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Nucleic Acids Research, 2000, Vol. 28, No. 14 2627-2633
© 2000 Oxford University Press

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

Satsuki Suzuki-Ishigaki, Keiko Numayama-Tsuruta, Atsuo Kuramasu, Eiko Sakurai, Yoko Makabe, Sanae Shimura1, Kunio Shirato1, Kazuhiko Igarashi2, Takehiko Watanabe and Hiroshi Ohtsu*

Department of Cellular Pharmacology, Tohoku University School of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575, Japan, 1Department of Internal Medicine I, Tohoku University School of Medicine, Seiryo-machi 1-1, Aoba-ku, Sendai 980-8574, Japan and 2Department of Biochemistry, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan

Received as resubmission May 27, 2000; Accepted May 29, 2000.

DDBJ/EMBL/GenBank accession no. AB039880.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning ~24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter–reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells leave the bone marrow as progenitors and complete their differentiation in peripheral tissue (1). However, little is known about the molecular mechanisms which control the differentiation of mast cells. One of the excellent markers for mast cell differentiation is L-histidine decarboxylase (HDC) (EC4.1.1.22). HDC is a unique enzyme that catalyzes the formation of histamine from L-histidine. In cells of hematopoietic lineage, the mouse HDC gene is expressed predominantly in mature mast cells and erythroblastic cells, but not in immature mast cells. The mouse immature mast cell line P815 is induced to mature and express high levels of HDC mRNA by peritoneal cavity incubation (2). Thus, HDC expression correlates well with the differentiation of mast cells. In light of these observations, it is important to clarify how HDC gene expression is regulated during the differentiation process.

The expression of genes conferring certain specific phenotypes during development and differentiation is often controlled by lineage-specific transcription factors. For instance, the mast cell carboxypeptidase A (MC-CPA), IL-4, high affinity IgE receptor {alpha} chain and IL-1 receptor-related T1 genes are activated by GATA factors (3–6) and the TNF-{alpha} gene in mast cells is activated by the Nrf1 transcription factor, one of the Cap ‘n’ collar basic leucine zipper (CNC-bZIP) members (7). In addition, we observed that NF-E2, another member of the CNC-bZIP family, transactivates the mouse HDC gene (2). These data imply that several transcription factors are not only expressed but are also functionally important in mast cells.

In addition to the control by specific transcription factors, DNA methylation plays a suppressive role in the regulation of gene expression during development. Two primary mechanisms are proposed by which this suppression is mediated: (i) methylation interferes with the initiation of transcription by preventing binding of the cellular factors AP-2 (8), c-Myc/Myn (9), E2F (10) and NF-{kappa}B (11); (ii) methylation attracts proteins which themselves mediate repression (12–15). We have previously characterized the structure of the human HDC gene (16) and suggested that an alteration of the DNA methylation state of this gene affects its expression (17).

In this report we have analysed the mouse HDC gene structure and the process of DNA methylation in the context of cell-type specific and inducible gene expression. Isolation of the mouse HDC gene allowed us to examine its promoter activity in the P815 peritoneal incubation system and to research the control mechanism of mast cell-specific gene expression. We showed that the promoter region was demethylated not only in the HDC-expressing cell lines but also in P815 cells when HDC expression was induced by peritoneal incubation. We also demonstrated that DNA methylation around the GC box suppressed the expression of a patch-methylated HDC promoter–reporter construct in stably transfected cell lines and that treatment with the demethylating agent 5-azaC restored expression of this construct. These observations suggest that demethylation of the promoter region is necessary for mouse HDC gene expression, and may lead to more general conclusions regarding the molecular control of transcription by methylation mechanisms in mast cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Genomic library screening and analysis of phage clones
Mouse genomic DNA clones were isolated from a 129Sv-derived E14 ES cell DNA P1 phagemid library by PCR screening with primers designed to produce a 147 bp PCR product (nt +1 to +147) within mouse cDNA (5' primer, GAG TGC ACA GCA CAG ACA AAG G; 3' primer, TCT AGC TCG GTA GTA TTC ACT). DNA was isolated from purified phage plaques according to the procedure described by Sambrook et al. (18) and fragments of interest were subcloned into the pBluescript KS(+) plasmid vector (Stratagene, San Diego, CA) for further analysis. The nucleotide sequences of the plasmid subclones containing the 5'-flanking region, exon 1 and intron 1 were determined by cycle sequencing using an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Foster City, CA) (DDBJ accession no. AB039880) (19).

Cell lines
The mouse mast cell line P815 (20) was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin (PS). The IL3-dependent mast cell progenitor line IC2 (21,22) and the mouse mast cell line MC9 (23,24) were maintained in RPMI 1640 medium supplemented with 50% WEHI-3B conditioned medium, 10% FBS and PS. The mouse erythroleukemia cell line DS19 and the mouse fibroblast cell line NIH 3T3 were maintained in Dulbecco’s minimum essential medium (D-MEM) supplemented with 10% FBS and PS.

Intraperitoneal induction and treatment with 5-azaC
P815 cells were introduced into the peritoneal cavities of BDF1 mice and recovered as previously described (2). Briefly, a suspension of 2 x 106 cells was injected into the peritoneal cavity of a BDF1 mouse; 1 week later, P815 cells were harvested from the peritoneal fluid. To assess the effect of demethylation of the promoter region on HDC transcription, P815, IC2 and NIH 3T3 cells were cultured with 5-azaC (Sigma, St Louis, MO) and harvested as follows. Initially, cells were cultured at 2 x 105 cells/ml and treated with 10 µM 5-azaC on the second and fifth days for 24 h each and were harvested on the seventh day.

RNA blot hybridization analysis
Total RNA was extracted by the acid guanidium thiocyanate/phenol–chloroform extraction method (25). Thirty micrograms of the RNA was resolved by electrophoresis on a 1.0% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech, Buckinghamshire, UK) by capillary transfer with 20x SSC. The membrane was hybridized with [32P]dCTP-labeled specific cDNA probes in hybridization buffer (50% formamide, 5x SSPE, 5x Denhardt’s solution, 0.5% SDS and 200 µg/ml salmon sperm) at 42°C overnight. After washing twice with 2x SSPE/0.1% SDS and once with 1x SSPE/0.1% SDS at 42°C, the membrane was exposed to X-ray film (Kodak, Rochester, NY) at –80°C for up to 5 days before the film was developed.

Reverse transcription (RT)–PCR
One microgram of total RNA was primed with random hexamer and reverse transcribed using Superscript II reverse transcriptase (Gibco BRL, Life Technologies, Inc., Rockville, MD) in a total volume of 20 µl. PCR was carried out in a total volume of 15 µl containing 0.5 µl of cDNA, 0.2 mM dNTP mixture, 100 mM specific sets of primers and 0.4 U AmpliTaq Gold (Perkin-Elmer). The PCR conditions were as follows: 9 min incubation at 94°C followed by 35 cycles of 94°C for 1 min, 55 (for HDC) or 65°C (for ß-actin) for 1 min and 72°C for 1 min. The primers used in PCR were as described previously (2). The histamine content of the cell lines was measured using a HPLC fluorometric system as previously described (2,26)

DNA blot hybridization analysis
Isolation of high molecular weight genomic DNA for Southern blot analysis was described elsewhere (18). The DNA was digested with various restriction enzymes and the resulting fragments were separated by electrophoresis on 1.0% agarose gels. After sequential treatment with 0.25 N HCl, 1.5 M NaCl/ 0.5 M NaOH and 1.5 M NaCl/0.5 M Tris–HCl (pH 7.2), the DNA fragments were transferred to nylon membranes. The membranes were hybridized with [32P]dCTP-labeled cDNA probes at 65°C overnight in hybridization buffer (5x SSPE, 5x Denhardt’s solution, 0.5% SDS and 20 µg/ml salmon sperm). The blots were washed twice with 2x SSPE/0.1% SDS and once with 1x SSPE/0.1% SDS at 65°C and the hybridization signals were visualized by autoradiography as described above.

Sodium bisulfite genomic sequencing
Bisulfite genomic sequencing was performed essentially as described (27–29). Ten micrograms of PstI-digested genomic DNA was denatured in 50 µl of 0.3 M NaOH at 37°C for 15 min. Next, sulfonation and hydrolytic deamination reactions were carried out by adding 450 µl of 2.5 M sodium metabisulfite (Na2S2O5)/10 mM hydroquinone (pH 5.0) to the digested genomic DNA and incubating the mixture at 50°C for 4 h in the dark. The DNA was purified using Glassmilk silica matrix (Bio 101, Joshua Way Vista, CA) and eluted from the matrix with TE buffer. Desulfonation was then performed in 200 µl of 0.3 M NaOH at 37°C for 15 min. The bisulfite-reacted DNA was precipitated, resuspended in 100 µl of water and stored at –20°C. The sequence of interest in the bisulfite-reacted DNA was amplified by PCR in a reaction mixture containing 5 µl bisulfite-reacted DNA, 0.5 µM primers, 200 µM dNTPs, 2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3) and 2.5 U AmpliTaq Gold in a final volume of 100 µl. Each amplification consisted of 9 min incubation at 94°C followed by 40 cycles of 94°C for 1 min, 52°C (sense strand) or 48°C (antisense strand) for 1 min and 72°C for 1 min. The primer sets used in this experiment were as follows. Sense strand: a1 (5'-GCT CTA GAC ACA AAC AAC AAT CAC AAA AAT AAA-3') and a2 (5'-GTT TCT AGA TTT GGG AGG TTA GGT GTT TTA ATT-3'); g1 (5'-AAA AAC CAA AAA TAA ACC AAA AAC ATT CAC-3') and g2 (5'-GGT ATA GAT AAT TGT TGT TAT TAG AAA GAT-3'). Antisense strand: b1 (5'-GCT CTA GAC TTA AAA AAC TAA ATA CCC CAA CTA-3') and b2 (5'-GCT CTA GAT ATA GGT AGT AAT TAT AGA AGT AGG-3'); h1 (5'-CAC CAA AAA AAT TTC CAT CTA ACA AAA TAA-3') and h2 (5'-TTT TTA GTT TAG AGG TTA GAA ATA GGT TAA-3'). Amplified PCR products were precipitated and sequenced directly by the cycle sequencing method using an ABI PRISM 377 DNA sequencer (Perkin-Elmer).

Patch-methylation of the reporter plasmid
The HDC–luciferase reporter plasmid pGLm-1099, the construction of which is described in detail elsewhere (2), contains a 1.1 kb promoter region (nt –1099 to +91) from the HDC gene. pGLm-1099 was digested with KpnI and HindIII to yield the 1.1 kb HDC promoter fragment, which was separated from the parent vector by agarose gel electrophoresis and recovered by electroelution according to the procedure described by Lida (30). This 1.1 kb promoter fragment was methylated with SssI methylase and finally ligated into KpnI/HindIII-digested pGL-2 plasmids to obtain the patch-methylated (PM+) plasmid. The mock-methylated (PM–) plasmid was constructed by the same procedure without methylase treatment. These constructs were linearized by digestion with SmaI for transfection.

Stable transfection assay
P815 cells (3 x 106) were incubated on ice for 10 min in 350 µl of PBS with a mixture of 1 µg linearized pGLm-1099 (PM+ or PM– plasmids or the linearized parental pGLm-1099 as a control) and 0.2 µg plasmid (pRcRSV) carrying the neo gene. Electroporation was then carried out with a Gene Pulser II electroporation device (Bio-Rad, Hercules, CA) set to 300 V/960 µF. The cell suspensions were dispensed into 96-well plates in 50 µl aliquots. After incubation at 37°C for 48 h, G418 (Gibco BRL, Life Technologies, Inc.) was added to each well at a final concentration of 500 µg/ml. The cell suspensions were incubated at 37°C for ~2 weeks to identify stable transfectants. We confirmed the presence of the transfected plasmid in two PM+ lines (P1M+ and P2M+), two PM– lines (P1M– and P2M–) and five control lines (C1–5). Cell lysates were prepared according to the protocol supplied with the luciferase assay kit (TOYO Ink, Tokyo, Japan) and luciferase activities were measured with a Biolumat luminometer (Berthold Japan, Tokyo, Japan). Data were expressed as the means of triplicate relative luciferase activities, normalized to total protein concentration. The protein concentrations of the cell lysates were measured using Bio-Rad protein assay solution (Bio-Rad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Structure of mouse HDC gene
We obtained two P1 plasmids by library screening using PCR and confirmed that both plasmids contained the entire cDNA sequence of mouse HDC (data not shown). The HDC gene, which was ~24 kb long, contains 12 exons, as does the human HDC gene (16). The structure of the mouse HDC gene is shown schematically in Figure 1A and B. The distances between exons were determined from the size of the PCR product with primers each of which was designed on adjacent exons. The nucleotide sequences of the exon–intron boundaries were determined by comparing genomic and cDNA sequence information (31) and these completely satisfied the GT/AG rule (32).



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  		Figure 1. Structure of the mouse HDC gene. (A) Schematic representation of the mouse HDC gene and mRNA. Open boxes show the amino acid coding region of the HDC mRNA. Exons are shown by closed boxes with their exon numbers. The gene is shown on a 1:10 scale relative to the mRNA. (B) Restriction sites: E, EcoRI; B, BamHI; P, PstI.

 
HDC expression in cell lines
RNA blot hybridization was used to analyze HDC expression in a range of cell lines (Fig 2A). No HDC expression was observed in the mast cell progenitor cell line IC2 or the fibro­blast cell line NIH 3T3. A low level of HDC expression was detected in the undifferentiated mast cell line P815. In contrast, a high level of expression was observed in the differentiated mast cell line MC9 and the erythroleukemia cell line DS19. As reported in a previous paper (2), HDC expression in P815 cells increased markedly after i.p. induction, indicating that HDC expression is not only cell-type specific but also inducible during mast cell differentiation. Basically, this expression pattern did not change when we used RT–PCR instead of RNA blot analysis (Fig. 2B), although we found trace signals in NIH 3T3 cells. Interestingly, the level of HDC mRNA also increased in P815 cells after treatment with the demethylating agent 5-azaC, suggesting that DNA methylation negatively regulates HDC expression in P815 cells.



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  		Figure 2. Expression of mouse HDC mRNA. (A) RNA blot analysis. Total RNA was resolved on an agarose/formaldehyde gel, transferred to a nylon membrane and probed with [32P]dCTP-labeled specific cDNA. Each lane contains 30 µg total RNA isolated from the following mouse cell lines: the mast cell line P815 without treatment (P815), after i.p. incubation (Induced P815) and after 5-azaC treatment (P815 with 5-azaC); the mast cell line IC2; MC9; the erythroleukemia cell line DS19; the fibroblast cell line NIH 3T3. (B) RT–PCR. The procedures of sample preparation and amplification are described in Materials and Methods.

 
HDC gene transcription and histamine production after 5-azaC treatment
The average histamine content per cell increased ~5-fold after 5-azaC treatment of P815 cells. Only a trace level was detected in IC2 and NIH 3T3 cells without 5-azaC treatment. In NIH 3T3 cells it was increased after 5-azaC treatment (Fig. 3A). The HDC gene expression profiles coincided with histamine production, suggesting that the 5-azaC effect is not limited only to transcription but also affects the product of this enzyme (Fig. 3B). To test this hypothesis that DNA methylation negatively regulates HDC expression in P815 cells, we next assessed the methylation state of the promoter region of the HDC gene by two methods, DNA blotting after methylation-sensitive endonuclease digestion and bisulfite genomic sequencing.



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  		Figure 3. Treatment of cell lines with 5-azaC. (A) Effect of 5-azaC on the histamine content of the three cell lines. The amount of histamine was measured as described in Materials and Methods. The histamine contents are expressed as means ± SEM (n = 3). (B) HDC and ß-actin gene expression assessed by RT–PCR.

 
Methylation state of the HDC gene
We analyzed the methylation state of the HDC gene by DNA blot hybridization after digestion with PstI and HpaII (methylation-sensitive) or MspI (methylation-insensitive) restriction enzymes (Fig. 4). To examine the methylation state in the 5'-region of the gene, we used a probe corresponding to the region between two MspI sites, –383 and +215 bp from the transcription start site (Fig. 4A). A 600 bp fragment was observed in all lanes with PstI/MspI digestion. PstI/HpaII-digested samples produced larger fragments than PstI/MspI-digested products in all cases. The fragment size produced with PstI/HpaII digestion was assigned to reflect the methylation states of the CCGG sites in each cell type. PstI/HpaII-digested samples produced smaller fragments in MC9 and DS19 cells than those in control P815, IC2 or NIH 3T3 cells, suggesting that the degree of DNA methylation was intense in P815, IC2 and NIH 3T3 cells (Fig. 4B). To further verify the correlation between HDC gene transcription and DNA methylation, we examined the DNA methylation state in P815 cells under different conditions using control cells, those incubated in the peritoneal cavity and those treated with 5-azaC (Fig. 4C). Although HDC expression increased greatly after peritoneal incubation (Fig. 2), we could not detect a shift to smaller fragments but, in contrast, we could detect smaller fragments after 5-azaC treatment (Fig. 4C). DNA blotting after methylation-sensitive restriction enzyme digestion can detect only gross changes in methylation state. Therefore, we used sodium bisulfite genomic sequencing to analyze the methylation state of each cytosine residue in the promoter (Fig. 5). The majority of these CpG sites were methyl­ated in IC2 and NIH 3T3 cells, which expressed no HDC, and in untreated P815 cells, which expressed only a low level of HDC. In contrast, CpG sites in the GC box were demethylated in HDC-expressing cell lines. In P815 cells after 5-azaC treatment, many CpG sites were partially demethylated by 5-azaC treatment. Thus, the number of demethylated CpG sites seemed generally to correlate with the level of HDC expression. It should be noted that one CpG site next to the GC box was found to be demethylated in P815 cells after i.p. incubation, although it was not detectable by Southern blot analysis. These observations indicate that the promoter region is demethylated in differentiated mast cells.



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  		Figure 4. Methylation state of the mouse HDC gene assessed with DNA blotting. (A) Restriction map around the transcription start site. The bent arrow indicates the transcription start site and vertical arrows indicate MspI recognition sequences (CCGG). Horizontal bars with numbers beside them indicate the expected hybridized fragments. (B and C) Aliquots of 15 µg of genomic DNA were digested with PstI (P), PstI and HpaII (P+H) or PstI and MspI (P+M), resolved by electrophoresis, transferred to nylon membranes and hybridized with [32P]dCTP-labeled probe (A). The names of the cell lines are indicated at the top of the lanes.

 


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  		Figure 5. Summary of methylation state using sodium bisulfite genomic sequencing. (A) Schematic map around the transcription start site is shown at the top. The bent arrow indicates the transcription start site and boxes represent the GC box and exon 1. Short vertical lines across the horizontal bar indicate CpG sites. MspI/HpaII recognition sequences (CCGG) are marked with asterisks. Filled, gray and open circles represent methylated, semi-methylated and unmethylated cytosine residues, respectively. (B) Typical sequencing data of the antisense strand of the CpG site on the 5'-side of the GC box are shown. The number with each trace corresponds to the CpG site indicated in (A). Only unmethylated cytosine (2) was converted to uracil and read as adenine (A).

 
Stable transfectants of HDC–reporter gene constructs
To further assess the effect of the methylated promoter on transcriptional activity of the HDC gene, we performed stable transfection assays using patch-methylated pGLm-1099. For this patch-methylation method, we first digested out the promoter fragment from pGLm-1099, methylated it in vitro and religated it into the reporter vector backbone. This patch-methylated construct was transfected into P815 cells with a selector plasmid to obtain stable transfectants. After picking up the stable transfectants, we assessed the methylation state by sodium bisulfite genomic sequencing and analyzed their luciferase activity (Fig. 6A). It seemed that some CpG sites drastically changed the methylation state after transfection (P2M–). Some lines (P2M+, P1M– and C1–5) showed high level luciferase activity. In these clones, the cytosine of the CpG site in the GC box (–57 bp) was unmethylated, suggesting again that the methylation state of the GC box is important in the regulation of expression of mouse HDC.



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  		Figure 6. The effect of demethylation of the promoter region on transcriptional activity. (A) P815 cells were stably transfected with patch-methylated pGLm-1099. The methylation state and transcriptional activities were assessed in those cells. PM+ and PM– represent cell lines stably transfected with patch-methylated and mock-methylated plasmid, respectively. C1–5 represents the mean data from control experiments using five different cell lines transfected with simply linearized plasmid. Filled, gray and open circles represent the methylation state as in Figure 5. The copy numbers of the transfected plasmids in the cells were basically similar in the PCR semi-quantitative analysis (data not shown). (B) The stably transfected P815 cell lines P1M+ and P2M– were cultured with 5-azaC. The relative luciferase activity [5-azaC (+)] was compared with that of the cell line without 5-azaC treatment [5-azaC (–)]. Results are expressed as means ± SEM (n = 3). (C) Analysis of the methylation state of the P1M+ line by sodium bisulfite reaction before and after 5-azaC treatment.

 
Forced demethylation experiment in stable transfectants
To further clarify the functional relationship between methyl­ation state and the transcriptional activity, we measured luciferase activity in the P1M+ and P2M– lines after 5-azaC treatment. Because the increase in luciferase activity was much higher in the P1M+ line than in the P2M– line (Fig. 6B), we assessed the demethylation state in the P1M+ line. The promoter activity increased by 5-fold after 5-azaC treatment (Fig. 6B) with a simultaneous demethylation of the promoter region (Fig. 6C). These data indicate that demethylation of the promoter region is functionally important for mouse HDC gene expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We cloned the mouse HDC gene to analyze transcriptional regulation along with the differentiation process of mast cells. Cloning also made it possible to monitor the methylation state of the promoter region of the HDC gene and to prepare and assay patch-methylated stable transfectants in P815 cells. By using these constructs we could show that transcription of the mouse HDC gene is functionally regulated by DNA methyl­ation. HDC expression is mast cell-specific and increases with cell differentiation. Our results suggest that DNA methylation plays an important role during such lineage- and stage-specific HDC gene expression. DNA demethylation has been shown to correlate with differentiation of a particular tissue or cell type (33,34). In a previous report about the human HDC gene, we also observed methylation events in the promoter region, but functional assays were insufficient (17). Therefore, in the present study we used 5-azaC to force demethylation of cytosines. 5-azaC has been shown to inhibit DNA methyltransferase activity by forming a covalent complex with this enzyme (35). The treatment resulted in increased levels of HDC mRNA expression, suggesting that demethylation is functionally important to activate transcription of the HDC gene in these cells.

The promoter region was found to be widely demethylated in HDC-expressing mast cell lines. In particular, the methylation states around the GC box appeared to differ among cell lines expressing different levels of HDC. Since the mouse promoter region is newly demethylated after peritoneal induction, demethylation is assumed to be involved not only in cell-type specific HDC expression but also in induction of HDC expression. Demethylation at a single specific CpG site just upstream of the GC box could first be clarified using sodium bisulfite genomic sequencing, because this method has a higher resolution than the authentic DNA blotting.

Our previous data revealed that patch-methylation of the human HDC gene promoter region reduced reporter activity in transient transfection assays using HeLa cells (17). In this study, we have studied the effects of patch-methylation on the mouse HDC promoter in a stable transfection system of P815 cells. The key advantages of this system are as follows. First, it is important to examine the expression of methylated plasmids using stable rather than transient transfection, because stably transfected DNA has a more chromatin-like configuration than transiently transfected DNA (36). Second, we used the mast cell line P815 instead of other cell lines. By establishing stable transfection the low efficiency of transient transfection could be circumvented. Third, we employed plasmids that harbor localized methylation generated in vitro. The effect of in vitro DNA methylation on gene expression has been analyzed in stable transfection experiments (37–39) and it was reported that in vitro methylation of a stably transfected plasmid containing a promoter–reporter gene cassette greatly inhibits gene expression. Since the plasmid is methylated indiscriminately in vitro, not only the promoter but also the reporter gene is affected. To avoid this complication, we prepared patch-methylated pGLm-1099 in which only the promoter region was methylated and this plasmid was stably transfected into P815 cells. Using such a system, we observed that the degree of methylation of the promoter region correlated well with silencing in stable transfection experiments. The stable transfectant P1M+ with the methylated promoter expressed low reporter activity. However, expression of the reporter gene increased after 5-azaC treatment. Sodium bisulfite genomic sequencing revealed that the HDC promoter region became demethylated when the transfectants were treated with 5-azaC. Therefore, these observations suggest that demethylation of the promoter region is important for expression of the mouse HDC reporter gene in chromatin.

The mouse HDC gene was found to be similar in structure and length to its human homolog. This conservation across species indicates the importance of this gene. Although there are many structural similarities between the human and mouse HDC genes, the mouse gene does not undergo alternative splicing, whereas two splicing variants of 2.4 and 3.4 kb are transcribed from the human HDC gene (16). The longer human HDC mRNA (3.4 kb) is generated by use of an alternative splice acceptor site in exon 12, which splices out one-third of the exon together with intron 11 and preserves the entire intron 7. In contrast, the mouse HDC gene produces only a single transcript of 2.4 kb (31). The exon–intron boundaries of the mouse HDC gene are generally similar to those of the human HDC gene and the nucleotide sequences of the exons of the mouse gene show 83.7–91.9% homology to their human counter­parts, although exons 1 and 12 are not so strongly conserved (63.8 and 74.9% homology, respectively). A consensus branch site and a branch site-like sequence were found in mouse intron 7 within 100 bp upstream of the acceptor site of exon 8, but there is only a single branch site-like sequence in human intron 7. The presence of a consensus branch site might explain why mouse intron 7 is always removed by splicing. The nucleotide sequence of the alternative splice acceptor site in human exon 12 is different from that of the mouse gene (human, CAG/CC; mouse, GAG/CC). This change (C->G) is observed in a small minority of cases as an exon–intron boundary (40). Moreover, one conserved branch site sequence and two branch site-like sequences can be found within 100 bp upstream of the second splice acceptor site in the last exon of the human gene, whereas no such sequences were found in the mouse gene. These sequence differences may be involved in the observed differences in splicing patterns between the two species.

In the present report, we have provided experimental results showing the structure of the mouse HDC gene, the process of DNA methylation in the context of cell-type specific and inducible gene expression and the effect of demethylation around the promoter region on transcriptional activity. Our observations suggest that demethylation of the promoter region is necessary for mouse HDC gene expression, and may lead to more general conclusions concerning the molecular control of transcription mechanisms in mast cells.


   ACKNOWLEDGEMENTS
 
We thank A. Ichikawa and S. Tanaka for providing the mouse mast cell line P815 and various suggestions about the experiment. We also thank members of the Department of Biochemistry and Cellular Pharmacology, Tohoku University School of Medicine for their help. This work was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan and a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 22 717 8058; Fax: +81 22 717 8058; Email: ohtsu@mail.cc.tohoku.ac.jp Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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