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Cytosine Methylation Does Not Affect Binding of Transcription Factor Spl (DNA Methylation/Metaliothionein Gene Expression/"GC Box") MAUREEN A

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Cytosine Methylation Does Not Affect Binding of Transcription Factor Spl (DNA Methylation/Metaliothionein Gene Expression/ Proc. Nati. Acad. Sci. USA Vol. 85, pp. 2066-2070, April 1988 Biochemistry Cytosine methylation does not affect binding of transcription factor Spl (DNA methylation/metaliothionein gene expression/"GC box") MAUREEN A. HARRINGTON*, PETER A. JONES*, MASAYOSHI IMAGAWAt, AND MICHAEL KARINt *Department of Biochemistry and Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, CA 90033; and tSchool of Medicine, Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093 Communicated by Peter B. Dervan, November 23, 1987 (received for review June 9, 1987) ABSTRACT DNA methylation may be a component of a the methylation signal. What remains unclear, however, is multilevel control mechanism that regulates eukaryotic gene whether methylation also alters the direct binding of specific expression. We used synthetic oligonucleotides to investigate transcription factors to defined promoter elements. the effect of cytosine methylation on the binding of the Tjian and coworkers originally described (14-16) and transcription factor Spl to its target sequence (a G+C-rich subsequently purified from HeLa cell nuclear extracts (17) a sequence known as a "GC box"). Concatemers of double- transcription factor, Spl, that specifically recognizes the stranded 14-mers containing a GC box successfully competed simian virus 40 early promoter. Subsequent studies revealed with the human metallothionein iHA promoter for binding to the decanucleotide consensus Spl binding sequence to be Spl in DNase I protection experiments. The presence of 5- methylcytosine in the CpG sequence of the GC box did not 5' TCGGCGGGGC 3' (the "GC box"). GC boxes have influence Spl binding. The result was confirmed using double- been identified in the 5' region of several eukaryotic genes, stranded 20-mers containing 16 base pairs of complementary including the human metallothionein (hMT) IIA, IA, and IB sequence. Electrophoretic gel retardation analysis of annealed genes (18-20); the Harvey ras-J protooncogene (21); the 28-mers containing a GC box incubated with an Spl-con- epidermal growth factor receptor gene (22); arnd the human taining HeLa cell nuclear extract demonstrated the formation immunodeficiency virus long terminal repeat (23). The ob- of DNA-protein complexes; formation of these complexes was servations that Spl interacts with GC-box sequences in the not inhibited when an oligomer without a GC box was used as major groove (16), that the GC box contains a potential a competitor. Once again, the presence of a 5-methylcytosine methylation site (CpG), and that the methyl group of 5- residue in.the GC box did not influence the binding of the methylcytosine extends into the major groove suggested that protein to DNA. The results therefore preclude a direct effect the protein might be able to sense the presence or absence of of cytosine methylation on Spl-DNA interactions. the methyl group. There is also evidence that both the mouse metallothionein I (24) and the hMT-1B (20) genes might be There is strong experimental support for the proposal that sensitive to methylation control. We therefore measured the DNA methylation may be a component of a multilevel effect of cytosine methylation on the interaction of Spl with control mechanism through which the expression of certain its target sequence. eukaryotic genes is regulated (1). Inhibition of genomic The hMT-IIA promoter region was selected as a substrate methylation by 5-azacytidine can result in reactivation of for Spl binding because the region contains at least five genes on the transcriptionally inactive X chromosome (2, 3), distinct control elements in the 5' flanking region (Fig. 1) (18, induction of tissue-specific gene expression (4, 5), and 25). Recently, Lee et al. (26) showed that the GC-box element expression of differentiated phenotypes in cultured cells in coordination with the basal-level elements (BLEs) regulate (6-8). Introduction of in vitro hypermethylated sequences basal levels of hMT-IIA transcription in vitro, and Karin et al. into recipient cells can preclude gene expression (9, 10). (25) showed that the same elements are required for optimal Despite these correlations the relationship between methyl- expression in vivo. The well-characterized nature of the ation patterns and expression of some genes remains unclear promoter region therefore provided an ideal systerh in which (11) and it is not known how changes in the methylation to study the effect of DNA sequence modification on the patterns of certain genes alter expression while the activities binding of Spl. of other genes remain unaffected. Recent reports strongly suggest that cytosine methylation MATERIALS AND METHODS may be involved in the structural ordering of chromatin (12, 13). Methylation of phage M13 constructs before transfec- Oligonucleotide Synthesis. Synthetic oligodeoxyribonu- tion into eukaryotic cells targets the assimilated DNA into cleotides were prepared with an Applied Biosystems oligo- DNase I-insensitive conformations. Buschhausen et al. (13) nucleotide synthesizer in the Microchemical Core Labora- showed that reconstitution of methylated thymidine kinase tory at the University of Southern California Cancer Center. DNA with histone octamers prior to microinjection resulted Oligonucleotides were purified by gel electrophoresis (7 M in biological inactivity even in the absence of integration into urea/20% acrylamide/1% NN'-methylenebisacrylamide in the host genome. In contrast, mock-methylated constructs 89 mM Tris/89 mM boric acid/2 mM EDTA buffer, pH 8.2) actively expressed thymidine kinase and the inhibitory effect and concentrated by passage through Sep-Pak C18 cartridges of methylation could be reversed by treatment with 5-aza- (Waters Associates), and organic contaminants were re- cytidine. These observations suggest that positioning of moved by Sephadex G-25 (Pharmacia) column chromatogra- structural nuclear proteins (histones, high-mobility group phy. The phosphoramidite of 5-methylcytosine was obtained proteins, or matrix proteins) may be involved in transducing from Applied Biosystems (Foster City, CA) and the occur- rence of the modified base in the synthetic oligonucleotides The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: hMT, human metallothionein; BLE, basal-level ele- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ment. Downloaded by guest on September 29, 2021 2066 Biochemistry: Harrington et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2067 mRNA -764 -256 -200 -150 -100 -50 0 +63 I I I 1 i Hind m 7 \ Bam HI GCCGGGGC GG GGCTTTTGCA FIG. 1. Map of the hMT-IIA promoter region. The start site for transcription is indicated by the horizontal arrow. dBLE, distal basal-level element; pBLE, proximal basal-level element; GC, GC-box element; MRE, metal-responsive element; GRE, glucocorticoid-responsive element; TATA, "TATA box." was verified by hydrolysis with formic acid followed by remove insoluble debris, and the supernatant was applied to HPLC (27). a heparin-agarose (Bio-Rad) column. Bound material was Fig. 2 shows the oligomers used to investigate Spl bind- eluted in a stepwise manner with TM buffer containing 0.1, ing. In addition, control experiments with the following 0.2, and 0.4 M KCl, in 0.5-ml fractions. The 0.4 M KCI annealed 26-mers were performed: 5' GATCCCATGAT- fractions were pooled and approximately 9 mg of protein was TGTGATTCTCACGAG 3' annealed to 5' GATCCTCGTG- recovered. Preliminary studies revealed that 4 ,ul of extract AGAATCACAATCATGG 3'. (8 ,ug) adequately prevented DNase I digestion of the GC Annealing Conditions. Oligonucleotides were combined in box in the hMT-IIA promoter (data not shown). 10 mM Tris HC/1 mM EDTA, pH 8.0, containing 0.3 M DNase I Protection Assays. The plasmid pH S1 (25) (see NaCl. Mixtures were heated to 90TC for 3 min and trans- Fig. 1) was cut with BamHI restriction endonuclease and ferred to a 65TC water bath, which was slowly brought to labeled at the 3' end by using the Klenow fragment of room temperature. Annealed oligonucleotides were then Escherichia coli DNA polymerase (United States Biochem- purified on Sephadrx G-25 columns and electrophoresed in ical, Cleveland, OH) and [a-32P]dNTPs or at the 5' end by 10% polyacrylamide gels (acrylamide/methylene-bisac- using calf intestinal alkaline phosphatase (Boehringer Mann- rylamide weight ratio 29.5:1) in an 89 mM Tris borate/1 mM heim) followed by bacteriophage T4 polynucleotide kinase EDTA buffer system. (New England Biolabs) and [y-32P]ATP. After digestion with HeLa Cell Nuclear Extracts. HeLa cell nuclear extracts HindIII (Boehringer Mannheim), the hMT-IIA promoter were prepared according to the method ofDignam et al. (28). region (nucleotides -764 to +63) inserts were purified by In brief, 3 x 109 HeLa cells were pelleted by centrifugation, gel electrophoresis. Individual assay mixtures contained 2 ng washed in phosphate-buffered saline, resuspended in 10 mM of labeled DNA (8000-10,000 cpm), 8 ,g of nuclear extract Hepes, pH 7.9/1.5 mM MgCl2/10 mM KCI/0.5 mM (based on protein), 2% (wt/vol) polyvinyl alcohol, 1 ,g of dithiothreitol/0.5 mM phenylmethylsulfonyl fluoride, and poly(deoxyinosinate-deoxycytidylate [poly(dI-dC), alternat- homogenized in a Dounce homogenizer by 10 passes of the ing copolymer], and the indicated concentrations of unla- tight plunger. Nuclei were pelleted and homogenized in 20 beled annealed synthetic oligonucleotides. Assay mixtures mM Hepes, pH 7.9/25% (vol/vol) glycerol/0.42 M NaCl/1.5 were incubated on ice for 15 min prior to the addition of mM MgCI2/0.2 mM EDTA/0.5 mM dithiothreitol/0.5 mM DNase 1 [Cooper, Malvern, PA; 4 x 104 units for 90 sec phenylmethylsulfonyl fluoride. Insoluble debris was re- at room temperature (22°C)] and reactions were terminated moved by centrifugation, and the supernatant was dialyzed by the addition of 100 1,l of a solution containing 200 mM against TM buffer (50 mM TrisHCI, pH 7.9/20% glyc- NaCl, 20 mM EDTA, 1% NaDodSO4, and 250 ,ug of yeast erol/12.5 mM MgCl2/i mM EDTA/1 mM dithiothreitol) tRNA per ml.
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