repression required a cofactor; we therefore screened for MZF-1-binding proteins and identified a four and a half LIM-only protein (FHL)3 as a repressive cofactor (18). FHL3 was predicted to act as an adaptor molecule recruiting unidentified other molecules to the MZF-1/DNA complex, because MZF-1 and FHL3 formed a very large m.w. complex in the nucleus (18). In contrast, GM-CSF was reported to induce MZF-1 expression (19). Moreover, we found that GM-CSF induced translocation of FHL3 from the cytoplasm to the nucleus (18), suggesting that both up-regulation of MZF-1 and nuclear translocation of FHL3 by GM-CSF facilitate formation of the MZF-1/FHL3 complex in the nucleus and reduce beta-chain gene expression through the element in the fourth intron. It was actually reported that GM-CSF decreased Fc{epsilon}RI expression on the surface of a human mast cell line HMC-1 (20, 21). In this study, we report that Fc{epsilon}RI beta-chain gene expression can be suppressed in the presence of GM-CSF through deacetylation of histones mediated by histone deacetylases (HDACs) which are recruited to the beta-chain gene through the complex including MZF-1/FHL3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

HMC-1 (a human mast cell line) was cultured in IMDM (Invitrogen Life Technologies) at 37°C in a humidified incubator with 5% CO2. KU812 (a human basophilic leukemia cell line) was cultured in RPMI 1640 (Sigma-Aldrich). Both media contained 10% (v/v) FBS (JRH Bioscience), 100 U/ml penicillin (Banyu Pharmaceutical), and 100 µg/ml streptomycin (Meiji Seika).

Plasmid construction

pBridge-MZF-1/FHL3 encoding human MZF-1 fused to GAL4 DNA binding domain (BD) under the constitutive alcohol dehydrogenase 1 (ADH1) promoter and human FHL3 under the conditional MET25 promoter was constructed as follows. A NdeI/SalI-digested fragment from pGBKT7-MZF1 (18) was ligated with SmaI/SalI-digested pBridge vector (BD Biosciences). Both ends of the resulting linear DNA were blunted by T4 polymerase and subsequently ligated for self-circularization to obtain plasmid A. In contrast, pCR3.1-FHL3 antisense, which carried human FHL3 cDNA in the reverse direction in pCR3.1 vector (Invitrogen Life Technologies), was prepared by the same method as previously described for construction of pCR3.1-FHL3 (18). A BamH I/NotI-digested fragment from pCR3.1-FHL3 antisense was ligated with BglII/NotI-digested pBridge vector. Adjustment of the codon frames in the yielding plasmid was achieved by digestion with NotI, blunting by T4 polymerase, and self-ligation. The resulting plasmid was digested with ScaI/SalI to obtain a 4.23-kb fragment. The fragment was ligated with a 4.77-kb fragment from ScaI/SalI-digested plasmid A to yield pBridge-MZF-1/FHL3. For in vitro transcription/translation of NFYC, human NFYC cDNA obtained by SmaI/XhoI digestion of pACT2-NFYC was inserted into pGADT7 vector (BD Clontech) which was digested with EcoRI, blunted by T4 polymerase and subsequently digested with XhoI to yield pGADT7-NFYC. To construct an expression plasmid of MZF-1 deletion mutant corresponding to the aa 214–485 region of human MZF-1, the MZF-1 aa 214–485 region was amplified by PCR using pGBKT7-MZF1 (18) as a template and synthetic oligonucleotides of 5'-ATGGGCGATCCCCCGGGCCCTGGCGCTA-3' and 5'-CTACTCGGCGCTGTGGACGCGCTGGTG-3' as primers. The amplified product was subcloned into pCR3.1 vector (Invitrogen Life Technologies). After confirming the direction and nucleotide sequence of the insert, the obtained plasmid was named pCR3.1-MZF-1 (aa 214–485).

Yeast three-hybrid assay

Matchmaker Gal4 Two-Hybrid System 3 (BD Clontech) was used according to the manufacturer’s instructions. Yeast AH109 was transformed with pBridge-MZF-1/FHL3. The cells were sequentially transformed with a human lymph node cDNA library in a pACT2 vector (BD Clontech) containing a GAL4 activation domain (AD) and an hemagglutinin (HA) tag. Cotransformed cells were allowed to grow on a minimal synthetic dropout medium lacking tryptophan and leucine (SD-Trp/-Leu), because pBridge carried a TRP1 gene and pACT2 carried a LEU2 gene. FHL3 was only expressed in the cells cultured on the medium without methionine. Protein interaction in the cells was detected by expression of three reporter genes (HIS3, ADE2, and MEL1) under the control of distinct GAL4 upstream activating sequences and TATA boxes to reduce false positives. After selecting the clones expressing HIS3 on SD-Trp/-Leu/-His/-Met plates, growing colonies were transferred onto SD-Trp/-Leu/-His/-Met/-Ade(adenine) plates containing X-{alpha}-Gal to select those expressing the other reporter genes of ADE2 and MEL1. X-{alpha}-Gal was purchased from BD Clontech. Furthermore, to obtain proteins interacting with MZF-1 not independent of FHL3 but dependent on FHL3, the cells which grew on the medium without methionine but hardly grew on the medium including methionine were selected. pACT2 plasmids carrying cDNA inserts were rescued from the selected clones and their nucleotide sequences were determined. The interaction among MZF-1, FHL3, and the candidate protein NFYC was retested by cotransformation of AH109 cells with pBridge-MZF-1/FHL3 and pACT2-NFYC. The cells were examined for expression of HIS3 and ADE2 reporter genes by testing their ability to grow on -His/-Ade medium in the absence (FHL3 was expressed) and presence (FHL3 was not expressed) of methionine.

Immunoprecipitation of in vitro-translated MZF-1, FHL3, and NFY

c-Myc-tagged MZF-1 (aa 1–217), FHL3, and HA-tagged NFYC were produced by in vitro transcription/translation method with TNT T7 Quick Coupled Transcription/Translation System (Promega) using pGBKT7-MZF1(aa 1–217) (18), pCR3.1-FHL3 (18), and pGADT7-NFYC. The products were mixed for immunoprecipitation with anti-c-Myc mAb (Santa Cruz Biotechnology) followed by immunoblotting with anti-c-Myc and anti-HA (Roche) mAbs.

Chromatin immunoprecipitation

Cells were exposed to 1% formaldehyde for 10 min to obtain cross-linked chromatins. After washing with ice-cold PBS containing 1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin, the cells were resuspended in ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM potassium chloride, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and incubated on ice for 10 min. Then, they were solubilized with 0.5% (v/v) Nonidet P-40 for an additional 15 min. After centrifugation at 6,000 x g for 1 min, nuclear pellets were resuspended in buffer S (50 mM Tris (pH 8.1), 1% SDS, 10 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and incubated on ice for 15 min. The lysates were sonicated with Biorupter (Cosmo Bio). After centrifuging at 15,000 x g for 10 min to remove debris, the supernatants were diluted 10-fold in buffer C (16.7 mM Tris (pH 8.1), 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and precleared with 80 µl of salmon sperm DNA/protein A agarose (Upstate Biotechnology) for 1 h at 4°C. The samples were then incubated with anti-NFYC, anti-HDAC1, anti-HDAC2, anti-acetylated histone H3 or anti-acetylated histone H4 Ab for 4 h at 4°C with rotation. All five Abs were rabbit polyclonal Abs, of which anti-NFYC and anti-HDAC2 Abs were purchased from Santa Cruz Biotechnology and the remaining Abs were obtained from Upstate Biotechnology. After addition of 60 µl of salmon sperm DNA/protein A agarose, the binding reactions were incubated for an additional 1 h at 4°C with rotation. The immunoprecipitated chromatins were washed sequentially with wash buffers 1, 2, 3, and 4 (1:50 mM Tris (pH 8.1), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1% Nonidet P-40, 2: 50 mM Tris (pH 8.1), 500 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1% Nonidet P-40, 3: 50 mM Tris (pH 8.1), 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholic acid, 1 mM EDTA, 4: 10 mM Tris (pH 8.0), 1 mM EDTA) and eluted from beads with elution buffer (1% SDS, 50 mM NaHCO3). After incubation in the elution buffer for 15 min at room temperature with rotation, supernatants were separated by centrifugation. To reverse cross-linking, the eluates were incubated at 65°C for 4 h in a buffer containing 200 mM NaCl and 1 µg of RNase A followed by treatment with 20 µg of proteinase K in 40 mM Tris (pH 6.5), 10 mM EDTA at 45°C for 1 h. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. The precipitates were resuspended in sterile water and subjected to PCR analyses using synthetic oligonucleotides of 5'-CTGGAATGTTGTCAATTATATCTGAAAGG-3' and 5'-CTGTTCTTCTTATCTTTTCAAGGATGGAC-3' specific for the beta-chain fourth intron region as primers. Primers of 5'-TGCCTAGGTCACCCACTAATG-3' and 5'-GTGGCCCGTGATGAAGGCTA-3' (22) that specifically recognize the beta-actin promoter sequences were used as control. A thermal cycle of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min was repeated 33 times. Sizes of the fragments amplified by the beta-chain intronic region-specific and beta-actin promoter region-specific primers were 99 and 160 bp, respectively.

Transfection of the cells

KU812 cells were suspended in the medium containing 20% FBS at the concentration of 5 x 106 cells/500 µl. The cells were transfected with 5 µg of pCR3.1-MZF-1(aa 214–485) or an empty vector pCR3.1-self (17) as control by electroporation at 300 V, 950 µF using Gene Pulser II (Bio-Rad) and cultured for 16 h. To select transfected cells, the cells were then cultured with 400 µg/ml G418 until they were subjected for the assay.

Quantitation of Fc{epsilon}RI beta-chain mRNA levels by real-time RT-PCR

Total RNA was isolated from cells cultured in the presence or absence of 10 ng/ml GM-CSF for 1–10 days with TRIzol reagent (Invitrogen Life Technologies) and was reverse transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems) using an oligo(dT)16 primer. Quantitative PCR was performed with TaqMan Universal PCR Master Mix without UNG AmpErase (Applied Biosystems) using human Fc{epsilon}RI beta-chain-specific and human beta-actin-specific primers and fluorescent probes (Applied Biosystems). Reactions were conducted with the conditions of 95°C for 10 min followed by 50 cycles of 95°C for 15 s and 60°C for 1 min by ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Relative expression levels of beta-chain and beta-actin were calculated respectively from the standard curve using a dilution series of template RNA. The expression levels of beta-chain were normalized by those of beta-actin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NFY forms a complex with MZF-1/FHL3

We at first tried to identify constituents of the large nuclear complex which contained MZF-1/FHL3 and suppressed human Fc{epsilon}RI beta-chain gene expression through the element in the fourth intron. A human cDNA library was screened for proteins forming a ternary complex with MZF-1 and FHL3 by yeast three-hybrid assay (Fig. 1, A and B). An expression plasmid which carried MZF-1 cDNA fused to GAL4BD under the constitutive ADH1 promoter (PADH1) and FHL3 cDNA under the conditional Met25 promoter (PMet25) repressed with methionine was constructed. Full-length MZF-1 fused to GAL4BD did not autonomously activate the reporter (18). The construct was introduced into a yeast AH109 strain with a human cDNA library-GAL4AD fusion expression plasmid to search proteins forming a ternary complex with MZF-1/FHL3. As represented in Fig. 1C, we screened 2.6 x 106 cDNA clones and obtained 188 positive clones expressing all three reporter genes of HIS3, ADE2, and MEL1. To distinguish the clones which encoded proteins interacting with MZF-1 in an FHL3-dependent manner from those encoded proteins directly binding MZF-1 independent of FHL3, the cells which grew on the medium without methionine, but hardly grew on the medium including methionine, were selected. Of 11 clones obtained, 10 clones encoded three different proteins and the remaining clone did not have a cDNA insert in frame.


Figure 1
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  		FIGURE 1. Screening of a human cDNA library by yeast three-hybrid assay. A, Structures of expression plasmids used for the yeast three-hybrid screen are shown. MZF-1 cDNA fused to GAL4BD and FHL3 cDNA are inserted into pBridge vector under the constitutive ADH1 promoter and the conditional MET25 promoter, respectively (pBridge-MZF-1/FHL3). A human cDNA library is fused to GAL4AD in pACT2 vector (pACT2-library). B, In the three-hybrid assay, FHL3 is expressed only in the absence of methionine. If a protein encoded by pACT2-library forms a ternary complex with FHL3 and MZF-1 as indicated in the figure, expression of reporter genes (HIS3, ADE2, MEL1) is activated. C, Yeast AH109 was sequentially transformed with pBridge-MZF-1/FHL3 and pACT2-library. After selecting the clones expressing HIS3 reporter on -Trp/-Leu/-His/-Met medium, growing colonies were then tested for expression of ADE2 and MEL1 reporters on -Trp/-Leu/-His/-Met/-Ade medium containing X-{alpha}-Gal. To obtain the clones which encoded proteins interacting with MZF-1 in an FHL3-dependent manner, the cells expressing all three reporter genes were further selected, based on their poor growth on -Trp/-Leu/-His/-Ade medium containing methionine.

 
The C subunit of the transcription factor NFY was one of the three identified proteins. To verify that NFYC interacts with MZF-1 through FHL3 to form a ternary complex, yeast AH109 cells were cotransformed with constructs carrying PADH1-BD-MZF-1/PMet25-FHL3 and PADH1-AD-NFYC, respectively. Growth of the transformed cells was examined on -His/-Ade medium to test the expression of two reporter genes of HIS3 and ADE2. The transformed cells grew on the -His/-Ade medium in the absence of methionine (FHL3 was expressed) but did not grew in the presence of methionine (FHL3 was not expressed) (Fig. 2A). To further confirm the interaction, c-Myc-tagged BD-MZF-1, FHL3, and HA-tagged AD-NFYC were produced by in vitro transcription/translation method. Their mixtures were immunoprecipitated with anti-c-Myc tag Ab. Because the full-length MZF-1 fused to BD was not expressed efficiently by in vitro transcription/translation, the aa 1–217 region of MZF-1 which could bind FHL3 (18) was used. BD-MZF-1 and AD-NFYC were coimmunoprecipitated in the presence of FHL3 but were not in the absence of FHL3 (Fig. 2B). Both of the results from Fig. 2 demonstrated that NFYC interacted with MZF-1 through FHL3 and formed a ternary complex, indicating that NFYC was a candidate constituting the large regulatory complex, which repressed human beta-chain gene expression through the element in the fourth intron, with MZF-1 and FHL3 in the nucleus.


Figure 2
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  		FIGURE 2. NFYC interacts with MZF-1 through FHL3. A, Yeast AH109 was cotransformed with pBridge-MZF-1/FHL3 and pACT2-NFYC to test interaction among MZF-1, FHL3, and NFYC. The cells were examined for expression of HIS3 and ADE2 reporter genes by testing their ability to grow on -Trp/-Leu/-His/-Ade medium in the absence (FHL3 was expressed, panel 7) and presence (FHL3 was not expressed, panel 5) of methionine. B, c-Myc-tagged MZF-1, FHL3, and HA-tagged NFYC were prepared by in vitro transcription/translation. These proteins were mixed with the indicated combination, immunoprecipitated with anti-c-Myc mAb, and subsequently blotted with anti-c-Myc and anti-HA mAbs.

 
NFY and HDAC interact with the fourth intron region of the human Fc{epsilon}RI beta-chain gene

We next analyzed whether NFYC was actually involved in the transcriptional regulation of the human Fc{epsilon}RI beta-chain gene. To examine whether NFYC interacts with the fourth intron region of the beta-chain gene in the context of chromatin in vivo, chromatin immunoprecipitation (ChIP) assays of the endogenous beta-chain gene in the human mast cell line HMC-1 were conducted. As shown in Fig. 3A, the fourth intron region of the beta-chain gene corresponding to nt 4133–4231 (represented nucleotide numbers begin at the transcription start site as position +1) which included the MZF-1-binding element was amplified from chromatins immunoprecipitated with anti-NFYC Ab but scarcely amplified from those immunoprecipitated with control rabbit IgG. In contrast, the beta-actin promoter region was amplified only at a low level from both chromatins immunoprecipitated with anti-NFYC Ab and control IgG. Relative band intensities to those from the input fraction were represented in the graphs. The band intensity of beta-chain intron region amplified from the fraction immunoprecipitated with anti-NFYC was significantly higher than that from the fraction immunoprecipitated with control IgG (p < 0.005), but the band intensities of the beta-actin promoter region from the fractions immunoprecipitated with anti-NFYC Ab and control IgG were not significantly different. These results indicated that NFYC specifically interacted with the beta-chain gene region including the element in the fourth intron.


Figure 3
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  		FIGURE 3. NFYC and HDAC interact with the fourth intron region of the beta-chain gene. A, ChIP assays of the endogenous beta-chain gene in HMC-1 cells were performed using anti-NFYC Ab. Rabbit IgG was used as control. The fourth intron region nt 4133/4231 of the beta-chain gene and the promoter region of the beta-actin gene as control were amplified by PCR from the immunoprecipitated chromatins. The lanes of "input" represent the results of PCR using diluted fractions of nonimmunoprecipitated chromatins as templates to quantitate the amount of DNA present in each sample before immunoprecipitation. Amplified fragments of the beta-chain intronic region and the beta-actin promoter region were 99 and 160 bp, respectively. B, ChIP assays of the endogenous beta-chain gene in HMC-1 cells were similarly performed using anti-HDAC1 and anti-HDAC2 Abs. The chromatins were immunodepleted with anti-NFYC Ab or control rabbit IgG before immunoprecipitation with anti-HDAC Abs. Data shown are representative of four (A) or three (B) independent experiments. Relative band intensities to those from input fractions are expressed in the graphs as means ± SD of the four (A) or three (B) experiments. In B, {blacksquare} and Figure 3 express the results of immunodepletion experiments with control IgG and anti-NFYC Ab, respectively. Statistically significant differences with the two-tailed Student t test are indicated by asterisks (**, p < 0.005 and *, p < 0.05).

 
NFY consists of A, B, and C subunits (23), of which the A subunit has been reported to bind HDACs (24). Therefore, we analyzed the interaction between HDACs and the intronic region of the beta-chain gene by ChIP assays using anti-HDAC1 and anti-HDAC2 Abs (Fig. 3B). The beta-chain intron region was significantly, more highly amplified from chromatins immunoprecipitated with anti-HDAC1 or anti-HDAC2 Ab than from those immunoprecipitated with rabbit IgG as control (p < 0.05). In contrast, amplification of the beta-actin promoter region from chromatins immunoprecipitated with anti-HDAC Abs was as low as that from chromatins immunoprecipitated with control IgG. Therefore, it was demonstrated that HDAC1 and HDAC2 interacted with the beta-chain gene region including the element in the fourth intron. Furthermore, pretreatment with anti-NFYC Ab resulted in remarkable decrease of amplification of the beta-chain intron region (p < 0.05, vs control IgG pretreatment), indicating that NFYC was actually required for the recruitment of HDACs to the beta-chain gene.

The experiments of Fig. 3 revealed that NFY and HDACs interacted with the beta-chain gene region including the element in the fourth intron in the context of chromatin in vivo. The results suggested a possibility that HDACs, which were recruited to the beta-chain gene through the NFY/FHL3/MZF-1 complex, deacetylated histones to repress the transcription. It was also suggested that the transcriptional repression of the beta-chain gene was promoted by GM-CSF which induced up-regulation of MZF-1 (19) and nuclear translocation of FHL3 (18), because it was predicted that recruitment of HDACs to the beta-chain gene was facilitated by increased formation of the large regulatory complex containing MZF-1 and FHL3 in the nucleus.

GM-CSF decreases histone acetylation of the human Fc{epsilon}RI beta-chain gene and suppresses beta-chain gene expression

To determine whether HDAC activities were involved in the transcriptional repression of the beta-chain gene through the element in the fourth intron, acetylation levels of histones were compared between the cells cultured in the presence and absence of GM-CSF for 1, 4, and 10 days. ChIP assays were conducted using anti-acetylated histone H3 and anti-acetylated histone H4 Abs to analyze whether the intronic region of the beta-chain gene associates with acetylated histones in HMC-1 cells (Fig. 4A). Amplification of the beta-chain fourth intron region from both of the chromatins immunoprecipitated with anti-acetylated histone H3 and anti-acetylated histone H4 Abs was not changed by GM-CSF at day 1 but significantly decreased by GM-CSF at days 4 and 10 (p < 0.05 vs without GM-CSF). The amounts of the amplified products of the beta-actin promoter were not affected by GM-CSF. It was thus demonstrated that acetylation of histones interacted with the fourth intron region of the beta-chain gene was decreased in HMC-1 cells cultured with GM-CSF for 4–10 days. Parallel to the decrease of histone acetylation, interaction of NFYC and HDACs with the beta-chain gene was increased at day 4 and more remarkably increased at day 10, suggesting that the decreased histone acetylation was mediated by HDACs which were recruited to the beta-chain gene through NFY.


Figure 4
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  		FIGURE 4. GM-CSF decreases acetylation of histones interacting with the fourth intron region of the beta-chain gene in an MZF1/FHL3-dependent manner. A, HMC-1 cells cultured with or without 10 ng/ml GM-CSF for 1, 4, and 10 days were subjected to ChIP assays. The chromatins immunoprecipitated with anti-NFYC, anti-HDAC1, anti-HDAC2, anti-acetylated histone H3 ({alpha}-AH3), and anti-acetylated histone H4 ({alpha}-AH4) Abs were amplified by PCR using primers specific for the intronic element of the beta-chain gene and the beta-actin promoter as control. Rabbit IgG was used as control. The lanes of "input" represent the results of PCR using diluted fractions of nonimmunoprecipitated chromatins as templates. B, An expression plasmid of MZF-1 deletion mutant ({Delta}MZF-1) which lacked the region binding FHL3 or an empty vector as control were respectively introduced into KU812 cells. After culturing the transfected cells with or without 10 ng/ml GM-CSF for 7 days, ChIP assays using anti-acetylated histone H3 and anti-acetylated histone H4 Abs were performed. Data shown are representative of three independent experiments. Relative band intensities to those from input fractions are expressed in the graphs as means ± SD of the three experiments. {blacksquare} and Figure 4 represent the results of cells cultured in the absence and presence of GM-CSF, respectively. Results of statistical analysis with the two-tailed Student t test between GM-CSF-treated and nontreated cells are as follows: *, p < 0.05 and **, p < 0.005.

 
In the previous report, we revealed that MZF-1 bound FHL3 through four zinc finger motifs at its N-terminal (18). An expression plasmid of the MZF-1 deletion mutant which lacked this FHL3-interacting region was constructed and introduced into a human basophilic leukemia cell line KU812 which was known to express mast cell-specific molecules, because HMC-1 cells gave very low transfection efficiency. By similar ChIP assays using anti-acetylated histone H3 and anti-acetylated histone H4 Abs, it was shown that association between the intronic region of the beta-chain gene and acetylated histones was not decreased in the cells cultured with GM-CSF for 7 days when the MZF-1 deletion mutant was overexpressed, while the association was significantly decreased in the mock-transfected cells by GM-CSF (Fig. 4B). The beta-chain mRNA expression was decreased to approximately one-half in KU812 cells cultured with GM-CSF for 7 days (data not shown). These results indicated that the decrease of histone acetylation in the presence of GM-CSF was mediated through the regulatory complex including MZF-1/FHL3/NFY.

We further analyzed whether Fc{epsilon}RI beta-chain expression was repressed in HMC-1 cells cultured in the presence of GM-CSF by real-time RT-PCR. As represented in Table I, the cells cultured with GM-CSF for 4 and 10 days expressed significantly smaller amounts of beta-chain mRNA compared with the cells cultured without GM-CSF (p < 0.05 at day 4 and p < 0.01 at day 10), although they expressed a slightly but reproducibly increased level of beta-chain mRNA at day 1 in the presence of GM-CSF (p < 0.05 vs without GM-CSF). These results showed that sustained exposure to GM-CSF, which decreased acetylation of histones interacting with the beta-chain gene, suppressed beta-chain gene expression.


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  		Table I. Effect of GM-CSF on Fc{epsilon}RIbeta-chain gene expressiona

 
Collectively, it was suggested that the repression of beta-chain gene expression in the presence of GM-CSF was dependent on deacetylation of histones by HDACs which were recruited to the beta-chain gene through the MZF-1/FHL3/NFY complex.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We revealed that Fc{epsilon}RI beta-chain gene expression was repressed in the cells cultured with GM-CSF and this transcriptional repression was correlated with histone deacetylation of the beta-chain gene. Fig. 5 describes mechanisms for the HDAC-mediated transcriptional repression which are suggested from our study. Increased histone deacetylation of the beta-chain gene in the presence of GM-CSF was thought to be dependent on enhanced formation of the HDAC/NFY/FHL3/MZF-1 complex in the nucleus, because expression of MZF-1 and nuclear translocation of FHL3 were induced by GM-CSF (18). Although the expression of NFY was not affected by GM-CSF (data not shown), there still remains a possibility that expression or subcellular localization of unidentified factors in the large regulatory complex including HDAC/NFY/FHL3/MZF-1 was influenced by GM-CSF.


Figure 5
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  		FIGURE 5. A schematic drawing of a model for histone deacetylation-mediated regulation of beta-chain gene expression. Histone deacetylation-mediated regulatory mechanisms of beta-chain gene expression suggested by our study are described schematically. Ac, acetyl group.

 
The cell surface expression of Fc{epsilon}RI has been reported to be suppressed by GM-CSF in HMC-1 cells where {alpha}-chain mRNA was decreased (20, 21). Because our study showed that beta-chain expression was also repressed, suppression of both {alpha} and beta-chain gene expression was thought to contribute to the decrease of Fc{epsilon}RI expression in the cells cultured with GM-CSF. GM-CSF is a hemopoietic cytokine which promotes differentiation and proliferation of myeloid cells. GM-CSF is also known to inhibit terminal differentiation of mast cells. These observations suggest that GM-CSF plays a particular role in myeloid cell differentiation by repressing the expression of Fc{epsilon}RI in the cells differentiating into eosinophils, neutrophils, or monocytes which express low or undetectable levels of Fc{epsilon}RI in their resting states. In addition to the effects on hemopoietic cell differentiation, GM-CSF plays a key role in peripheral inflammatory processes by enhancing functional activities of mature myeloid effector cells. Therefore, the regulatory mechanisms of Fc{epsilon}RI expression induced by GM-CSF will also contribute to the control of Fc{epsilon}RI-mediated functions of peripheral cells under specific circumstances.

Interestingly, the amount of beta-chain mRNA was once slightly increased before it was significantly decreased by GM-CSF (Table I). In the time course analyses in Fig. 4A and Table I, while the expression of beta-chain gene expression was decreased at days 4–10 parallel to the increased association of NFYC and HDACs with the beta-chain gene, neither significant difference of NFYC or HDAC interaction nor that of histone acetylation was observed between days 0 and 1. Therefore, it was thought that the slight increase of beta-chain gene expression observed at day 1 was not due to decreased association of NFY/HDAC with the fourth intron region of the beta-chain gene. However, we could not neglect the possibility that the change of NFY or HDAC interaction was small and not sufficient to be detected by ChIP assays. Although it is not clear what this phenomenon means at present, it may possibly reflect bidirectional regulation through the MZF-1-binding element, because we found that MZF-1 could activate beta-chain gene transcription with a different cofactor when the cofactor was overexpressed (K. Takahashi, N. Hayashi, and C. Ra, manuscript in preparation). The MZF-1-binding element in the fourth intron is thought to repress and activate beta-chain gene transcription depending on cofactor availability. The equilibrium will markedly incline to the transcriptional repression in the presence of GM-CSF, but nuclear translocation of FHL3 which was observed 24 h after the addition of GM-CSF (18) may not be sufficient to shift the equilibrium, judging from the fact that beta-chain mRNA was temporally increased before it was significantly decreased by GM-CSF.

This is the first report that revealed the binding between FHL3 and NFY. FHL3 is a member of four-and-a-half LIM only protein (FHL) family including FHL1, FHL2, FHL3, FHL4, and ACT (25, 26, 27, 28, 29, 30). They only contain a single N-terminal half-LIM domain followed by four sequential LIM domains, belonging to a subset of the LIM protein superfamily which possesses cysteine-rich double zinc finger motifs called LIM. FHL proteins are expressed in a cell- and tissue-specific manner and participate in various cellular processes by interacting with actin cytoskeleton in the cytoplasm and with transcriptional machinery in the nucleus. These studies suggest that FHL proteins function as adaptors or scaffolds to support the assembly of multimeric protein complex. Although there are several reports that FHL family proteins act as coactivators or corepressors of transcription factors (31, 32, 33, 34), precise mechanisms by which FHL proteins mediate the protein interaction and exert their functions as transcriptional cofactors remain to be elucidated. Our study presented just an example that FHL3 was involved in the transcriptional regulation by functioning as an adaptor molecule which linked the HDAC-containing protein complex with a transcription factor/DNA complex.

NFY is a heterotrimeric transcription factor which is known to bind CCAAT motifs in the proximal promoter of a wide variety of genes and activate transcription, particularly in TATA-less genes. Among the three subunits constituting NFY, A and B-subunits have been reported to bind HDAC and p300/CBP, respectively (24, 35). These reports indicate that NFY can directly interact with both histone acetyl transferase and HDAC, which are known to act as a transcriptional coactivator and corepressor, respectively. It has been actually reported that NFY functions not only as an activator but also as a repressor depending on promoter contexts and cell types (36, 37, 38, 39). Interestingly, NFY functions as a repressor by binding a DNA sequence different from its consensus-binding motif CCAAT (36), suggesting that NFY binds different DNA sequences through its different domains. It is thought that NFY changes its conformation by binding to the different DNA sequences via its different domains, which allows NFY to specifically bind histone acetyl transferase or HDAC. There is no typical CCAAT-binding motif in the human beta-chain gene, but NFY may bind to a noncanonical-binding motif in the beta-chain gene region apart from the element in the fourth intron and interact with the FHL3/MZF-1 complex. In that case, deacetylation of histones may not be limited in the region close to the element in the fourth intron but may spread out to a more broad region of the beta-chain gene.

In this study, we revealed the histone deacetylation-mediated mechanisms for transcriptional regulation of the human Fc{epsilon}RI beta-chain gene through the element in the fourth intron. The concrete and specific information about the molecules and their interactions which participate in the regulation of beta-chain gene expression like this will contribute to applications to the development of therapeutic and prophylactic drugs for allergy. More detailed study including the analyses on regulatory mechanisms through other elements of the beta-chain gene will reveal the entire regulatory mechanisms of Fc{epsilon}RI beta-chain gene expression.


   Acknowledgments
 
We thank the members of Division of Molecular Cell Immunology and Allergology for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Address correspondence and reprint requests to Dr. Chisei Ra, Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, 30-1 Oyaguchi Kamimachi, Itabashi-ku, Tokyo 173-8610, Japan. E-mail address: fcericra{at}med.nihon-u.ac.jp Back

3 Abbreviations used in this paper: MZF, myeloid zinc finger; FHL, four and a half LIM-only protein; HDAC, histone deacetylase; BD, binding domain; ADH, alcohol dehydrogenase; HA, hemagglutinin; AD, activation domain; ChIP, chromatin immunoprecipitation. Back

Received for publication March 15, 2006. Accepted for publication July 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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