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Cis-elements Required for the Demethylation of the Mouse M-lysozyme Downstream Enhancer

1997; Elsevier BV; Volume: 272; Issue: 33 Linguagem: Inglês

10.1074/jbc.272.33.20850

1083-351X

Alexander Schmitz, Marc L. Short, Ole Ammerpohl, Christian Asbrand, Joachim Nickel, Rainer Renkawitz,

RNA modifications and cancer

The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seenin vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site. The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seenin vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site. The mouse genome contains two lysozyme genes, a Paneth cell (P-lysozyme) 1The abbreviations used are: P-lysozyme, Paneth cell lysozyme; M-lysozyme, macrophage-specific lysozyme; kb, kilobase(s); HS, DNase I-hypersensitive; MLDE, M-lysozyme downstream enhancer (core); GABP, GA-binding protein; bp, base pair(s); tk, thymidine kinase; CAT, chloramphenicol acetyltransferase; LM-PCR, ligation-mediated polymerase chain reaction. 1The abbreviations used are: P-lysozyme, Paneth cell lysozyme; M-lysozyme, macrophage-specific lysozyme; kb, kilobase(s); HS, DNase I-hypersensitive; MLDE, M-lysozyme downstream enhancer (core); GABP, GA-binding protein; bp, base pair(s); tk, thymidine kinase; CAT, chloramphenicol acetyltransferase; LM-PCR, ligation-mediated polymerase chain reaction. and a macrophage-specific (M-lysozyme) gene generated by a gene duplication event (1Cross M. Renkawitz R. EMBO J. 1990; 9: 1283-1288Crossref PubMed Scopus (63) Google Scholar). The M- and P-lysozyme genes are arranged in tandem with the coding regions separated by 5 kb (Fig. 1). Analysis of the M-lysozyme gene domain by DNase I digestion identified multiple hypersensitive (HS) sites in the 5′ and 3′ M-lysozyme gene-flanking regions in macrophage and myeloid precursor cell lines (Fig.1) (2Möllers B. Klages S. Wedel A. Cross M. Spooncer E. Dexter T.M. Renkawitz R. Nucleic Acids Res. 1992; 20: 1917-1924Crossref PubMed Scopus (26) Google Scholar). Only a single site in the 3′-flanking region (HS3) was dependent on the differentiation state of the cell line and correlated with M-lysozyme gene expression (2Möllers B. Klages S. Wedel A. Cross M. Spooncer E. Dexter T.M. Renkawitz R. Nucleic Acids Res. 1992; 20: 1917-1924Crossref PubMed Scopus (26) Google Scholar). Transfection analysis of the flanking regions identified a single enhancer downstream of the M-gene which overlapped the HS3 site and is limited to the subregion HS3.2 (2Möllers B. Klages S. Wedel A. Cross M. Spooncer E. Dexter T.M. Renkawitz R. Nucleic Acids Res. 1992; 20: 1917-1924Crossref PubMed Scopus (26) Google Scholar, 3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). Analysis of the HS3 region methylation state in M-lysozyme-expressing and nonexpressing cells demonstrated a correlation between undermethylation of this region with both the presence of the HS3 site and expression of the M-lysozyme gene. Further fine mapping identified a central core enhancer (MLDE), which is bound by a heterotetrameric GABP complex (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). We found that GABP binding to the MLDE is methylation-sensitive (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). Thus, very likely, macrophage-specific demethylation of the single CpG dinucleotide within the MLDE is a mechanism to confer tissue-specific enhancer activity.Figure 1Mouse M- and P- lysozyme genes. The mouse lysozyme genes are located on a genomic region (solid line) about 24 kb in length. The macrophage- and P-specific genes are indicated with their exons (filled boxes) and the transcribed regions (dotted line with arrow). Macrophages and precursor cells contain multiple DNase I HS sites (HS) of various intensities (vertical arrows).HS 3/6 marks the 3′-enhancer region containing the core enhancer at HS site 3 (MLDE (Ref. 3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar)).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In other systems, methylation of CpG dinucleotides has been correlated with transcriptional inactivity as well (for review, see Refs. 5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar and6Eden S. Cedar H. Curr. Opin. Genet. Dev. 1994; 4: 255-259Crossref PubMed Scopus (280) Google Scholar). In two cases, DNA transfections have identified quite complex DNA regions required for tissue-specific demethylation (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Recent achievements in demethylating DNA in vitro (9Weiss A. Keshet I. Razin A. Cedar H. Cell. 1996; 86: 709-718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed an involvement of RNA and that tissue-specific proteins are required for the specificity of the reaction.Here, we wanted to analyze the mechanisms mediating macrophage-specific demethylation of the single CpG site within the mouse lysozyme enhancer core MLDE. We identified two short DNA regions of 163 and 79 bp which are required and sufficient for demethylation. The two fragments are overlapping, but the sequence in common is not sufficient for demethylation.DISCUSSIONMany examples have been identified linking sequence-specific DNA demethylation with differentiation of a particular tissue or cell type (for review, see Refs. 5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar and 6Eden S. Cedar H. Curr. Opin. Genet. Dev. 1994; 4: 255-259Crossref PubMed Scopus (280) Google Scholar). In these cases, it has been shown that the differentiation-dependent expression of a tissue-specific gene is correlated with the demethylation of flanking sequences. Such a demethylation is usually restricted to a specific region or at least to specific genes. For example, expression-linked demethylation has been seen for the genes coding for chicken vitellogenin, human dihydrofolate reductase, mouse collagen IV, rat α-actin, mouse κ chain, human estrogen receptor, human galectin-1, or mouse pyruvate dehydrogenase E1α subunit, to name only a few (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar,8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 17Philipsen J.N. Gruber M. Ab G. Biochim. Biophys. Acta. 1985; 826: 186-194Crossref PubMed Scopus (13) Google Scholar, 18Shimada T. Inokuchi K. Nienhuis A.W. Mol. Cell. Biol. 1987; 7: 2830-2837Crossref PubMed Scopus (17) Google Scholar, 19Burbelo P.D. Horikoshi S. Yamada Y. J. Biol. Chem. 1990; 265: 4839-4843Abstract Full Text PDF PubMed Google Scholar, 20Ferguson A.T. Lapidus R.G. Baylin S.B. Davidson N.E. Cancer Res. 1995; 55: 2279-2283PubMed Google Scholar, 21Benvenuto G. Carpentieri M.L. Salvatore P. Cindolo L. Bruni C.B. Chiariotti L. Mol. Cell. Biol. 1996; 16: 2736-2743Crossref PubMed Scopus (52) Google Scholar, 22Iannello R.C. Young J. Sumarsono S. Tymms M.J. Dahl H.M. Gould J. Hedger M. Kola I. Mol. Cell. Biol. 1997; 17: 612-619Crossref PubMed Scopus (60) Google Scholar). Other genes, not expressed in this particular tissue, remain methylated. Such a correlation has been found for the mouse M-lysozyme gene as well. The M-lysozyme gene is inactive in non-macrophage cells and shows a methylated CpG dinucleotide within the single HpaII recognition sequence of the downstream enhancer (3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). It has been shown that during the differentiation of the multipotent FTCP-A4 cell line toward macrophages the enhancer loses its methyl groups (3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). Similarly, the human lysozyme gene has been found to be demethylated depending on the differentiation as seen inex vivo cultures of hematopoetic progenitor cells, whereas the gene coding for myeloperoxidase showed unaltered demethylation (23Lubbert M. Brugger W. Mertelsmann R. Kanz L. Blood. 1996; 87: 447-455Crossref PubMed Google Scholar). Other myeloid-specific demethylation events have been shown as well to correlate with transcriptional activity, such as the c-fms gene and the regulatory region of the tumor necrosis factor-α gene (24Felgner J. Kreipe H. Heidorn K. Jaquet K. Heuss R. Zschunke F. Radzun H.J. Parwaresch M.R. Leukemia. 1992; 6: 420-425PubMed Google Scholar, 25Takei S. Fernandez D. Redford A. Toyoda H. Biochem. Biophys. Res. Commun. 1996; 220: 606-612Crossref PubMed Scopus (20) Google Scholar).Many of the published examples could not distinguish between a role for the demethylation being required for transcriptional activity or a possible demethylation-inducing function mediated by transcription. In some cases, methylation-dependent repressor binding or inhibition of transcription factor binding could be demonstrated (5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar,26Boyes J. Bird A. Cell. 1991; 64: 1123-1134Abstract Full Text PDF PubMed Scopus (581) Google Scholar, 27Jost J.P. Saluz H.P. Pawlak A. Nucleic Acids Res. 1991; 19: 5771-5775Crossref PubMed Scopus (40) Google Scholar, 28Saluz H.P. Feavers I.M. Jiricny J. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6697-6700Crossref PubMed Scopus (39) Google Scholar, 29Robertson K.D. Hayward S.D. Ling P.D. Samid D. Ambinder R.F. Mol. Cell. Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (109) Google Scholar). For the mouse lysozyme gene, it was shown that within the core part of the downstream enhancer the heterotetrameric transcription factor GABP is required for full enhancer activity (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). In addition, it was demonstrated that the methylation of a single CpG dinucleotide within the enhancer core region inhibits in vitro DNA binding of GABP (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). A similar sensitivity in methylated DNA binding was demonstrated for GABP and other ETS proteins in the context of binding sites that differ from the mouse lysozyme GABP binding site (30Yokomori N. Kobayashi R. Moore R. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1995; 15: 5355-5362Crossref PubMed Scopus (81) Google Scholar, 31Gaston K. Fried M. Gene ( Amst. ). 1995; 157: 257-259Crossref PubMed Scopus (25) Google Scholar, 32Desmet C. Debacker O. Faraoni I. Lurquin C. Brasseur F. Boon T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7149-7153Crossref PubMed Scopus (464) Google Scholar). This suggests that demethylation of this enhancer is a prerequisite for enhancer activity and therefore, for transcription. Here we show that the cis-element-dependent demethylation can be observed independent of the type of the neighboring DNA or promoter context. The 224-bp fragment HS3.2 can be stably transfected either fused to a eukaryotic reporter gene or to a prokaryotic vector and will be demethylated in both cases. Therefore, the lysozyme downstream enhancer confers at least two functions. One function is to mediate the demethylation during differentiation, and another function is to activate gene transcription by the bound enhancer factors. A similar dual activity has been demonstrated for the α-actin promoter and the κ enhancer (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar).Our transfection data suggest that the MLDE fragment within the downstream enhancer is not sufficient to mediate demethylation. This fragment harbors the single HpaII site and binds the heterotetrameric GABP factor in the absence of methylation. Here we have shown that even a single methyl group on the lower strand is sufficient to inhibit GABP binding, whereas the hemimethylated upper strand does not interfere with binding. Since GABP binding is impaired even by hemimethylated DNA, this factor cannot be the cause for demethylation. This is in contrast to the mechanism observed in the context of Sp1 binding (15Macleod D. Charlton J. Mullins J. Bird A.P. Genes Dev. 1994; 8: 2282-2292Crossref PubMed Scopus (514) Google Scholar). Sp1 is able to bind methylated DNA and after replication prevents the maintenance methylase from modifying the newly synthesized DNA strand. Such a Sp1-like activity could have been envisioned to be utilized by an unknown factor that has been found to bind to the GABP response element even in the case of a fully methylated DNA (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). If this factor would indeed play such a role, this function would not be sufficient for demethylation, since we have shown that the methylated MLDE fragment is not demethylated (Fig. 6).Within the group of fragments mediating demethylation, there seems to be a bias in demethylation efficiency: all of the fragments extending up to the position 224 (Fig. 7; i.e. fragments HS3/6, HS3.2, 82–224, 146–224) mediate 90–100% demethylation. In contrast, fragments with a downstream deletion (Fig. 7;i.e. fragments 1–181, 1–163) mediate demethylation for only 70–80% of the molecules. Nevertheless, two different sets of fragments, overlapping in the GABP site only, confer demethylation. Computer analysis of the sequences flanking the GABP site did not reveal any consensus in common, which otherwise might have been an indication for trans-acting proteins required for demethylation. In addition, in vitro footprinting showed only one protected region in addition to the GABP site (Fig. 3 C).One of the minimal regions required and sufficient for demethylation is only 78 bp in length (HS3.2(146–224)) (Fig. 7). None of the sequences analyzed for conferring demethylation in the other model systems could be delineated to such a small fragment. For both the α-actin promoter and the κ enhancer, DNA fragments of 800 bp to more than 1000 bp in length were required for demethylation (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar,8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Despite the complexity of the κ enhancer it could be shown that the absence of a single enhancer factor (nuclear factor-κB) leads to the failure of this fragment to undergo selective demethylation (33Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergman Y. Nat. Genet. 1996; 13: 435-441Crossref PubMed Scopus (208) Google Scholar). Previous results on in vitro demethylation of hemimethylated DNA by chicken embryonic extracts suggested a glycosylase activity (34Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4684-4688Crossref PubMed Scopus (146) Google Scholar,35Jost J.P. Siegmann M. Sun L. Leung R. J. Biol. Chem. 1995; 270: 9734-9739Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Ras-induced overall demethylation in mouse embryonal P19 cells could be followed in vitro as well (36Szyf M. Theberge J. Bozovic V. J. Biol. Chem. 1995; 270: 12690-12696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In contrast to embryonic cells and tissues, demethylation specific for differentiation and for particular sites may utilize a different mechanism. Recent achievements with in vitro demethylation showed that RNA is involved in sequence-specific demethylation (9Weiss A. Keshet I. Razin A. Cedar H. Cell. 1996; 86: 709-718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Nevertheless, these authors showed that the tissue specificity of the demethylation reaction involves proteins as well.Taken together, these and our results demonstrate that the demethylation activity is mediated via enhancer elements and may be organized in a manner similar to that of the enhancer elements; that is, specific modules within the enhancer regions are either sufficient by themselves to mediate demethylation or have to act in combination with other modules. The mouse genome contains two lysozyme genes, a Paneth cell (P-lysozyme) 1The abbreviations used are: P-lysozyme, Paneth cell lysozyme; M-lysozyme, macrophage-specific lysozyme; kb, kilobase(s); HS, DNase I-hypersensitive; MLDE, M-lysozyme downstream enhancer (core); GABP, GA-binding protein; bp, base pair(s); tk, thymidine kinase; CAT, chloramphenicol acetyltransferase; LM-PCR, ligation-mediated polymerase chain reaction. 1The abbreviations used are: P-lysozyme, Paneth cell lysozyme; M-lysozyme, macrophage-specific lysozyme; kb, kilobase(s); HS, DNase I-hypersensitive; MLDE, M-lysozyme downstream enhancer (core); GABP, GA-binding protein; bp, base pair(s); tk, thymidine kinase; CAT, chloramphenicol acetyltransferase; LM-PCR, ligation-mediated polymerase chain reaction. and a macrophage-specific (M-lysozyme) gene generated by a gene duplication event (1Cross M. Renkawitz R. EMBO J. 1990; 9: 1283-1288Crossref PubMed Scopus (63) Google Scholar). The M- and P-lysozyme genes are arranged in tandem with the coding regions separated by 5 kb (Fig. 1). Analysis of the M-lysozyme gene domain by DNase I digestion identified multiple hypersensitive (HS) sites in the 5′ and 3′ M-lysozyme gene-flanking regions in macrophage and myeloid precursor cell lines (Fig.1) (2Möllers B. Klages S. Wedel A. Cross M. Spooncer E. Dexter T.M. Renkawitz R. Nucleic Acids Res. 1992; 20: 1917-1924Crossref PubMed Scopus (26) Google Scholar). Only a single site in the 3′-flanking region (HS3) was dependent on the differentiation state of the cell line and correlated with M-lysozyme gene expression (2Möllers B. Klages S. Wedel A. Cross M. Spooncer E. Dexter T.M. Renkawitz R. Nucleic Acids Res. 1992; 20: 1917-1924Crossref PubMed Scopus (26) Google Scholar). Transfection analysis of the flanking regions identified a single enhancer downstream of the M-gene which overlapped the HS3 site and is limited to the subregion HS3.2 (2Möllers B. Klages S. Wedel A. Cross M. Spooncer E. Dexter T.M. Renkawitz R. Nucleic Acids Res. 1992; 20: 1917-1924Crossref PubMed Scopus (26) Google Scholar, 3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). Analysis of the HS3 region methylation state in M-lysozyme-expressing and nonexpressing cells demonstrated a correlation between undermethylation of this region with both the presence of the HS3 site and expression of the M-lysozyme gene. Further fine mapping identified a central core enhancer (MLDE), which is bound by a heterotetrameric GABP complex (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). We found that GABP binding to the MLDE is methylation-sensitive (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). Thus, very likely, macrophage-specific demethylation of the single CpG dinucleotide within the MLDE is a mechanism to confer tissue-specific enhancer activity. In other systems, methylation of CpG dinucleotides has been correlated with transcriptional inactivity as well (for review, see Refs. 5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar and6Eden S. Cedar H. Curr. Opin. Genet. Dev. 1994; 4: 255-259Crossref PubMed Scopus (280) Google Scholar). In two cases, DNA transfections have identified quite complex DNA regions required for tissue-specific demethylation (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Recent achievements in demethylating DNA in vitro (9Weiss A. Keshet I. Razin A. Cedar H. Cell. 1996; 86: 709-718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed an involvement of RNA and that tissue-specific proteins are required for the specificity of the reaction. Here, we wanted to analyze the mechanisms mediating macrophage-specific demethylation of the single CpG site within the mouse lysozyme enhancer core MLDE. We identified two short DNA regions of 163 and 79 bp which are required and sufficient for demethylation. The two fragments are overlapping, but the sequence in common is not sufficient for demethylation. DISCUSSIONMany examples have been identified linking sequence-specific DNA demethylation with differentiation of a particular tissue or cell type (for review, see Refs. 5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar and 6Eden S. Cedar H. Curr. Opin. Genet. Dev. 1994; 4: 255-259Crossref PubMed Scopus (280) Google Scholar). In these cases, it has been shown that the differentiation-dependent expression of a tissue-specific gene is correlated with the demethylation of flanking sequences. Such a demethylation is usually restricted to a specific region or at least to specific genes. For example, expression-linked demethylation has been seen for the genes coding for chicken vitellogenin, human dihydrofolate reductase, mouse collagen IV, rat α-actin, mouse κ chain, human estrogen receptor, human galectin-1, or mouse pyruvate dehydrogenase E1α subunit, to name only a few (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar,8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 17Philipsen J.N. Gruber M. Ab G. Biochim. Biophys. Acta. 1985; 826: 186-194Crossref PubMed Scopus (13) Google Scholar, 18Shimada T. Inokuchi K. Nienhuis A.W. Mol. Cell. Biol. 1987; 7: 2830-2837Crossref PubMed Scopus (17) Google Scholar, 19Burbelo P.D. Horikoshi S. Yamada Y. J. Biol. Chem. 1990; 265: 4839-4843Abstract Full Text PDF PubMed Google Scholar, 20Ferguson A.T. Lapidus R.G. Baylin S.B. Davidson N.E. Cancer Res. 1995; 55: 2279-2283PubMed Google Scholar, 21Benvenuto G. Carpentieri M.L. Salvatore P. Cindolo L. Bruni C.B. Chiariotti L. Mol. Cell. Biol. 1996; 16: 2736-2743Crossref PubMed Scopus (52) Google Scholar, 22Iannello R.C. Young J. Sumarsono S. Tymms M.J. Dahl H.M. Gould J. Hedger M. Kola I. Mol. Cell. Biol. 1997; 17: 612-619Crossref PubMed Scopus (60) Google Scholar). Other genes, not expressed in this particular tissue, remain methylated. Such a correlation has been found for the mouse M-lysozyme gene as well. The M-lysozyme gene is inactive in non-macrophage cells and shows a methylated CpG dinucleotide within the single HpaII recognition sequence of the downstream enhancer (3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). It has been shown that during the differentiation of the multipotent FTCP-A4 cell line toward macrophages the enhancer loses its methyl groups (3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). Similarly, the human lysozyme gene has been found to be demethylated depending on the differentiation as seen inex vivo cultures of hematopoetic progenitor cells, whereas the gene coding for myeloperoxidase showed unaltered demethylation (23Lubbert M. Brugger W. Mertelsmann R. Kanz L. Blood. 1996; 87: 447-455Crossref PubMed Google Scholar). Other myeloid-specific demethylation events have been shown as well to correlate with transcriptional activity, such as the c-fms gene and the regulatory region of the tumor necrosis factor-α gene (24Felgner J. Kreipe H. Heidorn K. Jaquet K. Heuss R. Zschunke F. Radzun H.J. Parwaresch M.R. Leukemia. 1992; 6: 420-425PubMed Google Scholar, 25Takei S. Fernandez D. Redford A. Toyoda H. Biochem. Biophys. Res. Commun. 1996; 220: 606-612Crossref PubMed Scopus (20) Google Scholar).Many of the published examples could not distinguish between a role for the demethylation being required for transcriptional activity or a possible demethylation-inducing function mediated by transcription. In some cases, methylation-dependent repressor binding or inhibition of transcription factor binding could be demonstrated (5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar,26Boyes J. Bird A. Cell. 1991; 64: 1123-1134Abstract Full Text PDF PubMed Scopus (581) Google Scholar, 27Jost J.P. Saluz H.P. Pawlak A. Nucleic Acids Res. 1991; 19: 5771-5775Crossref PubMed Scopus (40) Google Scholar, 28Saluz H.P. Feavers I.M. Jiricny J. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6697-6700Crossref PubMed Scopus (39) Google Scholar, 29Robertson K.D. Hayward S.D. Ling P.D. Samid D. Ambinder R.F. Mol. Cell. Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (109) Google Scholar). For the mouse lysozyme gene, it was shown that within the core part of the downstream enhancer the heterotetrameric transcription factor GABP is required for full enhancer activity (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). In addition, it was demonstrated that the methylation of a single CpG dinucleotide within the enhancer core region inhibits in vitro DNA binding of GABP (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). A similar sensitivity in methylated DNA binding was demonstrated for GABP and other ETS proteins in the context of binding sites that differ from the mouse lysozyme GABP binding site (30Yokomori N. Kobayashi R. Moore R. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1995; 15: 5355-5362Crossref PubMed Scopus (81) Google Scholar, 31Gaston K. Fried M. Gene ( Amst. ). 1995; 157: 257-259Crossref PubMed Scopus (25) Google Scholar, 32Desmet C. Debacker O. Faraoni I. Lurquin C. Brasseur F. Boon T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7149-7153Crossref PubMed Scopus (464) Google Scholar). This suggests that demethylation of this enhancer is a prerequisite for enhancer activity and therefore, for transcription. Here we show that the cis-element-dependent demethylation can be observed independent of the type of the neighboring DNA or promoter context. The 224-bp fragment HS3.2 can be stably transfected either fused to a eukaryotic reporter gene or to a prokaryotic vector and will be demethylated in both cases. Therefore, the lysozyme downstream enhancer confers at least two functions. One function is to mediate the demethylation during differentiation, and another function is to activate gene transcription by the bound enhancer factors. A similar dual activity has been demonstrated for the α-actin promoter and the κ enhancer (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar).Our transfection data suggest that the MLDE fragment within the downstream enhancer is not sufficient to mediate demethylation. This fragment harbors the single HpaII site and binds the heterotetrameric GABP factor in the absence of methylation. Here we have shown that even a single methyl group on the lower strand is sufficient to inhibit GABP binding, whereas the hemimethylated upper strand does not interfere with binding. Since GABP binding is impaired even by hemimethylated DNA, this factor cannot be the cause for demethylation. This is in contrast to the mechanism observed in the context of Sp1 binding (15Macleod D. Charlton J. Mullins J. Bird A.P. Genes Dev. 1994; 8: 2282-2292Crossref PubMed Scopus (514) Google Scholar). Sp1 is able to bind methylated DNA and after replication prevents the maintenance methylase from modifying the newly synthesized DNA strand. Such a Sp1-like activity could have been envisioned to be utilized by an unknown factor that has been found to bind to the GABP response element even in the case of a fully methylated DNA (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). If this factor would indeed play such a role, this function would not be sufficient for demethylation, since we have shown that the methylated MLDE fragment is not demethylated (Fig. 6).Within the group of fragments mediating demethylation, there seems to be a bias in demethylation efficiency: all of the fragments extending up to the position 224 (Fig. 7; i.e. fragments HS3/6, HS3.2, 82–224, 146–224) mediate 90–100% demethylation. In contrast, fragments with a downstream deletion (Fig. 7;i.e. fragments 1–181, 1–163) mediate demethylation for only 70–80% of the molecules. Nevertheless, two different sets of fragments, overlapping in the GABP site only, confer demethylation. Computer analysis of the sequences flanking the GABP site did not reveal any consensus in common, which otherwise might have been an indication for trans-acting proteins required for demethylation. In addition, in vitro footprinting showed only one protected region in addition to the GABP site (Fig. 3 C).One of the minimal regions required and sufficient for demethylation is only 78 bp in length (HS3.2(146–224)) (Fig. 7). None of the sequences analyzed for conferring demethylation in the other model systems could be delineated to such a small fragment. For both the α-actin promoter and the κ enhancer, DNA fragments of 800 bp to more than 1000 bp in length were required for demethylation (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar,8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Despite the complexity of the κ enhancer it could be shown that the absence of a single enhancer factor (nuclear factor-κB) leads to the failure of this fragment to undergo selective demethylation (33Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergman Y. Nat. Genet. 1996; 13: 435-441Crossref PubMed Scopus (208) Google Scholar). Previous results on in vitro demethylation of hemimethylated DNA by chicken embryonic extracts suggested a glycosylase activity (34Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4684-4688Crossref PubMed Scopus (146) Google Scholar,35Jost J.P. Siegmann M. Sun L. Leung R. J. Biol. Chem. 1995; 270: 9734-9739Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Ras-induced overall demethylation in mouse embryonal P19 cells could be followed in vitro as well (36Szyf M. Theberge J. Bozovic V. J. Biol. Chem. 1995; 270: 12690-12696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In contrast to embryonic cells and tissues, demethylation specific for differentiation and for particular sites may utilize a different mechanism. Recent achievements with in vitro demethylation showed that RNA is involved in sequence-specific demethylation (9Weiss A. Keshet I. Razin A. Cedar H. Cell. 1996; 86: 709-718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Nevertheless, these authors showed that the tissue specificity of the demethylation reaction involves proteins as well.Taken together, these and our results demonstrate that the demethylation activity is mediated via enhancer elements and may be organized in a manner similar to that of the enhancer elements; that is, specific modules within the enhancer regions are either sufficient by themselves to mediate demethylation or have to act in combination with other modules. Many examples have been identified linking sequence-specific DNA demethylation with differentiation of a particular tissue or cell type (for review, see Refs. 5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar and 6Eden S. Cedar H. Curr. Opin. Genet. Dev. 1994; 4: 255-259Crossref PubMed Scopus (280) Google Scholar). In these cases, it has been shown that the differentiation-dependent expression of a tissue-specific gene is correlated with the demethylation of flanking sequences. Such a demethylation is usually restricted to a specific region or at least to specific genes. For example, expression-linked demethylation has been seen for the genes coding for chicken vitellogenin, human dihydrofolate reductase, mouse collagen IV, rat α-actin, mouse κ chain, human estrogen receptor, human galectin-1, or mouse pyruvate dehydrogenase E1α subunit, to name only a few (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar,8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 17Philipsen J.N. Gruber M. Ab G. Biochim. Biophys. Acta. 1985; 826: 186-194Crossref PubMed Scopus (13) Google Scholar, 18Shimada T. Inokuchi K. Nienhuis A.W. Mol. Cell. Biol. 1987; 7: 2830-2837Crossref PubMed Scopus (17) Google Scholar, 19Burbelo P.D. Horikoshi S. Yamada Y. J. Biol. Chem. 1990; 265: 4839-4843Abstract Full Text PDF PubMed Google Scholar, 20Ferguson A.T. Lapidus R.G. Baylin S.B. Davidson N.E. Cancer Res. 1995; 55: 2279-2283PubMed Google Scholar, 21Benvenuto G. Carpentieri M.L. Salvatore P. Cindolo L. Bruni C.B. Chiariotti L. Mol. Cell. Biol. 1996; 16: 2736-2743Crossref PubMed Scopus (52) Google Scholar, 22Iannello R.C. Young J. Sumarsono S. Tymms M.J. Dahl H.M. Gould J. Hedger M. Kola I. Mol. Cell. Biol. 1997; 17: 612-619Crossref PubMed Scopus (60) Google Scholar). Other genes, not expressed in this particular tissue, remain methylated. Such a correlation has been found for the mouse M-lysozyme gene as well. The M-lysozyme gene is inactive in non-macrophage cells and shows a methylated CpG dinucleotide within the single HpaII recognition sequence of the downstream enhancer (3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). It has been shown that during the differentiation of the multipotent FTCP-A4 cell line toward macrophages the enhancer loses its methyl groups (3Klages S. Möllers B. Renkawitz R. Nucleic Acids Res. 1992; 20: 1925-1932Crossref PubMed Scopus (29) Google Scholar). Similarly, the human lysozyme gene has been found to be demethylated depending on the differentiation as seen inex vivo cultures of hematopoetic progenitor cells, whereas the gene coding for myeloperoxidase showed unaltered demethylation (23Lubbert M. Brugger W. Mertelsmann R. Kanz L. Blood. 1996; 87: 447-455Crossref PubMed Google Scholar). Other myeloid-specific demethylation events have been shown as well to correlate with transcriptional activity, such as the c-fms gene and the regulatory region of the tumor necrosis factor-α gene (24Felgner J. Kreipe H. Heidorn K. Jaquet K. Heuss R. Zschunke F. Radzun H.J. Parwaresch M.R. Leukemia. 1992; 6: 420-425PubMed Google Scholar, 25Takei S. Fernandez D. Redford A. Toyoda H. Biochem. Biophys. Res. Commun. 1996; 220: 606-612Crossref PubMed Scopus (20) Google Scholar). Many of the published examples could not distinguish between a role for the demethylation being required for transcriptional activity or a possible demethylation-inducing function mediated by transcription. In some cases, methylation-dependent repressor binding or inhibition of transcription factor binding could be demonstrated (5Tate P.H. Bird A.P. Curr. Opin. Genet. Dev. 1993; 3: 226-231Crossref PubMed Scopus (597) Google Scholar,26Boyes J. Bird A. Cell. 1991; 64: 1123-1134Abstract Full Text PDF PubMed Scopus (581) Google Scholar, 27Jost J.P. Saluz H.P. Pawlak A. Nucleic Acids Res. 1991; 19: 5771-5775Crossref PubMed Scopus (40) Google Scholar, 28Saluz H.P. Feavers I.M. Jiricny J. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6697-6700Crossref PubMed Scopus (39) Google Scholar, 29Robertson K.D. Hayward S.D. Ling P.D. Samid D. Ambinder R.F. Mol. Cell. Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (109) Google Scholar). For the mouse lysozyme gene, it was shown that within the core part of the downstream enhancer the heterotetrameric transcription factor GABP is required for full enhancer activity (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). In addition, it was demonstrated that the methylation of a single CpG dinucleotide within the enhancer core region inhibits in vitro DNA binding of GABP (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). A similar sensitivity in methylated DNA binding was demonstrated for GABP and other ETS proteins in the context of binding sites that differ from the mouse lysozyme GABP binding site (30Yokomori N. Kobayashi R. Moore R. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1995; 15: 5355-5362Crossref PubMed Scopus (81) Google Scholar, 31Gaston K. Fried M. Gene ( Amst. ). 1995; 157: 257-259Crossref PubMed Scopus (25) Google Scholar, 32Desmet C. Debacker O. Faraoni I. Lurquin C. Brasseur F. Boon T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7149-7153Crossref PubMed Scopus (464) Google Scholar). This suggests that demethylation of this enhancer is a prerequisite for enhancer activity and therefore, for transcription. Here we show that the cis-element-dependent demethylation can be observed independent of the type of the neighboring DNA or promoter context. The 224-bp fragment HS3.2 can be stably transfected either fused to a eukaryotic reporter gene or to a prokaryotic vector and will be demethylated in both cases. Therefore, the lysozyme downstream enhancer confers at least two functions. One function is to mediate the demethylation during differentiation, and another function is to activate gene transcription by the bound enhancer factors. A similar dual activity has been demonstrated for the α-actin promoter and the κ enhancer (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Our transfection data suggest that the MLDE fragment within the downstream enhancer is not sufficient to mediate demethylation. This fragment harbors the single HpaII site and binds the heterotetrameric GABP factor in the absence of methylation. Here we have shown that even a single methyl group on the lower strand is sufficient to inhibit GABP binding, whereas the hemimethylated upper strand does not interfere with binding. Since GABP binding is impaired even by hemimethylated DNA, this factor cannot be the cause for demethylation. This is in contrast to the mechanism observed in the context of Sp1 binding (15Macleod D. Charlton J. Mullins J. Bird A.P. Genes Dev. 1994; 8: 2282-2292Crossref PubMed Scopus (514) Google Scholar). Sp1 is able to bind methylated DNA and after replication prevents the maintenance methylase from modifying the newly synthesized DNA strand. Such a Sp1-like activity could have been envisioned to be utilized by an unknown factor that has been found to bind to the GABP response element even in the case of a fully methylated DNA (4Nickel J. Short M.L. Schmitz A. Eggert M. Renkawitz R. Nucleic Acids Res. 1995; 23: 4785-4792Crossref PubMed Scopus (33) Google Scholar). If this factor would indeed play such a role, this function would not be sufficient for demethylation, since we have shown that the methylated MLDE fragment is not demethylated (Fig. 6). Within the group of fragments mediating demethylation, there seems to be a bias in demethylation efficiency: all of the fragments extending up to the position 224 (Fig. 7; i.e. fragments HS3/6, HS3.2, 82–224, 146–224) mediate 90–100% demethylation. In contrast, fragments with a downstream deletion (Fig. 7;i.e. fragments 1–181, 1–163) mediate demethylation for only 70–80% of the molecules. Nevertheless, two different sets of fragments, overlapping in the GABP site only, confer demethylation. Computer analysis of the sequences flanking the GABP site did not reveal any consensus in common, which otherwise might have been an indication for trans-acting proteins required for demethylation. In addition, in vitro footprinting showed only one protected region in addition to the GABP site (Fig. 3 C). One of the minimal regions required and sufficient for demethylation is only 78 bp in length (HS3.2(146–224)) (Fig. 7). None of the sequences analyzed for conferring demethylation in the other model systems could be delineated to such a small fragment. For both the α-actin promoter and the κ enhancer, DNA fragments of 800 bp to more than 1000 bp in length were required for demethylation (7Paroush Z. Keshet I. Yisraeli J. Cedar H. Cell. 1990; 63: 1229-1237Abstract Full Text PDF PubMed Scopus (128) Google Scholar,8Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Despite the complexity of the κ enhancer it could be shown that the absence of a single enhancer factor (nuclear factor-κB) leads to the failure of this fragment to undergo selective demethylation (33Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergman Y. Nat. Genet. 1996; 13: 435-441Crossref PubMed Scopus (208) Google Scholar). Previous results on in vitro demethylation of hemimethylated DNA by chicken embryonic extracts suggested a glycosylase activity (34Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4684-4688Crossref PubMed Scopus (146) Google Scholar,35Jost J.P. Siegmann M. Sun L. Leung R. J. Biol. Chem. 1995; 270: 9734-9739Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Ras-induced overall demethylation in mouse embryonal P19 cells could be followed in vitro as well (36Szyf M. Theberge J. Bozovic V. J. Biol. Chem. 1995; 270: 12690-12696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In contrast to embryonic cells and tissues, demethylation specific for differentiation and for particular sites may utilize a different mechanism. Recent achievements with in vitro demethylation showed that RNA is involved in sequence-specific demethylation (9Weiss A. Keshet I. Razin A. Cedar H. Cell. 1996; 86: 709-718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Nevertheless, these authors showed that the tissue specificity of the demethylation reaction involves proteins as well. Taken together, these and our results demonstrate that the demethylation activity is mediated via enhancer elements and may be organized in a manner similar to that of the enhancer elements; that is, specific modules within the enhancer regions are either sufficient by themselves to mediate demethylation or have to act in combination with other modules. We thank M. Cross for technical hints in macrophage transfections, H. Wahn for excellent technical assistance, and A. Baniahmad for critically reading the manuscript.