INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

In vertebrates, including the mammals, the chromosomal DNAs are modified by C methylation at a limited number of CpG dinucleotides, resulting in m⁵CpG, methylated at position 5 of the C residues. This methylation at CpG has been implicated in the regulation of a number of genetic activities during mammalian cell differentiation and embryo development. These activities include tissue-specific gene transcription, X chromosome inactivation, genomic imprinting, cellular defense against viral agents, and tumorigenesis (references 1, 2, 11, 13, 15, and 34 and references therein). The level of m⁵CpG in the vertebrate cells is presumably balanced by the combined actions of DNA cytosine (C-5) methyltransferases (CpG MTases) (2, 26) and DNA demethylases (3).

It is generally accepted that regulation of the different cellular activities by DNA methylation is modulated mainly through the m⁵CpG residues. Indeed, m⁵CpG could be recognized by a specific class of proteins that consist of the methylated-DNA binding domain (MBD) (21). These "MBD proteins," in particular MeCP2 and MBD2/3, could preferentially bind to m⁵CpG-containing DNA, recruit histone deacetylase (HDAC)-containing complexes, and thus cause chromatin condensation in the vicinity of m⁵CpG (12, 22, 23, 33). This is very likely one major, but not the only, scheme involved in the functioning of vertebrate DNA methylation.

Unlike the vertebrates, several invertebrate species, including Drosophila melanogaster (27, 31), do not have apparent DNA methylation in their genomes. Nor has CpG MTase been reported to occur in these invertebrate animals. It was thus surprising to find mammalian CpG MTase-related proteins as well as MBD proteins expressed in Drosophila. A Drosophila protein, DmMTR1, is characteristically similar to the vertebrate maintenance CpG MTase, dnmt1, in several aspects (9). In particular, DmMTR1 molecules are located outside the nuclei during interphase of the syncytial Drosophila embryos, as is dnmt1 in the mouse blastocyst (19). However, DmMTR1 molecules appear to be rapidly transported into and then out of the nuclei again, as the embryos undergo the mitotic waves (9). Immunofluorescence data further indicate that DmMTR1 molecules "paint" the whole set of condensed Drosophila chromosomes throughout the mitotic phase, suggesting that it may play an essential role in the cell cycle-regulated condensation of the Drosophila chromosomes. In addition to DmMTR1, another Drosophila polypeptide, DmMT2, exhibiting high sequence homology to mammalian dnmt2, a relatively short homolog of dnmt1 but without detectable methylation activities (25), has been identified through a database search (9, 30). Expression of DmMT2 in Drosophila is developmentally regulated (9).

Interestingly, several recent reports have also noted the identification of an MBD protein encoded by the Drosophila genome (9, 30, 33). Like the mammalian MBD (12, 22, 23, 33), the Drosophila MBD protein, which was termed the "Drosophila MBD-like sequence" (33) or dMBD2/3 (30), interacts with HDAC in vitro and in vivo (30). However, it did not appear to recognize m⁵CpG (30). Here we report the complete genomic structure of the Drosophila dMBD2/3 gene, which encodes two alternatively spliced and developmentally regulated MBD proteins. We also provide the first evidence that the Drosophila MBD protein(s) could recognize m⁵CpG-containing DNA, albeit with a lower affinity than the mammalian MBD proteins. Remarkably, the protein(s) could also function as a corepressor via an HDAC-independent pathway.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Recombinant clones. The three P1 clones containing the dMBD2/3 gene were identified by the hybridization of filter-containing Drosophila P1 genomic clones (Genome Systems Inc.) with an expression sequence tag (EST) clone, LD03777. The nucleotide sequences of the P1 clones, LD03777, and another EST clone, LD46808, were determined by automated sequencing of both strands.

Northern blot analysis. Total Drosophila RNAs were isolated from different stages using the TRIZOL reagent (Life Technologies). Poly(A) RNAs were then isolated with a kit from Qiagen. One microgram of the RNAs was loaded in each lane, blotted onto nitrocellulose filter paper, and hybridized with ³²P-labeled LD03777 probe. 1 tubulin RNA patterns were used as the internal control for the Northern blot analysis.

EMSA. The sequence of the -TATA oligonucleotide used in the electrophoretic mobility shift assay (EMSA) is 5'-GATCTCACTGTGTCGACCTTGGGCATAAAAGGCAGAGCACTGGAGCTGCTGCTTAA-3' (complement, 3'-CTAGAGTGACACAGCTGGAACCCGTATTTTCCGTCTCGTGACCTC GACGACGAATT-5'). To obtain methylated DNA, the oligonucleotide or pBluescript II KS (pBSKS; Stratagene) was fully methylated with SssI methylase according to the instructions of the manufacturer (New England Biolabs). The oligonucleotides were labeled with ³²P at their 5' ends and gel purified prior to EMSA. The GST fusion proteins were all generated from recombinant pGEX-4T plasmids (Amersham Pharmacia Biotech) transformed into Escherichia coli BL21 cells. The proteins were purified with glutathione-Sephadex beads (Amersham Pharmacia Biotech). The expression plasmid pGST-ankyrin was a gift from Xin Chen. For EMSA, different amounts of the fusion proteins were preincubated with 100 ng of pBSKS and 8 µg of bovine serum albumin at room temperature for 5 min in 20 mM HEPES (pH 7.9)-3 mM MgCl₂-1 mM EDTA-10 mM -mercaptoethanol-0.1% Triton X-100-10% glycerol in a total volume of 20 µl. The labeled oligonucleotides (40,000 cpm, approximately 1 ng) were then added, and the samples were further incubated for 20 min at room temperature. To separate the protein-DNA complexes, the reaction mixtures were loaded onto a running nondenaturing 4% polyacrylamide gel, which had been prerun in 0.5× Tris-borate-EDTA buffer for 30 min at 4°C and 200 V. Electrophoresis was further carried out at 4°C and 200 V for 2 1/2 h. Gels were dried and exposed on X-ray film at 80°C. For competition experiments, a 100-fold molar excess of the competitor DNAs was included in the binding reactions.

Transient DNA transfection. The human kidney epithelial cell line 293T was grown in Dulbecco modified Eagle medium-10% fetal calf serum and transfected at 20% confluence in 2-cm "Cell Wells." The DNA samples for transfection were introduced as a calcium phosphate coprecipitate consisting of 1 µg of a reporter plasmid (pG0-TK-CAT, pG5-TK-CAT, pG0--Luc, pG5--Luc, p21, or p21-GCmut), 0.3 µg of pCMV--gal (18), 10 µg of salmon sperm DNA as the carrier, and different amounts of an expression plasmid [pSG424-dMBD2/3, pSG424-MBD2b, pCI-Gal4, pCI-Gal4-dMBD2/3, pCI-Gal4-dMBD2/3 (36-226), pCI-Gal4-MBD2b, or pSG424-p45]. pSG424 and/or pCI-Gal4 was used as the control plasmid(s), and either was also added as required to keep the total amount of DNA used in each transfection the same. Sixteen hours after transfection, the medium was changed and cells were incubated for another 48 h. The chloramphenicol acetyltransferase (CAT), luciferase, and -galactosidase assays were performed as described previously.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Genomic organization of dMBD2/3, a Drosophila gene encoding two MBD-containing polypeptides. Multiple EST clones were identified by Blast search using the mammalian MBD2b sequence (8) as the bait. One of the clones (LD03777) originating from a Drosophila embryonic cDNA library was first sequenced. Its insert is 865 bp long, including the poly(A) tail. LD03777 bears an open reading frame corresponding to 226 aa, which has been referred to previously as the "Drosophila MBD-like sequence" (33) or dMBD2/3 (30). The protein contains an MBD-like sequence in the N-terminal domain and a coiled-coil motif near the C terminus. It is apparently the ortholog of the vertebrate MBD2/MBD3 family (8, 30, 33).

View larger version (96K): [in this window] [in a new window]   FIG. 1.   Structure and expression of the Drosophila dMBD2/3 gene. (A) Genomic and cDNA organization of dMBD2/3. The chromosomal location (85D) of the gene and the three P1 clones spanning the region are indicated. Shown below the P1 clones are the cDNAs for dMBD2/3 and dMBD2/3, which were derived from the cDNA clones LD46808 and LD03777, respectively. The base numbering of the two cDNAs corresponds to that of the genomic sequence of dMBD2/3 shown in panel B. (B) Genomic sequence of dMBD2/3. The sequence of the dMBD2/3 gene was deduced from the three P1 clones. The putative site of transcriptional initiation, as inferred from the sequences of the two cDNA clones, is designated +1. The two introns and the regions upstream and downstream of the gene are shown with lowercase letters. The corresponding amino acids are also shown, with those homologous to the conserved amino acids among the vertebrate MBDs indicated by bold letters. The putative TATA box in the upstream promoter region and the poly(A) signal, AATAAA, are underlined. (C) Expression of dMBD2/3 and dMBD2/3 mRNA. Drosophila poly(A) RNAs isolated from different stages of development were analyzed by Northern blot hybridization with the cDNA clone LD03777 as the probe. The two transcripts (1,270 and 890 nt long) are indicated.

Developmental regulation of the alternative splicing of dMBD2/3. The two dMBD2/3 mRNAs are differentially expressed during Drosophila development, as shown by Northern blot analysis using LD03777 as the hybridization probe (Fig. 1C). In overnight embryos and in adult flies, two mRNAs of lengths similar to those of dMBD2/3 (890 nucleotides [nt]) and dMBD2/3 (1,270 nt), respectively, were present (Fig. 1C, lanes 2 and 6). On the other hand, only the long transcript was expressed in embryos younger than 2 h (Fig. 1C, lane 1), and the short mRNA was the predominant species at the other stages of development (Fig. 1C, lanes 3 through 5). The Northern blotting data suggest that the alternative splicing scheme generating dMBD2/3 and dMBD2/3 mRNAs is developmentally regulated and that the two proteins play different roles during development. dMBD2/3 is likely an essential gene for the early development of Drosophila, since a line with P-element insertion in the putative promoter region of dMBD2/3 is lethal to homozygous embryos (M.-S. Hung, unpublished data).

dMBD2/3 could preferentially recognize m⁵CpG-containing DNA. Whether the Drosophila MBD protein binds to m⁵CpG is an interesting question. All vertebrate MBD proteins identified thus far could recognize and preferentially bind to DNA containing m⁵CpG (reference 4 and reference therein). Close sequence examination indicates that the MBD in dMBD2/3 is similar to those in vertebrates but with many amino acid differences, including relatively long deletions (Fig. 1B) (30, 33). Recombinant GST fusions of different MBD proteins (Fig. 2A) were thus compared for their abilities to bind methylated or unmethylated DNA (Fig. 2B). As expected from the previous studies (8), little binding of mouse MBD2b to unmethylated probe could be detected (Fig. 2B, lanes 1 through 3). On the other hand, it bound to the methylated DNA probe with relatively high affinity (Fig. 2B, compare lanes 4 through 6 to lanes 1 through 3).

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Preferential binding of Drosophila and mammalian MBD proteins to m5CpG-containing DNA. (A) Coomassie blue staining patterns of recombinant GST fusion polypeptides. One microgram each of GST fusions with the intact dMBD2/3 (lane 2), dMBD2/3 (36-226) (lane 3), a mouse ankyrin polypeptide (lane 4), and mouse MBD2b (lane 6) was electrophoresed on a denaturing sodium dodecyl sulfate-polyacrylamide gel and stained with Coomassie blue. Also run on the gel were molecular mass markers (lane 1) and bovine serum albumin (lane 5). (B) EMSA of recombinant GST fusions. The abilities of the Drosophila dMBD2/3 and mouse MBD2b proteins to recognize m5CpG were compared by EMSA. A 56-bp DNA probe (-TATA) containing the human  globin TATA box with a single CpG was electrophoresed after incubation with competitor DNA and increasing amounts of proteins (2, 20, and 200 ng). Another probe, -TATAm, which contains the same sequence as -TATA but with the CpG site methylated at position 5 of the C residues, was analyzed in the same way. The oligonucleotides -TATA and -TATAm were used as the probes in an EMSA after binding with increasing amounts (2, 20, and 200 ng) of recombinant GST fusions of MBD2b or dMBD2/3. The arrow points to the complex formed between the -TATAm oligonucleotide and both MBD2b and dMBD2/3. The GST protein did not form a complex either with -TATA or with -TATAm (data not shown).

MBD is required for dMBD2/3 binding to methylated DNA. The specificity of the dMBD2/3-DNA complex was further confirmed by experiments shown in Fig. 3. First, as exemplified for a GST-ankyrin fusion polypeptide (Fig. 3A, lanes 1 and 2), proteins without MBD do not bind to the probe, methylated or unmethylated. Similarly, GST alone could not form a complex with the same set of DNA oligonucleotides (data not shown). Second, the complex was effectively competed away upon the addition of a 100-fold molar excess of methylated probe or methylated pBluescript DNA (Fig. 3A, compare lane 3 to lanes 4 and 6) but not upon addition of the same amount of unmethylated competitors (Fig. 3A, lanes 5 and 7). Finally, removal of aa 1 to 35 from dMBD2/3, which comprise a major portion of the MBD-like sequence, abolished its ability to bind methylated DNA, as demonstrated by EMSA (Fig. 3B, compare lanes 2 and 3) and by Southwestern analysis (data not shown). It should be noted here that Tweedie et al. (30) have found no methyl-CpG-binding activity for either dMBD2/3 or dMBD2/3 in their EMSA. However, in those cases, a different DNA oligonucleotide was used. It is likely that the recognition of m⁵CpG by these two Drosophila MBD proteins is dependent on the DNA sequence context.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   (A) Oligonucleotide competition EMSA of dMBD2/3. -TATA and -TATAm oligonucleotides were allowed to form complexes with 200 ng of the GST fusion of dMBD2/3, 20 ng of the GST fusion of MBD2b, or 200 ng of the GST fusion of a mouse ankyrin polypeptide. The binding reactions were carried out without () or with the inclusion of 100 ng of competitor DNA oligonucleotide -TATA, -TATAm, plasmid pBSKS, or pBSKS methylated at all of the CpG sites (pBSKSm). The reaction products were then analyzed by EMSA. (B) Requirement of the MBD of dMBD2/3 for complex formation. The oligonucleotides were allowed to form a complex with 200 ng of the GST fusion of intact dMBD2/3 or with 200 ng of the GST fusion of a truncated dMBD2/3.

dMBD2/3 and mouse MBD2b could function as transcription corepressors or repressors in human 293T cells. The mammalian MBD2a protein repressed promoter activity in a cotransfection assay (23). To examine whether the Drosophila MBD protein(s) could also act as a transcriptional corepressor, we first carried out side-by-side comparison of the effects of MBD2b, which is an isoform of mouse MBD2a, and of Drosophila dMBD2/3 on the expression in human 293T cells of a cotransfected TK promoter with five Gal4-binding sites (Fig. 4B). The Gal4 fusions of either protein significantly repressed the TK promoter in a dosage-dependent manner (Fig. 4B, see data for pSG424-MBD2b and pSG424-dMBD2/3) when being recruited to the promoter via the upstream Gal4-binding sites in pG5-TK-CAT. In contrast, expression of only the Gal4 (Fig. 4B, see data for pSG424) had no obvious effect and that of a fusion polypeptide between Gal4 and the activation domain of p45, a subunit of the erythroid enriched factor NF-E2, stimulated the TK promoter activity (Fig. 4B, see data for pSG424-p45).

View larger version (125K): [in this window] [in a new window]   FIG. 4.   Transcription repression by Drosophila and mammalian MBD proteins in human cells. (A) Maps of reporter and expression plasmids used in the cotransfection assays. For more details of the plasmids, see Materials and Methods. (B) Relative CAT activities from pG5-TK-CAT in human 293T cells cotransfected with increasing amounts of different recombinant expression plasmids. The CAT activities were standardized with the -galactosidase activity from cotransfected pCMV--gal. Activity with 3.5 µg of cotransfected pSG424 is defined as 1. The bars represent standard deviations of two sets of transfection carried out in duplicate. (C) Relative luciferase activities from pG5--Luc or pG0--Luc in human 293T cells cotransfected with increasing amounts of different recombinant expression plasmids. The activation from pG5--Luc or pG0--Luc was measured and standardized with the -galactosidase activity from cotransfected pCMV--gal. The luciferase activity from pG5--Luc without cotransfected expression plasmid is defined as 1 (lane 1). Note that in contrast with activity shown in panel B, the expression of the Gal fusions in this set of experiments was directed by a CMV promoter in the pCI vector.

TSA treatment could not inhibit the repressor or corepressor function of dMBD2/3. Whether the corepressor and repressor functions of dMBD2/3 or MBD2b are mediated through HDAC(s) has been tested with the use of TSA. In general, TSA treatment could increase the activities of many endogenous and transfected promoters (10, 32). We have observed the same phenomenon. As shown in Fig. 5A, the activity of p21, the promoter of the murine p21^waf1 gene, was greatly increased in a TSA concentration-dependent manner. This is consistent with the finding by Xiao et al. (35). Furthermore, a mutant promoter, p21-GCmut, with a mutation in the proximal GC box of p21, exhibited a much smaller magnitude of TSA inducibility (Fig. 5A). Similar to p21^waf1, the activities of the TK promoter in plasmid pG0-TK-CAT or pG5-TK-CAT (Fig. 5B) and the  globin promoter in pG0--Luc or pG5--Luc (Fig. 5C) were all increased after TSA treatment, 2- to 4-fold and 15- to 16-fold, respectively. However, it is obvious from Fig. 5B and C that TSA treatment could not relieve the repression by dMBD2/3, whether or not the reporter plasmids contained the Gal4-binding sites.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Effects of TSA treatment on the repression function of dMBD2/3. (A) Activity increases of the p21waf1 and p21-GCmut promoters after treatment with the indicated concentrations of TSA. Increases relative to the promoter activity in the absence of TSA treatment are indicated above the bars. (B) Activity increases of the TK promoter by TSA treatment. The blank bars represent the TK promoter activities in the presence of exogenously expressed Gal4 protein, and the black bars represent activities with the Gal4 fusion of dMBD2/3. (C) Activity increases of the human  globin promoter caused by TSA treatment.

dMBD2/3 also could effectively repress transcription in Drosophila cells. To test whether dMBD2/3 could function as a corepressor and/or repressor in a Drosophila cellular environment, we carried out DNA transfection in an SL2 cell culture. As in those experiments performed for Fig. 4B and C, pG5--Luc or pG0--Luc was cotransfected with pSG424 or pSG424-dMBD2/3. In addition, pPacSp1 was also cotransfected since high-level activities of both the  globin promoter in pG5--Luc or pG0--Luc and the simian virus 40 promoter in pSG424 require the binding of Sp1 factor, which Drosophila cells lack (6).

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Transcriptional repression by dMBD2/3 in Drosophila SL2 cells. The relative luciferase activities from pG5--Luc or pG0--Luc in SL2 cells cotransfected with pSG424 or pSG424-dMBD2/3 were calculated. Activity with 1 µg of cotransfected pSG424 is defined as 1. The  globin promoter and simian virus 40 promoter in pG0--Luc and pG5--Luc and in pSG424, respectively, were driven by Sp1 factor expressed by cotransfected pPacSp1. Indeed, the luciferase activities were at least 10 times lower without cotransfection with pPacSp1 (data not shown).

Conservation of Drosophila proteins related to factors involved in the vertebrate DNA methylation program. As already mentioned, the end product of the DNA methylation reaction, m⁵CpG, has been recognized as the key determinant responsible for the functioning of CpG MTases and DNA demethylases in vertebrate cells (reference 13 and reference therein). Among the possible mechanisms, m⁵CpG serves as the binding site for MBD proteins, many of which appear to reside within chromatin remodeling complexes that contain HDACs (12, 22, 23, 30). The identification of Drosophila proteins (DmMTR1 [9] and DmMT2 [9, 30]) related to two of the vertebrate CpG MTase family members and of the Drosophila MBD proteins (9, 30, 33) has thus raised interesting questions regarding the mechanisms of functioning of these proteins in vertebrates as well as in Drosophila (9, 30). Two scenarios could be envisioned to explain the evolutionary conservation of the vertebrate CpG MTase-related proteins in Drosophila. First, DNA methylation-demethylation processes actually occur in Drosophila but only transiently at specific stages of the development or cell cycle, thereby escaping previous methods of detection (27, 31). Alternatively, these proteins and their mammalian counterparts carry out currently unknown functions not requiring the enzymatic reactions. Our data on dMBD2/3 have interesting implications for the above models.