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• W2071476046 abstract "The maize opaque-2 locus (o2) has an endosperm-specific expression and is positively autoregulated by its gene product, a b-Zip protein, to a TGACGTTG motif. The genomic sequencing method was used here to describe, in leaf and endosperm, the methylation pattern of a 390-base pair region of the o2 promoter. In leaf, 96% of the C residues are methylated, whereas in endosperm the 5-methylcytosine content is 84%. Comparison of these methylation patterns indicates that the o2 tissue-specific expression does not result from the demethylation of any specific C residue and that, in vivo, O2 interacts with a methylated target sequence. Consistently, gel-shift experiments using a CpG-methylated, partially methylated, and hemimethylated o2 promoter fragments showed that, in vitro, the O2 protein binds to the major groove of a methylated target sequence, although its binding activity decreases at increasing levels of C-methylation and is more effectively reduced by methylation of the lower strand than of the upper strand of the DNA. Using partially purified endosperm cell extracts, we also show that, besides the O2 protein, other proteins specifically bind to a partially methylated o2 promoter fragment, therefore indicating that in plants a subset of different proteins may mediate the expression of a naturally occurring methylated o2 promoter. The maize opaque-2 locus (o2) has an endosperm-specific expression and is positively autoregulated by its gene product, a b-Zip protein, to a TGACGTTG motif. The genomic sequencing method was used here to describe, in leaf and endosperm, the methylation pattern of a 390-base pair region of the o2 promoter. In leaf, 96% of the C residues are methylated, whereas in endosperm the 5-methylcytosine content is 84%. Comparison of these methylation patterns indicates that the o2 tissue-specific expression does not result from the demethylation of any specific C residue and that, in vivo, O2 interacts with a methylated target sequence. Consistently, gel-shift experiments using a CpG-methylated, partially methylated, and hemimethylated o2 promoter fragments showed that, in vitro, the O2 protein binds to the major groove of a methylated target sequence, although its binding activity decreases at increasing levels of C-methylation and is more effectively reduced by methylation of the lower strand than of the upper strand of the DNA. Using partially purified endosperm cell extracts, we also show that, besides the O2 protein, other proteins specifically bind to a partially methylated o2 promoter fragment, therefore indicating that in plants a subset of different proteins may mediate the expression of a naturally occurring methylated o2 promoter. DNA methylation has been implicated in mediating several fundamental cellular processes in both prokaryotes and eukaryotes. In higher plants, 5-methylcytosine (m5C) 1The abbreviations used are: m5C, 5-methylcytosine; CREB, cAMP response element; PCR, polymerase chain reaction; bp, base pair(s); EMSA, electrophoretic mobility shift assay; wt, wild-type; mt, mutagenized. can constitute as much as one-third of the cytosine residues of nuclear DNA and has been found to control transposable element activity (1Fedoroff N.V. Chandler V. Paszkowski J. Homologous Recombination and Gene Silencing in Plants. Kluwer Academic Publishers, The Netherlands1994: 349-385Google Scholar), genomic imprinting (2Matzke M.A. Matzke A.J.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 53-76Google Scholar), gene silencing (3Matzke M.A. Matzke A.J.M. Plant Physiol. 1995; 107: 679-685Google Scholar, 4Flavell R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3490-3496Google Scholar), inhibition of transcription (3Matzke M.A. Matzke A.J.M. Plant Physiol. 1995; 107: 679-685Google Scholar), and inheritance of a variety of epigenetic phenomena (5Martienssen R.A. Richards E.J. Curr. Opin. Genet. & Dev. 1995; 5: 234-242Google Scholar). More recently, Ronemuset al. (6Ronemus M.J. Galbiati M. Ticknor C. Chen J. Dellaporta S.L. Science. 1996; 273: 654-657Google Scholar) showed that DNA methylation is an essential component in establishing or maintaining cellular processes resulting in phase transition and meristem determinacy in transgenic Arabidopsis plants expressing an antisense cytosine methyltransferase gene. In animals, DNA methylation appears to influence the tissue-specific expression of developmentally regulated genes by affecting the interactions of DNA with chromatin proteins and transcription factors (4Flavell R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3490-3496Google Scholar). In plants, methylation of the C residue embedded within the consensus sequence for mammalian CREB factors (cAMP response element, TGACGTCA) was found to inhibit in vitro DNA binding activity of CREB-like factors present in nuclear extracts of pea, wheat, soybean, and cauliflower (7Inamdar N.M. Ehrich K.C. Ehrlich M. Plant Mol. Biol. 1991; 17: 111-123Google Scholar). However, binding of plant nuclear proteins to hemimethylated target sequences has been reported for the tobacco CG-1 nuclear factor(s) (8Staiger D. Kaulen H. Schell J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6930-6934Google Scholar) and the maize ac-encoded transposase (9Kunze R. Starlinger P. EMBO J. 1989; 8: 3177-3185Google Scholar). Interestingly, binding of the actransposase occurs only to hemimethylated sequences with a methylated lower strand and not vice versa. A method based upon chemical treatment of genomic DNA (10Frommer M. McDonald L.E. Millar D.S. Collis C.M. Watt F. Grigg G.W. Molloy P.L. Paul C.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1827-1831Google Scholar) has allowed detailed analysis of the methylation state of several genes in mammals, fungi, and plants and has led to the discovery of m5C outside the canonical symmetrical CpG and CpNpG sites (11Selker E.U. Fritz D.E. Singer M.J. Science. 1993; 262: 1724-1728Google Scholar, 12Clark S.J. Harrison J. Paul C.L. Frommer M. Nucleic Acids Res. 1994; 22: 2990-2997Google Scholar, 13Meyer P. Niedenhof I. ten Lohuis M. EMBO J. 1994; 13: 2084-2088Google Scholar). In transgenic plants, the application of this method clearly revealed the association between a high level of m5C observed within a silenced CaMV35S promoter and the transcriptional inactivation of the transgene (13Meyer P. Niedenhof I. ten Lohuis M. EMBO J. 1994; 13: 2084-2088Google Scholar, 14Park Y.-D. Papp I. Moscone E.A. Iglesias V.A. Vaucheret H. Matzke A.J.M. Matzke M.A. Plant J. 1996; 9: 183-194Google Scholar). In maize, analysis of the methylation pattern ofac transposable element ends suggested a model to explain the association of element transposition with replication (15Wang L. Heinlein M. Kunze R. Plant Cell. 1996; 8: 747-758Google Scholar). However, at present little is known about the methylation state of endogenous plant promoters, thus correlations between their methylation pattern and gene activity can not be clearly asserted. Although a positive correlation between the expression level of the maize Scutellar node (sn) gene and the methylation content of its promoter has been observed (16Ronchi A. Petroni K. Tonelli C. EMBO J. 1995; 14: 5318-5328Google Scholar), no information is available about whether the methylation pattern of a plant promoter may mediate its tissue-specific expression. Similarly, the exact degree of C-methylation of an endogenous plant promoter has never been estimated in a statistically significant manner. To address these questions we have detailed the methylation pattern of the maizeopaque-2 (o2) locus in an expressing and non-expressing tissue, endosperm and leaf, respectively. In maize, o2 regulates the expression of b-32,cyppdk1, and the 22- and 14-kDa zein gene families (17Motto, M., Thompson, R. D., Salamini, F., Cellular and Molecular Biology of Plant Seed Development, Larkins, B. A., Vasil, I. A., 1997, Kluwer Academic Publishers, Dordrecht, The Netherlands.Google Scholar). Theo2 locus has been cloned and found to be expressed only in developing endosperm (18Schmidt R.J. Burr F.A. Burr B. Science. 1987; 238: 960-963Google Scholar, 19Motto M. Maddaloni M. Ponziani G. Brembilla M. Marotta R. Di Fonzo N. Soave C. Thompson R.D. Salamini F. Mol. Gen. Genet. 1988; 212: 488-494Google Scholar). o2 encodes for a member of the basic domain/leucine zipper (b-Zip) class of transcriptional activators (20Hartings H. Maddaloni M. Lazzaroni N. Di Fonzo N. Motto M. Salamini F. Thompson R.D. EMBO J. 1989; 8: 2795-2801Google Scholar, 21Schmidt R.J. Burr F.A. Aukerman M.J. Burr B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 46-50Google Scholar). Similarly to other plant b-Zip factors (22Ramachandran S. Hiratsuka K. Chua N.-H. Curr. Opin. Genet. & Dev. 1994; 4: 642-646Google Scholar), the O2 protein target sequence, TGACGTTG, contains an ACGT core. Multiple copies of this sequence are present in the promoter region of the genes that are under o2 transcriptional control. Interestingly, strong in vitro binding of the O2 protein to the odd base palindromic C-box sequence, ATGAGTCAT, found in the pea lectin promoter has been reported (23de Pater S. Katagiri F. Kijne J. Chua N.-H. Plant J. 1994; 6: 133-140Google Scholar). The O2 protein can also transiently activate the expression of its own promoter, indicating that it is positively autoregulated (24Lohmer S. Maddaloni M. Motto M. Di Fonzo N. Hartings H. Salamini F. Thompson R.D. EMBO J. 1991; 10: 617-624Google Scholar). Furthermore, O2 binds in vitro to an o2 promoter fragment and protects from DNaseI treatment a single region encompassing its consensus binding sequence (24Lohmer S. Maddaloni M. Motto M. Di Fonzo N. Hartings H. Salamini F. Thompson R.D. EMBO J. 1991; 10: 617-624Google Scholar). In this study we used the genomic sequencing method to determine the methylation state of the o2 promoter in endosperm and leaf cells. Our data show a different methylation content of the o2 promoter in these tissues, less methylated in endosperm than in leaf. However, in endosperm the high m5C level observed, 84%, correlates with a low level of expression of the o2 gene in this tissue (25Hartings H. Lazzaroni N. Rossi V. Riboldi G.R. Thompson R.D. Salamini F. Motto M. Genet. Res. Camb. 1995; 65: 11-19Google Scholar). Comparison of the two methylation patterns also revealed that endosperm-specific expression of the o2 promoter is not mediated by the demethylation of a specific C residue. Because the methylation pattern of the o2 promoter in endosperm indicates the occurrence of m5C within the O2 protein binding site and its flanking sequences, gel-shift experiments were performed to investigate the DNA binding activity of O2 protein to DNA fragments whose methylation state mimics the o2 promoter methylation pattern. Results show that unlike the plant transcriptional activators analyzed so far, O2 binding activity is not impaired by CpG methylation. However, we have observed that the pattern and average level of C-methylation can modulate DNA binding affinity of the O2 protein to the tested substrates. Finally, we provide evidence showing that other proteins isolated from immature endosperm can specifically bind to a partially methylated o2 promoter, indicating that factors other than O2 may bind to methylated DNA. Taken together these data suggest the possibility that in plants DNA methylation may control plant gene expression level by modulating transcription factor's DNA binding affinity to their cognate sequences. The maize line used throughout this study is the same line used for cloning and sequencing the o2 gene (20Hartings H. Maddaloni M. Lazzaroni N. Di Fonzo N. Motto M. Salamini F. Thompson R.D. EMBO J. 1989; 8: 2795-2801Google Scholar). From a single selfed plant, young leaves and immature endosperm (20 days after pollination) were harvested, immediately frozen in liquid nitrogen, and stored at −80 °C. Maize nuclear genomic DNA was purified from 10 g of leaves or endosperm using a previously described method (26Pellegrini L. Rohfritsch O. Fritig B. Legrand M. Plant Physiol. 1994; 106: 877-886Google Scholar). DNA was cleaved with EcoRI and then subjected to bisulfite treatment as described by Frommer et al. (10Frommer M. McDonald L.E. Millar D.S. Collis C.M. Watt F. Grigg G.W. Molloy P.L. Paul C.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1827-1831Google Scholar) with the following modifications: 2 μg of DNA were denatured with 50 mmNaOH, neutralized by addition of 1 volume of 1 m Tris pH 7.5, and desalted on a Sephadex G-50 spin column. Eluted DNA was immediately denatured at 98 °C for 10 min and processed as described by Meyer et al. (13Meyer P. Niedenhof I. ten Lohuis M. EMBO J. 1994; 13: 2084-2088Google Scholar). At the end of the bisulfite treatment, DNA was recovered using a QIAEX II extraction kit (QIAGEN). In the control reaction, 20 pg of linearized plasmid (pLPO2) containing the −1020, +58 region of the o2 promoter was methylated in vitro with SssI CpG-methylase (New England Biolabs) and mixed with 2 μg of salmon sperm DNA. In 50 μl total volume, the first polymerase chain reaction (PCR) amplifications contained 100 ng of bisulfite-treated DNA, 25 pmol of each external primer, 200 μm of each dNTP, 1 × Promega PCR buffer (50 mm KCl, 10 mm Tris-HCl, pH 9, 0.1% Triton X-100), 5 mm MgCl2, and 2.5 units of Taq DNA polymerase (Promega). One μl of first PCR amplification was then used as template in a second round of PCR that differed for the sets of internal primers and magnesium concentration (2.5 mm MgCl2). For the top strand a 30-mer LP18-A2E (5′-gttgagatttttgtttatgttttttttttg-3′) and a 33-mer LP19-A1E (5′-cccaataataaataactatcaatcaataattcc-3′) were used as external primers, and a 33-mer LP3-A2 (5′-cgggatccgtttaatttggatgtagattatttg-3′) and a 37-mer LP4-A1 (5′-gcggtaccatcaaccttacactcaatataaacctaca-3′) were used as internal primers. For the bottom strand a 34-mer LP9-B1E (5′-aacatataaactaaaatttctatttatactttcC-3′) and a 28-mer LP10-B2E (5′-tgagttaattatgttagttgtttgaagg-3′) were used as external primers and a 35-mer LP5B1 (5′-cgggtacctaatttcaaacaacaaattacttatac-3′) and a 34-mer LP6B2 (5′-cgggatccgtatttaatgtgaatttgtagggtag-3′) were used as internal primers. PCR reactions consisted of an initial denaturation step of 2 min at 94 °C followed by 30 cycles of amplification (1 min at 94 °C, 2 min at 46 °C, and 1 min at 72 °C). The annealing temperature of the second PCR amplification was 50 °C. Amplified fragments were digested with KpnI and BamHI, gel-purified, and cloned into pBluescript KS+ (Stratagene). Plasmid DNA was purified, and individual clones were sequenced using an ABI373A DNA sequencer (Applied Biosystem). Data were analyzed by standard analysis of variance and Student's t significant difference was used to compare means. All statistic analyses were performed using the “Statistica Software Package” (Stat Soft Inc., 1995). O2 protein was overexpressed in E. coli strain BL21/lDE3 harboring pLys and pET3cO2 (27Izawa T. Foster R. Chua N.-H. J. Mol. Biol. 1993; 230: 1131-1144Google Scholar), which contains the O2 cDNA fused to the T7 promoter. An overnight culture was grown at 37 °C in LB media containing chloramphenicol (25 μg/ml) and ampicillin (100 μg/ml). Cells were diluted into 50 ml of fresh medium and grown till theA600 = 0.5, and O2 expression was then induced by addition of isopropyl-1-thio-β-d-galactopyranoside to 0.4 mm. After 4 h, the cells were harvested by centrifugation, washed in water, and resuspended in 0.1 volume of 1 × binding buffer (20 mm Hepes, pH 7.5, 40 mm KCl, 1 mm EDTA, 88 μg/ml phenylmethylsulfonyl fluoride, 10% glycerol). The cell suspension was aliquoted into Eppendorf tubes, frozen in liquid nitrogen, and stored at −80 °C. For experiments, cells were thawed and centrifuged, and then the supernatant was recovered and filtered with disposable 0.45-μm filters. Filtered protein extracts were concentrated with Ultrafree-15 (50,000 MW; Millipore), to obtain a final protein concentration, estimated by the Bradford assay, of approximately 1.5 μg/μl. For the band shift experiments, the control extract consisted of E. coli cells transformed with the pET3c plasmid lacking of the O2 cDNA insert. Immatureo2-m(r) kernels were harvested at 16 days after pollination and frozen in liquid nitrogen. Pericarps and embryos were eliminated, and 15 g of endosperm was ground in 10 ml of extraction buffer (30 mm Tris, pH 7.4, 88 μg/ml phenylmethylsulfonyl fluoride). The homogenate was centrifuged, and the supernatant filtered onto disposable 0.45-μm filters. Endosperm protein extract was applied onto a pre-equilibrated HiTrap SP column (Pharmacia Biotech Inc.), using a peristaltic pump, and washed with 5 column volumes of extraction buffer. Four fractions were eluted by applying 6 ml of extraction buffer containing increasing KCl concentrations (75, 150, 225, and 300 mm KCl). Eluted fractions were concentrated with Ultrafree-15 (50,000 MW; Millipore), to obtain a final protein concentration, estimated by the Bradford assay, of approximately 1 μg/μl. A 188-bp long o2 promoter fragment spanning from −399 to −211 relative to the transcription initiation site of the o2 locus was cloned in the EcoRV site of pBluescript KS(+) to make pLP190. The P188 DNA fragment was excised from pLP190 with HindIII andPstI, gel-purified, recovered by electroelution, and end-labeled. The P188-CG probe was prepared by methylating the P188 probe with SssI CpG DNA methylase (New England Biolabs) following the manufacturer's recommendation. Completeness of CpG-methylation was assessed by restriction digestion analysis of the P188-CG probe with the methylation-sensitive restriction enzymeHhaI. The P188–50% probe was prepared by PCR amplification of the P188 DNA fragment cloned in pLP190 using KS and SK primers. PCR reaction mix consisted of 10 ng of pLP190, 1 × Promega buffer, 2.5 mm MgCl2, 200 μm dATP, 200 μm dGTP, 200 μm dTTP, 100 μmdCTP, 100 μm m5-dCTP, 1 μm each oligonucleotide primer, and 0.5 units of Taq DNA polymerase (Promega). PCR products were digested with XbaI, gel-purified, and spectrophotometrically quantified. Thirty ng of DNA was labeled at one end by end-filling using Klenow DNA polymerase and [32P]dCTP; probes were purified from unincorporated nucleotides with Sephadex-G50 spin columns. The P188–100%, P188–30%, and P188–70% probes were produced the same as for P188–50% except that the molar ratios of m5-dCTP:dCTP were 1:0, 3:7, and 7:3 respectively, in the PCR reaction. Each hemimethylated fragment was prepared by combining methylated and unmethylated single-stranded complementary DNA. Annealing was achieved by heating the DNA mix at 95 °C for 10 min, followed by slow cooling to room temperature. Annealed DNA was gel-purified and labeled as indicated above. Single-stranded DNA was prepared as follows. For the upper strand, SstI-digested pLP190 was subjected to two rounds of 50 elongation steps using the KS primer, 1 × Promega buffer, 200 μm of each dNTP, and 1 unit of Taqpolymerase. For the lower strand, KpnI-digested pLP190 was used in combination with the SK primer. A methylated strand was obtained by including m5-dCTP in the reaction mix. p36wt and p36mt probes were made by annealing 1 μg of primers (p36wt: 5′-atacaatacatgttgacgttggtaaggca-3′; 5′-gacaccatgccttaccaacgtcaacatg-3′. p36mt: 5′-atacaatacatgttgatgttggtaaggca-3′; 5′-gacaccatgccttaccaacatcaacatg-3′), and end-filling of 3 ng of oligonucleotide duplexes with Klenow enzyme in the presence of [32P]dCTP. The radiolabeled probes were purified from unincorporated nucleotides with Sephadex-G50 spin columns. p61wt primers (5′-cagaaagtgcaatacaatacatgttgacgttgg-3′; 5′-ggctttgtgtgctagacaccatgccttaccaacgtcaacatg-3′), and p61mt primers (5′-cagaaagtgcaatacaatacatgttgatgttgg-3′; 5′-ggctttgtgtgctagacaccatgccttaccaacatcaacatg-3′) were labeled at their 5′ ends with T4 kinase. P61wt and P61mt probes were made by annealing equimolar amounts (1 pmol) of labeled primers and then extended with Klenow enzyme in the presence of cold dNTPs. Radiolabeled probes were purified with Sephadex-G50 spin columns. All oligonucleotides used throughout this study were designed from the genomic sequence of the o2 gene (20Hartings H. Maddaloni M. Lazzaroni N. Di Fonzo N. Motto M. Salamini F. Thompson R.D. EMBO J. 1989; 8: 2795-2801Google Scholar) and synthesized by Pharmacia Biotech Inc. Their integrity was verified by gel electrophoresis on acrylamide gels. Binding reactions were performed in a final volume of 20 μl of binding buffer containing 100 ng/μl poly[d(I-C)]. The amount of E. colipET3cO2 or endosperm cell extract employed varied from 2.5 to 4 μg, whereas 3.2 × 104 cpm of end-labeled probe were routinely used. The binding reaction was incubated at 20 °C for 10 min, and 10 μl was then loaded onto a 4–7% Tris borate/EDTA acrylamide gel (29:1). Gels were run at 8 °C at 15 V/cm, dried, and autoradiographed. Previous analysis of the methylation state of the o2 promoter region in immature endosperm and young leaf tissue with methylation-sensitive restriction enzymes indicated the occurrence of different methylation patterns within the −510, −990 region. Also, in both tissues we observed the presence of m5C residues close the O2 binding site. 2V. Rossi and L. Pellegrini, unpublished observations. To gain more insight into the methylation status of the o2 promoter in these tissues, we undertook a strategy aimed to detail the m5C distribution by means of the bisulfite treatment of maize genomic DNA (10Frommer M. McDonald L.E. Millar D.S. Collis C.M. Watt F. Grigg G.W. Molloy P.L. Paul C.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1827-1831Google Scholar). This method is based upon a chemical treatment that converts unmethylated C residues into U residues that appear as T residues after PCR of specific, single-stranded DNA. Conversely, all m5C residues remain unmodified. Separate PCR amplifications were performed for the upper and lower strands to produce DNA fragments that were subsequently cloned and sequenced. CpG-methylated plasmid DNA bearing the o2 promoter was used as a control for complete conversion of unmethylated C residues into U residues (data not shown). Fig. 1 A shows the methylation pattern of a 390-bp region of the o2 promoter (from −436 to −46) in endosperm. We cloned and sequenced 13 upper strand and 16 lower strand PCR products. All clones displayed different methylation patterns and thus were derived from individual genomic O2 sequences. Noteworthy is the considerable level of C-methylation (84%) present in the o2 promoter at the time and place where it leads to gene expression. Moreover, all the C residues embedded within the O2 binding site (boxed in Fig. 1 A), were methylated. The methylation pattern of the lower strand differed markedly from that of the upper strand. Statistical analysis (Student's t test) of the mean methylation values obtained from the collected data indicated that, with p = 1%, the lower strand was significantly less methylated than the upper strand (80% m5Cversus 89%). Interestingly, an unequal m5C distribution was observed, and the lower strand was less methylated at the most distal part of the region analyzed. We have also applied the Student's t analysis to compare the methylation level of each C residue within their sequence context. Accordingly, we found that in endosperm, for both strands, CpT and CpG dinucleotides were significantly less methylated than CpA and CpC (p= 1 and 5%, respectively). The methylation pattern of the −436, −46 o2 promoter region was also analyzed in leaf by sequencing 10 upper strand and 10 lower strand clones (Fig. 1 B). Again, different methylation patterns were observed, indicating that the clones were derived from individual genomic O2 sequences. Analysis (Student's t test) was again used to compare the mean methylation values of DNA obtained from leaf and endosperm. Data indicated that with p = 1% the level of C-methylation in leaf was significantly higher than in endosperm (96% m5C versus 84%). An unequal m5C distribution was also observed in the lower strand of the most distal part (−436, −370) of DNA from leaves. Taken together these findings indicate that the endosperm-specific expression of theo2 gene is not associated with the specific and complete demethylation of any C residue present within the promoter region analyzed. The expression of the o2 gene appears to be positively autoregulated by its gene product, possibly by binding to the target sequence, TGACGTTG, which is found in its own promoter and in the promoter of several o2-controlled genes (24Lohmer S. Maddaloni M. Motto M. Di Fonzo N. Hartings H. Salamini F. Thompson R.D. EMBO J. 1991; 10: 617-624Google Scholar, 17Motto, M., Thompson, R. D., Salamini, F., Cellular and Molecular Biology of Plant Seed Development, Larkins, B. A., Vasil, I. A., 1997, Kluwer Academic Publishers, Dordrecht, The Netherlands.Google Scholar). In immature endosperm, genomic sequencing revealed the occurrence of m5C within the CpG site embedded in the ACGT core. Thus, unlike CG-1 and the CREB-like MIB-1 transcription factor (8Staiger D. Kaulen H. Schell J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6930-6934Google Scholar, 7Inamdar N.M. Ehrich K.C. Ehrlich M. Plant Mol. Biol. 1991; 17: 111-123Google Scholar), the O2 protein is likely to bind in vivo to its cognate sequence even if, in both strands, the CpG dinucleotide is methylated. To further confirm this possibility, we performed in vitro gel-shift assays using extracts from E. colicells expressing a full-length O2 cDNA (pET3cO2; Ref. 27Izawa T. Foster R. Chua N.-H. J. Mol. Biol. 1993; 230: 1131-1144Google Scholar) and anin vitro CpG-methylated o2 promoter fragment (P188-CG; spanning from −399 to −211). For the gel-shift analysis a negative control consisted of E. coli cell extracts from cells transformed with pET3c (not expressing O2). Consistent with earlier data (27Izawa T. Foster R. Chua N.-H. J. Mol. Biol. 1993; 230: 1131-1144Google Scholar), the control extracts never resulted in a band shift when tested with the P188-CG probe (not shown). A total of seven methylated CpG sequences are present in the P188-CG probe, one within the O2 target sequence (see Fig. 1). Under the conditions used, the P188-CG probe resulted in a single retarded band (Fig. 2 A, lane 2), and at higher extract concentrations all the probe was shifted (data not shown). Consistently, a 100:1 molar excess of unlabeled P188-CG was needed to compete with the unmethylated P188 probe and abolish the O2·P188 retarded complex (Fig. 2 B, lane 1). Addition of distamycin A, a transcriptional inhibitor that binds to the minor groove of DNA (28Dickinson L.A. Joh T. Kohwi Y. Kohwi-Shigematsu T. Cell. 1992; 70: 631-645Google Scholar), did not impair O2 binding to the P188-CG probe; this indicated that the interaction of O2 protein occurs to the major groove of the DNA (Fig. 2 B, lanes 3–5). Formation of the O2·P188-CG complex indicated that neither the methylation pattern generated by the SssI DNA methylase nor the methylation at the C residue located within the O2 binding site were able to impair the in vitro binding activity of the O2 protein to its cognate DNA. Because the pattern and level of C-methylation generated by SssI DNA methylase on the P188 promoter fragment were different from that found in endosperm, we investigated the highest methylation content resulting in a detectable O2 in vitrobinding activity. EMSA experiments were performed using o2 promoter fragments generated with different m5C contents. Briefly, partially methylated DNA was prepared by PCR amplification of the P188 promoter fragment by addition of appropriate molar ratios of m5-dCTP and dCTP in the reaction mix. Probes with an average m5C content of 30, 50, 70, and 100% were generated (named P188–30%, P188–50%, P188–70%, and P188–100%) and used in gel shift and competition experiments with the O2 protein. Assuming an equal rate of nucleotide incorporation, each PCR reaction generates DNA fragments with a C-methylation content that can be plotted as a Gaussian distribution. Accordingly, restriction digestion analyses of the P188–50% probe with a methylation-sensitive enzyme resulted in the digestion of approximately 50% of a labeled PCR product (data not shown). Thus, it is conceivable to assume that, for each partially methylated probe, the most abundant fraction of labeled DNA fragments has an average C-methylation content of 30, 50, and 70%, respectively. However, among these DNA fragments the methylation pattern is stochastically represented. First, we tested O2 binding activity using a fully methylated DNA. Under the conditions tested, no band shift was observed (Fig. 2 A, lane 3). Even after the addition of twice the amount of pET3cO2 extract and longer autoradiogram exposures, no band shift was detected (data not shown). As an additional test, we performed competition experiments between a fully methylated probe and a labeled, unmethylated one. A 100-fold molar excess of unlabeled P188–100% did not eliminate formation of the O2 protein-P188 complex (Fig. 2 A, lane 4), indicating that, in vitro, complete C-methylation inhibits O2 binding to its cognate DNA. Similar results were obtained using the P188–70% probe (data not shown). In addition, we tested O2 binding to the P188–30% and P188–50% probes using several different O2 protein concentrations in the binding reaction. As shown in Fig. 3, at high O2 concentrations the P188–30% and P188–50% probes were retarded (Fig. 3, lanes 2 and 5); this indicated that DNA fragments with this methylation content and pattern are suitable substrates for O2 binding activity. Consistently, excess of unlabeled competitor eliminated the band shift (Fig. 3, lanes 3 and 6). It is noteworthy that we have never observed retardation of the O2·P188–50% complex as a sharp band but rather as a limited smear (Fig. 3, lane 5). Similar results have also been observed using a P188" @default.
• W2071476046 created "2016-06-24" @default.
• W2071476046 creator A5009985541 @default.
• W2071476046 creator A5013045721 @default.
• W2071476046 creator A5054942236 @default.
• W2071476046 date "1997-05-01" @default.
• W2071476046 modified "2023-10-18" @default.
• W2071476046 title "Analysis of the Methylation Pattern of the Maize Opaque-2 (O2) Promoter and in Vitro Binding Studies Indicate That the O2 B-Zip Protein and Other Endosperm Factors Can Bind to Methylated Target Sequences" @default.
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