MINIREVIEW
A Role for DNA Methylation in Vertebrate Gene Expression?
Jeannine S. Strobl
Methyl-5-cytosine (m5C) represents 0.75-2.4 mol% of all vertebrate DNA bases and is localized within transcriptionally inactive DNA (1, 2). This review addresses the distribution and formation of m5C in vertebrate DNA in regard to its functional role in the control of gene expression. Early studies established that transcriptionally inactive mouse satellite DNA contains two to four times more m5C than bulk DNA and is enriched in methylated CpT, TpC, and CpC dinucleotides. The distribution of m5C within gene coding sequences different than the generalized, heavy methylation of satellite DNA. Methyl-5-cytosine is concentrated in the CpG dinucleotides of transcriptionally inactive genes. Transcriptionally active genes are depleted in m5C and are associated with methylation-free CpG islands, DNA sequences 500-2000 base pairs in length with an overall G + C content of 50-70% and a near random frequency of CpG dinucleotides. CpG islands are now known to occur in housekeeping genes that are expressed in all cell types and in genes whose expression is limited to specialized cell types (3). Methylation-free CpG islands frequently predict the 5'-end of transcriptionally active DNA sequences. Thus in many instances, tissue-specific heterogeneity in the methylation status of CpG islands can account for the association between m5C and tissue-specific gene repression established by the traditional method of digestion of genomic DNA with methylation-sensitive restriction endonucleases, followed by Southern hybridization analyses with genespecific probes. It is generally accepted that m5C plays a role in determining transcriptionally inactive chromatin structure^) though no experiments define precisely how. Gene transfer studies show eucaryotic genes with m5C completely substituted for cytosine undergo stable integration into the genome as DNasel-insensitive chromatin. These same genes when unmethylated integrate into chromatin structures possessing both DNasel-sensitive and -hypersensitive sites (4). No gross differences in nucleosome structure have been detected by in vitro
reconstitution experiments with methylated and unmethylated plasmid DNA (5). Nevertheless, reconstitution of chicken core histones with Herpes simplex virus thymidine kinase DNA methylated at /-/pall sites results in histone octamers which are transcriptionally inactive upon microinjection into mammalian cells. When microinjected alone the same methylated DNA is active for approximately 8 h, until incorporated into chromatin. These observations suggest that m5C can direct the incorporation of viral DNA into transcriptionally inactive chromatin and that core histone-DNA interactions are involved (6). Methylated DNA is also enriched in histone H1 in vivo (7). Additional proteins may play a role in chromatin formation with methylated DNA. For example, a methyl-CpG binding protein that binds preferentially to DNA sequences containing clustered m5CpG dinucleotides recently has been identified in mouse tissues (8). Finally, extensive replacement of cytosine by m5C may contribute to the regional inactivation of chromatin directly via local elevations in the Tm of the DNA helix or by stabilization of H- and Z-form DNA (1, 2,9). Selective sites of m5C located 5' or 3' of eucaryotic structural gene sequences can play a dominant role in the control of gene expression in vivo (10). Furthermore, in converse experiments using the blocker of methylation 5-azacytidine, selective sites of demethylation required for gene reexpression have been identified (11). Gene regulation by these methylation events is unlikely to operate by mechanisms that, influence chromatin structure in general. There is more evidence that sitespecific methylation of cytosine plays a role in regulation by influencing the interactions between transcription factors and cis-active regulatory regions of DNA. Methyl-5-cytosine projects a methyl group into the major groove of B-form DNA, and this rather subtle change in DNA sequence is sufficient to interfere with regulatory protein binding to DNA. Examples where methylation blocks the site-specific binding of transcription factors include sites in the far upstream region of the rat tyrosine aminotransferase gene (12) and in the cAMPresponse element of the human glycoprotein a-subunit (13).
0888-8809/90/0181 -0183$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
The inhibition of transcription factor binding to DNA 181
Downloaded from https://academic.oup.com/mend/article-abstract/4/2/181/2713983 by guest on 29 January 2019
Department of Pharmacology and Toxicology West Virginia University Health Sciences Center Morgantown, West Virginia 26506
MOL ENDO-1990 182
present in a nonhistone type 4 preparation. In contrast, methylation of this CpG site results in the binding of a distinctly different protein, providing the first association between a methylation-dependent binding protein and transcriptional repression (18). Thus, studies of the avian vitellogenin II gene promoter region indicate that transcription activity related differences in m5C at specific CpG dinucleotides is the result of a highly dynamic situation involving the binding and dissociation of multiple transcription factors. The generality of these observations has not been established, possibly due to the technical demands of genomic sequencing. Saluz and Jost (19) have recently published a modification of the genomic sequencing technique that employs a Tagl polymerase amplification of the DNA chemical cleavage products in the presence of radiolabeled oligonucleotide primers and gel electrophoresis. The relative ease of this method may encourage more widespread use of genomic footprinting and sequencing. The underlying mechanisms responsible for changes in DNA methylation states may be approached biochemically through studies of DNA methyltransferases. These enzymes have been isolated from a wide variety of mammalian sources and exhibit remarkably similar physical and enzymatic properties (20). All mammalian DNA methyltransferases studied to date catalyze both de novo DNA methylation, the addition of methyl groups to previously unmethylated single-stranded and doublestranded DNA, and maintenance DNA methylation reactions, in which methyl groups are added to the unmethylated strand of a hemi-methylated doublestranded DNA template. Maintenance and de novo methylation occur under similar reaction conditions, however reaction rates differ with the configuration of the DNA template. Rates of maintenance methylation proceed 10-100 times faster than de novo methylation on a double-stranded hemi-methylated DNA templates, providing a rational explanation for the rapid maintenance methylation of newly synthesized DNA at the DNA replication fork in vivo. In contrast, de novo methylation occurs preferentially on single-stranded DNA templates, consistent with a model in which local denaturation of the helix could promote the de novo reaction. De novo methylation also exhibits some DNA sequence specificity in vitro, and can generate reproducible, sequence-specific sites of hemi-methylated DNA (21). Biochemical analyses have not provided any insight into how tissue-specific patterns of DNA methylation are established. Mammalian DNA methyltransferases purified from different tissues exhibit indistinguishable physical properties and DNA template specificities in vitro, suggesting that additional cellular components are required to establish the specific patterns of DNA methylation observed in vivo. Modulation of the in vivo activity of DNA methyltransferase by DNA conformation is one possibility (22). Evidence for accessory proteins that regulate the activity of DNA methyltransferase in vivo has also been reported (23).
Downloaded from https://academic.oup.com/mend/article-abstract/4/2/181/2713983 by guest on 29 January 2019
by m5C is not universal, and for some housekeeping genes binding of constitutive transcription factors may prevent DNA methylation and create methylation-free CpG islands. In particular, Sp1 is a constitutive transcription factor that binds to a consensus sequence (GC box) 5' of several eucaryotic housekeeping genes. Methylation of the central CpG dinucleotide within the Sp1 GC box has no effect on either Sp1 binding or in vitro transcription (14). Conversely, sequence-specific m5C-protein recognition events also occur. The methylated DNA binding protein isolated from human placenta binds a consensus sequence that includes m5C (or T) but not C (15). The biological consequences of methylated DNA binding protein binding are unknown. Activation of RNA transcription during tissue differentiation or hormonal stimulation is frequently accompanied by demethylation at specific sites within the 5'or 3'-flanking region of genes illustrating that dynamic changes in DNA methylation take place. Genomic sequencing has been used to ascertain complete, strandspecific patterns of DNA methylation as they occur in vivo. Genomic sequencing is well suited to the analysis of dynamic changes in DNA methylation in vivo, and when used in concert with the dimethyl sulfate genomic footprinting technique, can identify methylation-dependent alterations in DNA-protein interactions. Sequencing of avian vitellogenin II has revealed that estradiol activation of vitellogenin gene transcription is accompanied by three CpG demethylation events near the consensus estrogen receptor binding site, approximately 500 base pairs 5' of the transcription initiation site (16). Demethylation proceeds through the generation of three hemimethylated sites within the coding strand which appear simultaneously with the activation of mRNA synthesis. Demethylation of the noncoding strand follows approximately 24 h later, to yield three completely unmethylated CpG sites. The generation of these sites proceeds in the absence of DNA synthesis and in some instances, in the absence of vitellogenin mRNA synthesis as well, suggesting that the binding of the estrogen-receptor complex to its consensus sequence initiates some active mechanism for DNA demethylation. Earlier evidence for a DNA synthesis-independent demethylating activity in mouse erythroleukemia cells appeared in 1982 (17), but it is still a process that is poorly understood. A fourth CpG dinucleotide within the avian vitellogenin II gene promoter region is maintained in a hemimethylated state before gene activation; it undergoes complete demethylation beginning 24 h after gene activation. The existence of hemi-methylated sites in vivo can only be demonstrated using genomic sequencing. Interestingly, this data is inconsistent with conventional models of DNA methyltransferase which in vitro, preferentially methylates hemi-methylated DNA substrates. Tissue-specific and estrogen-dependent expression of vitellogenin II in the avian liver is also associated in vivo with a symmetrical demethylation of a CpG site +10 relative to the transcription initiation site. The unmethylated, transcriptionally active +10 region of the vitellogenin II gene binds to at least two ubiquitous proteins
Vol 4 No. 2
MINIREVIEW
To conclude, evidence has been presented that DNA methylation plays multiple roles in the control of vertebrate gene expression by modulating protein-DNA interactions. Histone interactions with heavily methylated DNA facilitate the formation of transcriptionally inactive nucleosomes, and limited, site-selective methylation events interfere with the binding of specific transcription factors to gene promoters. Genomic sequencing of the avian vitellogenin II and tyrosine aminotransferase genes has provided important information on specific and dynamic changes in DNA methylation that impinge upon transcription factor binding to DNA. More genes need to be analyzed at this level of detail. Finally, of increasing importance is the elucidation of regulatory mechanisms controlling DNA methyltransferase and DNA demethylating activities.
8.
9.
10. 11.
12. 13.
14.
15.
16.
17. 18.
Acknowledgment Address requests for reprints to: Jeannine S. Strobl, Department of Pharmacology and Toxicology, West Virginia University Health Sciences Center, Morgantown, West Virginia 26506.
19.
REFERENCES 1. Razin A, Cedar H, Riggs AD (eds) 1984 DNA Methylation, Biochemistry and Biological Significance. Springer Verlag, New York 2. Adams RLP, Burdon RH 1985 Molecular Biology of DNA Methylation. Springer-Verlag, New York 3. Gardiner-Garden M, Frommer M 1987 CpG islands in vertebrate genomes. J Mol Biol 196:261-282 4. Keshet I, Lieman-Hurwitz J, Cedar H 1986 DNA methylation affects the formation of active chromatin. Cell 44:535-543 5. Drew HR, McCall MJ 1987 Structural analysis of a reconstituted DNA containing three histone octamers and histone H5. J Mol Biol 197:485-511 6. Buschhausen G, Wittig B, Graessmann M, Graesmann A 1987 Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci 84:1177-1181 7. Ball DJ, Gross DS, Garrard WT 1983 5-Methylcytosine is
20.
21.
22. 23.
24.
localized in nucleosomes that contain histone H1. Proc Natl Acad Sci 80:5490-5494 Meehan RR, Lewis JD, McKay S, Kleiner EL, Bird AP 1989 Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58:499-507 Lee JS, Woodsworth ML, Latimer LJP, Morgan AR 1984 Poly(pyrimidine)-poly-(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. Nucleic Acids Res 12:6603-6614 Gaido ML, Strobl JS 1989 Inhibition of rat growth hormone promoter activity by site-specific DNA methylation. Biochim Biophys Acta 1008:234-242 Hansen RS, Ellis NA, Gartler SM 1988 Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression. Mol Cell Biol 8:4682-4699 Becker PB, Ruppert S, Schutz G 1987 Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors. Cell 51:435-443 Tguchi-Ariga SMM and Schaffner W 1989 CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev 3:612-619 Holler M, Westin G, Jiricny J, Schaffner W 1988 Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated. Genes Dev 2:1127-1135 Zhang X-Y, Supakar P, Khan R, Ehrlich K, Ehrlich M 1989 Related sites in human and herpes virus DNA recognized by methylated DNA-binding protein from human placenta. Nucleic Acids Res 17:1459-1474 Saluz HP, Jiricny J, Jost JP 1986 Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc Natl Acad Sci 83:7167-7171 Gjerset RA, Martin DW 1982 Presence of a DNA demethylating activity in the nucleus of murine erythroleukemic cells. J Biol Chem 257:8581-8583 Saluz HP, Feavers IM, Jiricny J, Jost JP 1988 Genomic sequencing and in vivo footprinting of an expressionspecific DNasel-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA. Proc Natl Acad Sci 85:6697-6700 Saluz H, Jost JP 1989 A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes. Proc Natl Acad Sci 86:2602-2606 Pfeifer GP, Kohlmaier L, Tomassetti A, Schleicher R, Follman H, Pfohl-Leszkowicz A, Dirheimer G, Drahovsky D 1989 Polypeptide composition and an immunological analysis of DNA methyltransferases from different species. Arch Biochem Biophys 268:388-392 Bolden AH, Ward CA, Nalin CM, Weissbach A 1986 The primary DNA sequence determines in vitro methylation by mammalian DNA methyltransferases. Prog Nucleic Acids Res 33:231-250 Bestor T1987 Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase. Nucleic Acids Res 15:3835-3843 Tomassetti A, Driever PH, Pfeifer GP, Drahovsky D 1988 Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase. Biochim Biophys Acta 951:201-212 Bestor T, Laudano A, Mattaliano R, Ingram V 1988 Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. J Mol Biol 203:971-983
Downloaded from https://academic.oup.com/mend/article-abstract/4/2/181/2713983 by guest on 29 January 2019
Molecular cloning and DNA sequence analyses will provide the data necessary to detect tissue-specific differences if any, in mammalian DNA methyltransferase genes. Bestor et al. (24) have cloned and sequenced the first vertebrate DNA methyltransferase from mouse erythroleukemia cells, and as predicted, all mouse tissues tested express cross-hybridizing mRNAs of an identical size by Northern gel analyses. Interestingly, the amino terminal domain of the mouse DNA methyltransferase gene encodes a cysteine-rich region that theoretically can adopt a metal binding configuration reminiscent of that proposed for vertebrate steroid hormone receptors and shown in certain yeast regulatory proteins. The authors speculate that such a metal binding domain could participate in protein-protein interactions in vivo to direct the formation of tissue-specific patterns of DNA methylation.
183
A Role for DNA Methylation in Vertebrate Gene Expression?
Jeannine S. Strobl
Methyl-5-cytosine (m5C) represents 0.75-2.4 mol% of all vertebrate DNA bases and is localized within transcriptionally inactive DNA (1, 2). This review addresses the distribution and formation of m5C in vertebrate DNA in regard to its functional role in the control of gene expression. Early studies established that transcriptionally inactive mouse satellite DNA contains two to four times more m5C than bulk DNA and is enriched in methylated CpT, TpC, and CpC dinucleotides. The distribution of m5C within gene coding sequences different than the generalized, heavy methylation of satellite DNA. Methyl-5-cytosine is concentrated in the CpG dinucleotides of transcriptionally inactive genes. Transcriptionally active genes are depleted in m5C and are associated with methylation-free CpG islands, DNA sequences 500-2000 base pairs in length with an overall G + C content of 50-70% and a near random frequency of CpG dinucleotides. CpG islands are now known to occur in housekeeping genes that are expressed in all cell types and in genes whose expression is limited to specialized cell types (3). Methylation-free CpG islands frequently predict the 5'-end of transcriptionally active DNA sequences. Thus in many instances, tissue-specific heterogeneity in the methylation status of CpG islands can account for the association between m5C and tissue-specific gene repression established by the traditional method of digestion of genomic DNA with methylation-sensitive restriction endonucleases, followed by Southern hybridization analyses with genespecific probes. It is generally accepted that m5C plays a role in determining transcriptionally inactive chromatin structure^) though no experiments define precisely how. Gene transfer studies show eucaryotic genes with m5C completely substituted for cytosine undergo stable integration into the genome as DNasel-insensitive chromatin. These same genes when unmethylated integrate into chromatin structures possessing both DNasel-sensitive and -hypersensitive sites (4). No gross differences in nucleosome structure have been detected by in vitro
reconstitution experiments with methylated and unmethylated plasmid DNA (5). Nevertheless, reconstitution of chicken core histones with Herpes simplex virus thymidine kinase DNA methylated at /-/pall sites results in histone octamers which are transcriptionally inactive upon microinjection into mammalian cells. When microinjected alone the same methylated DNA is active for approximately 8 h, until incorporated into chromatin. These observations suggest that m5C can direct the incorporation of viral DNA into transcriptionally inactive chromatin and that core histone-DNA interactions are involved (6). Methylated DNA is also enriched in histone H1 in vivo (7). Additional proteins may play a role in chromatin formation with methylated DNA. For example, a methyl-CpG binding protein that binds preferentially to DNA sequences containing clustered m5CpG dinucleotides recently has been identified in mouse tissues (8). Finally, extensive replacement of cytosine by m5C may contribute to the regional inactivation of chromatin directly via local elevations in the Tm of the DNA helix or by stabilization of H- and Z-form DNA (1, 2,9). Selective sites of m5C located 5' or 3' of eucaryotic structural gene sequences can play a dominant role in the control of gene expression in vivo (10). Furthermore, in converse experiments using the blocker of methylation 5-azacytidine, selective sites of demethylation required for gene reexpression have been identified (11). Gene regulation by these methylation events is unlikely to operate by mechanisms that, influence chromatin structure in general. There is more evidence that sitespecific methylation of cytosine plays a role in regulation by influencing the interactions between transcription factors and cis-active regulatory regions of DNA. Methyl-5-cytosine projects a methyl group into the major groove of B-form DNA, and this rather subtle change in DNA sequence is sufficient to interfere with regulatory protein binding to DNA. Examples where methylation blocks the site-specific binding of transcription factors include sites in the far upstream region of the rat tyrosine aminotransferase gene (12) and in the cAMPresponse element of the human glycoprotein a-subunit (13).
0888-8809/90/0181 -0183$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
The inhibition of transcription factor binding to DNA 181
Downloaded from https://academic.oup.com/mend/article-abstract/4/2/181/2713983 by guest on 29 January 2019
Department of Pharmacology and Toxicology West Virginia University Health Sciences Center Morgantown, West Virginia 26506
MOL ENDO-1990 182
present in a nonhistone type 4 preparation. In contrast, methylation of this CpG site results in the binding of a distinctly different protein, providing the first association between a methylation-dependent binding protein and transcriptional repression (18). Thus, studies of the avian vitellogenin II gene promoter region indicate that transcription activity related differences in m5C at specific CpG dinucleotides is the result of a highly dynamic situation involving the binding and dissociation of multiple transcription factors. The generality of these observations has not been established, possibly due to the technical demands of genomic sequencing. Saluz and Jost (19) have recently published a modification of the genomic sequencing technique that employs a Tagl polymerase amplification of the DNA chemical cleavage products in the presence of radiolabeled oligonucleotide primers and gel electrophoresis. The relative ease of this method may encourage more widespread use of genomic footprinting and sequencing. The underlying mechanisms responsible for changes in DNA methylation states may be approached biochemically through studies of DNA methyltransferases. These enzymes have been isolated from a wide variety of mammalian sources and exhibit remarkably similar physical and enzymatic properties (20). All mammalian DNA methyltransferases studied to date catalyze both de novo DNA methylation, the addition of methyl groups to previously unmethylated single-stranded and doublestranded DNA, and maintenance DNA methylation reactions, in which methyl groups are added to the unmethylated strand of a hemi-methylated doublestranded DNA template. Maintenance and de novo methylation occur under similar reaction conditions, however reaction rates differ with the configuration of the DNA template. Rates of maintenance methylation proceed 10-100 times faster than de novo methylation on a double-stranded hemi-methylated DNA templates, providing a rational explanation for the rapid maintenance methylation of newly synthesized DNA at the DNA replication fork in vivo. In contrast, de novo methylation occurs preferentially on single-stranded DNA templates, consistent with a model in which local denaturation of the helix could promote the de novo reaction. De novo methylation also exhibits some DNA sequence specificity in vitro, and can generate reproducible, sequence-specific sites of hemi-methylated DNA (21). Biochemical analyses have not provided any insight into how tissue-specific patterns of DNA methylation are established. Mammalian DNA methyltransferases purified from different tissues exhibit indistinguishable physical properties and DNA template specificities in vitro, suggesting that additional cellular components are required to establish the specific patterns of DNA methylation observed in vivo. Modulation of the in vivo activity of DNA methyltransferase by DNA conformation is one possibility (22). Evidence for accessory proteins that regulate the activity of DNA methyltransferase in vivo has also been reported (23).
Downloaded from https://academic.oup.com/mend/article-abstract/4/2/181/2713983 by guest on 29 January 2019
by m5C is not universal, and for some housekeeping genes binding of constitutive transcription factors may prevent DNA methylation and create methylation-free CpG islands. In particular, Sp1 is a constitutive transcription factor that binds to a consensus sequence (GC box) 5' of several eucaryotic housekeeping genes. Methylation of the central CpG dinucleotide within the Sp1 GC box has no effect on either Sp1 binding or in vitro transcription (14). Conversely, sequence-specific m5C-protein recognition events also occur. The methylated DNA binding protein isolated from human placenta binds a consensus sequence that includes m5C (or T) but not C (15). The biological consequences of methylated DNA binding protein binding are unknown. Activation of RNA transcription during tissue differentiation or hormonal stimulation is frequently accompanied by demethylation at specific sites within the 5'or 3'-flanking region of genes illustrating that dynamic changes in DNA methylation take place. Genomic sequencing has been used to ascertain complete, strandspecific patterns of DNA methylation as they occur in vivo. Genomic sequencing is well suited to the analysis of dynamic changes in DNA methylation in vivo, and when used in concert with the dimethyl sulfate genomic footprinting technique, can identify methylation-dependent alterations in DNA-protein interactions. Sequencing of avian vitellogenin II has revealed that estradiol activation of vitellogenin gene transcription is accompanied by three CpG demethylation events near the consensus estrogen receptor binding site, approximately 500 base pairs 5' of the transcription initiation site (16). Demethylation proceeds through the generation of three hemimethylated sites within the coding strand which appear simultaneously with the activation of mRNA synthesis. Demethylation of the noncoding strand follows approximately 24 h later, to yield three completely unmethylated CpG sites. The generation of these sites proceeds in the absence of DNA synthesis and in some instances, in the absence of vitellogenin mRNA synthesis as well, suggesting that the binding of the estrogen-receptor complex to its consensus sequence initiates some active mechanism for DNA demethylation. Earlier evidence for a DNA synthesis-independent demethylating activity in mouse erythroleukemia cells appeared in 1982 (17), but it is still a process that is poorly understood. A fourth CpG dinucleotide within the avian vitellogenin II gene promoter region is maintained in a hemimethylated state before gene activation; it undergoes complete demethylation beginning 24 h after gene activation. The existence of hemi-methylated sites in vivo can only be demonstrated using genomic sequencing. Interestingly, this data is inconsistent with conventional models of DNA methyltransferase which in vitro, preferentially methylates hemi-methylated DNA substrates. Tissue-specific and estrogen-dependent expression of vitellogenin II in the avian liver is also associated in vivo with a symmetrical demethylation of a CpG site +10 relative to the transcription initiation site. The unmethylated, transcriptionally active +10 region of the vitellogenin II gene binds to at least two ubiquitous proteins
Vol 4 No. 2
MINIREVIEW
To conclude, evidence has been presented that DNA methylation plays multiple roles in the control of vertebrate gene expression by modulating protein-DNA interactions. Histone interactions with heavily methylated DNA facilitate the formation of transcriptionally inactive nucleosomes, and limited, site-selective methylation events interfere with the binding of specific transcription factors to gene promoters. Genomic sequencing of the avian vitellogenin II and tyrosine aminotransferase genes has provided important information on specific and dynamic changes in DNA methylation that impinge upon transcription factor binding to DNA. More genes need to be analyzed at this level of detail. Finally, of increasing importance is the elucidation of regulatory mechanisms controlling DNA methyltransferase and DNA demethylating activities.
8.
9.
10. 11.
12. 13.
14.
15.
16.
17. 18.
Acknowledgment Address requests for reprints to: Jeannine S. Strobl, Department of Pharmacology and Toxicology, West Virginia University Health Sciences Center, Morgantown, West Virginia 26506.
19.
REFERENCES 1. Razin A, Cedar H, Riggs AD (eds) 1984 DNA Methylation, Biochemistry and Biological Significance. Springer Verlag, New York 2. Adams RLP, Burdon RH 1985 Molecular Biology of DNA Methylation. Springer-Verlag, New York 3. Gardiner-Garden M, Frommer M 1987 CpG islands in vertebrate genomes. J Mol Biol 196:261-282 4. Keshet I, Lieman-Hurwitz J, Cedar H 1986 DNA methylation affects the formation of active chromatin. Cell 44:535-543 5. Drew HR, McCall MJ 1987 Structural analysis of a reconstituted DNA containing three histone octamers and histone H5. J Mol Biol 197:485-511 6. Buschhausen G, Wittig B, Graessmann M, Graesmann A 1987 Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci 84:1177-1181 7. Ball DJ, Gross DS, Garrard WT 1983 5-Methylcytosine is
20.
21.
22. 23.
24.
localized in nucleosomes that contain histone H1. Proc Natl Acad Sci 80:5490-5494 Meehan RR, Lewis JD, McKay S, Kleiner EL, Bird AP 1989 Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58:499-507 Lee JS, Woodsworth ML, Latimer LJP, Morgan AR 1984 Poly(pyrimidine)-poly-(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. Nucleic Acids Res 12:6603-6614 Gaido ML, Strobl JS 1989 Inhibition of rat growth hormone promoter activity by site-specific DNA methylation. Biochim Biophys Acta 1008:234-242 Hansen RS, Ellis NA, Gartler SM 1988 Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression. Mol Cell Biol 8:4682-4699 Becker PB, Ruppert S, Schutz G 1987 Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors. Cell 51:435-443 Tguchi-Ariga SMM and Schaffner W 1989 CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev 3:612-619 Holler M, Westin G, Jiricny J, Schaffner W 1988 Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated. Genes Dev 2:1127-1135 Zhang X-Y, Supakar P, Khan R, Ehrlich K, Ehrlich M 1989 Related sites in human and herpes virus DNA recognized by methylated DNA-binding protein from human placenta. Nucleic Acids Res 17:1459-1474 Saluz HP, Jiricny J, Jost JP 1986 Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc Natl Acad Sci 83:7167-7171 Gjerset RA, Martin DW 1982 Presence of a DNA demethylating activity in the nucleus of murine erythroleukemic cells. J Biol Chem 257:8581-8583 Saluz HP, Feavers IM, Jiricny J, Jost JP 1988 Genomic sequencing and in vivo footprinting of an expressionspecific DNasel-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA. Proc Natl Acad Sci 85:6697-6700 Saluz H, Jost JP 1989 A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes. Proc Natl Acad Sci 86:2602-2606 Pfeifer GP, Kohlmaier L, Tomassetti A, Schleicher R, Follman H, Pfohl-Leszkowicz A, Dirheimer G, Drahovsky D 1989 Polypeptide composition and an immunological analysis of DNA methyltransferases from different species. Arch Biochem Biophys 268:388-392 Bolden AH, Ward CA, Nalin CM, Weissbach A 1986 The primary DNA sequence determines in vitro methylation by mammalian DNA methyltransferases. Prog Nucleic Acids Res 33:231-250 Bestor T1987 Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase. Nucleic Acids Res 15:3835-3843 Tomassetti A, Driever PH, Pfeifer GP, Drahovsky D 1988 Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase. Biochim Biophys Acta 951:201-212 Bestor T, Laudano A, Mattaliano R, Ingram V 1988 Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. J Mol Biol 203:971-983
Downloaded from https://academic.oup.com/mend/article-abstract/4/2/181/2713983 by guest on 29 January 2019
Molecular cloning and DNA sequence analyses will provide the data necessary to detect tissue-specific differences if any, in mammalian DNA methyltransferase genes. Bestor et al. (24) have cloned and sequenced the first vertebrate DNA methyltransferase from mouse erythroleukemia cells, and as predicted, all mouse tissues tested express cross-hybridizing mRNAs of an identical size by Northern gel analyses. Interestingly, the amino terminal domain of the mouse DNA methyltransferase gene encodes a cysteine-rich region that theoretically can adopt a metal binding configuration reminiscent of that proposed for vertebrate steroid hormone receptors and shown in certain yeast regulatory proteins. The authors speculate that such a metal binding domain could participate in protein-protein interactions in vivo to direct the formation of tissue-specific patterns of DNA methylation.
183
