Periodic DNA Methylation in Maize Nucleosomes and Demethylation by Environmental Stress
2002; Elsevier BV; Volume: 277; Issue: 40 Linguagem: Inglês
10.1074/jbc.m204050200
ISSN1083-351X
AutoresNicolas Steward, Mikako Ito, Yube Yamaguchi, Nozomu Koizumi, Hiroshi Sano,
Tópico(s)Genomics and Chromatin Dynamics
ResumoWhen maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990)Mol. Gen. Genet. 220, 441–447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence. When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990)Mol. Gen. Genet. 220, 441–447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence. 5-methylcytosine 4′,6-diamidino-2-phenylindoleDAPI DNA of higher eukaryotes is characterized by the presence of 5-methylcytosine (m5C)1 nucleotides, comprising up to 30% of the total cytosines. In vertebrates, m5C is located almost exclusively in CpG, whereas in plants it occurs in both CpG and CpNpG (1Gruenbaum Y. Naveh-Many T. Cedar H. Razin A. Nature. 1981; 292: 860-862Crossref PubMed Scopus (593) Google Scholar). The distribution within the genome is non-random and varies depending on the tissue and the developmental stages. The physiological function of m5C is essentially to silence gene expression, which is important for host DNA defenses against incorporation of "parasitic" DNA (2Yoder J.A. Walsh C.P. Bestor T.H. Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1501) Google Scholar, 3Colot V. Rossignol J.L. Bioessays. 1999; 21: 402-411Crossref PubMed Scopus (271) Google Scholar). Two systems are involved: one directly blocks transcriptional machinery attached to promoter regions of genes by altering DNA structure and the other indirectly interferes with transcription by influencing nucleosome conformation and stability (4Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1541) Google Scholar). Recent studies indicated the latter to occur frequently, as shown by various disorders due to abnormal chromosome structures that are caused by defective DNA methylation (5Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (869) Google Scholar). One hallmark of cancer cells is local hypermethylation and global hypomethylation of chromosomal DNA (6Smith S.S. J. Mol. Biol. 2000; 302: 1-7Crossref PubMed Scopus (42) Google Scholar). Abnormal methylation in the promoter regions of regulatory genes may indeed result in cancer development (7Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1513) Google Scholar). Using antisense inhibition of DNMT1, a maintenance type DNA methyltransferase, about 10% of all genes in cultured mouse cells were found to be activated (8Jackson-Grusby L. Beard C. Possemat R. Tudoe M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (562) Google Scholar). These observations confirmed that DNA methylation functions as a global repressor of gene expression (9Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5330) Google Scholar). The reverse case, i.e. global demethylation has also been inferred to be critical during embryogenesis in mammals (9Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5330) Google Scholar, 10Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2395) Google Scholar). What controls the on-off switch for DNA methylation, however, is still largely unclear. While a set of DNA methyltransferases has been identified in various organisms, including plants, the presence of DNA demethylases is controversial (7Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1513) Google Scholar). Among several candidate proteins, 5-methylcytosine DNA glycosylase was shown to induce genome-wide demethylation upon transfection into mouse myoblasts (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar). Whatever the mechanism may be, reprogramming of DNA methylation appears to be fundamental in normal development (10Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2395) Google Scholar).Epigenetic inheritance is defined as change in gene expression without base sequence alteration (12Riggs A.D. Porter T.N. Russo V.E.A. Martienssen R.A. Riggs A.D. Epigenetic Mechanisms of Gene Regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 29-45Google Scholar). This typically occurs during somatic cell differentiation, in which the clonal expansion of a single cell leads to a diversity of cell types (13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar). Such a cellular inheritance is common during ontogeny but is usually erased before gametes are produced (14Reik W. Walter J. Nat. Rev. Genet. 2001; 2: 21-32Crossref PubMed Scopus (1806) Google Scholar). In plants, however, it has long been known that epigenetically acquired traits can be sexually transmitted, as exemplified by flax (Linum) (15Smith J.M. J. Theor. Biol. 1990; 143: 41-53Crossref PubMed Scopus (72) Google Scholar). In this case, epigenetic changes were induced by external factors such as nutrients and temperature (16Durrant A. Heredity. 1962; 17: 27-61Crossref Scopus (148) Google Scholar, 17Cullis C.A. CRC Crit. Rev. Plant Sci. 1983; 1: 117-131Crossref Scopus (42) Google Scholar). Consequently, the idea has been proposed that an environmental stimulus can induce heritable chromatin modifications as an adaptive response (18Jablonka E. Lamb M.J. J. Theor. Biol. 1989; 139: 69-83Crossref PubMed Scopus (186) Google Scholar). It is established that some clonal epigenetic changes are mediated through DNA methylation (6Smith S.S. J. Mol. Biol. 2000; 302: 1-7Crossref PubMed Scopus (42) Google Scholar,13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar), but evidence is limited for involvement of the latter in inheritance of acquired characteristics (19Martienssen R.A. Colot V. Science. 2001; 293: 1070-1074Crossref PubMed Scopus (401) Google Scholar). We have previously shown that a single exposure of germinated rice seeds to the DNA demethylating agent, 5-azadeoxycytidine, induced dwarfism at maturity (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). Genomic DNA isolated from dwarf plants showed a 16% reduction in the m5C content in comparison with DNA from untreated plants. Both hypomethylation and dwarfism were transmitted to progeny for at least three generations (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). Thus, the acquired phenotype due to acquired changes in DNA methylation was heritable. However, whether or not such an epigenetic inheritance occurs under natural condition remained to be solved.In this report we provide support for the conclusion that levels of DNA methylation change in the cores of nucleosomes in maize root tissues in response to cold stress or environmental cues. This finding allowed us to speculate that DNA methylation may function as a common switch of gene expression and that naturally induced changes in DNA methylation may result in heritable epigenetic modification of gene expression.DISCUSSIONNucleosomes are organized into precise positions on DNA by signals in the sequence (31Davey C. Pennings S. Allan J. J. Mol. Biol. 1997; 267: 276-288Crossref PubMed Scopus (82) Google Scholar). One such signal has been proposed to be m5C, but the experimental evidence is controversial. For example, opposite results as to preferential binding of histone H1 to methylated DNA have been reported (32McArthur M. Thomas J.O. EMBO J. 1996; 15: 1705-1714Crossref PubMed Scopus (62) Google Scholar, 33Campoy F.J. Meehan R.R. McKay S. Nixon J. Bird A. J. Biol. Chem. 1995; 270: 26473-26481Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and currently the interaction between histones and methylated DNA is in general not considered to be specific. However, under certain circumstances, nucleosome assembly appears to depend on CpG methylation (31Davey C. Pennings S. Allan J. J. Mol. Biol. 1997; 267: 276-288Crossref PubMed Scopus (82) Google Scholar, 34Godde J.S. Kass S.U. Hirst M.C. Wolffe A.P. J. Biol. Chem. 1996; 271: 24325-24328Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This suggests a dynamic interaction between methylated DNA and proteins that constitute chromatin. Methylation of histones was shown to be essential for triggering DNA methylation in filamentous fungi, and it was suggested that propagation of DNA methylation patterns is dependent on feed-back loops between modification of chromatin proteins and DNA (35Tamaru H. Selker E.U. Nature. 2001; 414: 277-283Crossref PubMed Scopus (849) Google Scholar). Our result showing periodic oscillation of DNA methylation within the nucleosomal structure is consistent with this view and further suggests a positive role for methylation in determining the chromatin infrastructure. Our findings also substantiate occurrence of demethylation upon environmental stress.During differentiation of mammalian cells, genome-wide loss of DNA methylation occurs, for which active or passive mechanisms have been proposed (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar, 36Zhu B. Benjamin D. Zheng Y. Angliker H. Thiry S. Siegmann M. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5031-5036Crossref PubMed Scopus (69) Google Scholar). In active demethylation, m5C is enzymatically removed from DNA, and in passive demethylation, newly replicated DNA is not methylated (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar). Our results with maize indicate that genome-wide demethylation occurs, possibly through active demethylation, because chilled tissues immediately ceased DNA replication (28Steward N. Kusano T. Sano H. Nucleic Acids Res. 2000; 28: 3250-3259Crossref PubMed Scopus (123) Google Scholar). A rapid and active decrease in global DNA methylation was also observed during seed germination of Silene latifolia (37Zluvova J. Janousek B. Vyskot B. J. Exp. Bot. 2001; 52: 2265-2273Crossref PubMed Scopus (81) Google Scholar). Active demethylation is catalyzed by 5-methylcytosine DNA glycosidase (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar, 36Zhu B. Benjamin D. Zheng Y. Angliker H. Thiry S. Siegmann M. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5031-5036Crossref PubMed Scopus (69) Google Scholar), although such activity has not yet been found in plants. A specific feature of our findings was that demethylation predominantly occurred at the nucleosome core regions as revealed by direct methylation mapping. A question arises, then, as to how such a differential demethylation pattern is formed. Currently we have no explanation for this, but some clues for speculation are available. For example, nucleosome cores are proposed to be located outside the 30-nm fiber, which is composed of a unit of six nucleosomes. Linker regions are connected by histone H1 inside the fiber. This structure makes 5-methylcytosine DNA glycosylase more accessible to the core than to the linker. Alternatively, 5-methylcytosine DNA glycosylase may be selectively recruited to the core by specific protein(s) such as, for example, 5-methylcytosine binding proteins. Another distinct finding of the direct methylation mapping is ubiquitous demethylation at CpG, CpNpG and other sites, suggesting no restriction to only one strand of DNA. This means that, for remethylation, de novo methyltransferase activity is necessary. If this enzyme is absent in quiescent cells, for example, in root tissues, the methylated status can not be restored. This could account for the apparently progressive demethylation in cold-chased seedlings.While the average methylation level of total genomic DNA in untreated root tissues was ∼18%, that of ZmMI1 was 38%, 2-fold higher. This indicates a heterogeneous distribution of m5C in the genome, consistent with the histochemical observations, and suggests ZmMI1 to be located in heterochromatin. However, because demethylation occurred throughout the genome, including theZmMI1 sequence, the periodic methylation pattern found in the latter may be common to other parts of genome. If so, the occurrence of demethylation mainly in the core regions of nucleosomes could induce an alteration of chromatin structure and thus influence gene expression. Increase in ZmMI1 transcripts substantiates this hypothesis. The advantage of this feature is that expression of many genes could be simultaneously controlled. Indeed, it was estimated that plants may express as many as several hundred genes upon cold stress (38Thomashow M.F. Meyerowitz E.M. Somerville C.R. Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 807-834Google Scholar). It is attractive to speculate that plants have developed the methylation/demethylation system to simultaneously regulate a vast number of genes at once, rather than to individually regulate each gene. Alternatively, because ∼70% of the maize genome is composed of retroposon-like sequences (39Bennetzen J.L. Plant Mol. Biol. 2000; 42: 251-269Crossref PubMed Scopus (605) Google Scholar) and because demethylation has been shown to reactivate transposons (40Dennis E.S. Brettell R.I. Philos. Trans. R. Soc. Lond-Biol. Sci. 1990; 362: 217-229Google Scholar), the global demethylation possibly transcriptionally activates such sequences to translocate, resulting in secondary activation/inactivation of other genes (41Miura A. Yonebayashi S. Watanabe K. Toyama T. Shimada H. Kakutani T. Nature. 2001; 411: 212-214Crossref PubMed Scopus (465) Google Scholar). However, the reason why such demethylation occurs only in root tissues is not clear. One explanation might be that above-ground parts are equipped with a more complete self-defense system than those underground, as exemplified by the presence and absence of maintenance-type DNA methyltransferase in the respective tissues (28Steward N. Kusano T. Sano H. Nucleic Acids Res. 2000; 28: 3250-3259Crossref PubMed Scopus (123) Google Scholar).Heredity of epigenetic modification, or acquired traits, in response to environmental conditions has long been speculated, and DNA methylation has been proposed as one of its promoters (13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar). In animal cells, however, DNA methylation patterns are strictly regulated in somatic cells so that upon gametogenesis those that are acquired are usually completely erased and not transmitted to the progeny (14Reik W. Walter J. Nat. Rev. Genet. 2001; 2: 21-32Crossref PubMed Scopus (1806) Google Scholar). In plants, the distinction between somatic and germ cells is less obvious in comparison with animals, making possible transmission of acquired DNA methylation patterns. We have experimentally shown this by treatment of germinated rice seeds with 5-azadeoxycytidine, a chemical that powerfully induces demethylation of DNA in vivo (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). At maturity, plants exhibited a global demethylation and altered phenotypes, including dwarfism. The acquired traits and demethylation patterns were inherited for up to at least six generations. 2N. Steward, M. Ito, Y. Yamaguchi, N. Koizumi, and H. Sano, unpublished observation. Thus alteration of DNA methylation can induce changed expression of some genes, resulting in a new phenotype, both of which are heritable (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar).Our above-mentioned experiments suggested that the acquired trait due to an acquired methylation pattern is sexually transmittable, but evidence of methylation patterns changing depending on the environment was missing. The present study showed that this can indeed occur. However, maize roots are differentiated tissues consisting of somatic cells and they do not form germ cells. In this context, the demethylation pattern would not be transmittable to progeny. The fact that no obvious demethylation was observed in stem mesocotyl tissues, which contain cells with the potentiality to develop into germ cells, suggests that these latter are well protected against environmental stresses in terms of DNA methylation. However, if a DNA methylation pattern did change, it could result in heritable epimutations. If such mutations were advantageous for survival, they might persist for generations. An example is the flower morphology change inLinaria vulgaris, considered to be the result of hypermethylation of the Lcyc gene which occurred 250 years ago (42Cubas P. Vincent C. Coen E. Nature. 1999; 401: 157-161Crossref PubMed Scopus (901) Google Scholar). Also, in plants, vegetative reproduction is not rare as seen with tuber propagation, making it possible to directly transmit altered methylation patterns to the next generation. We therefore speculate that Lamarckian inheritance does exist, being mediated through DNA methylation. DNA of higher eukaryotes is characterized by the presence of 5-methylcytosine (m5C)1 nucleotides, comprising up to 30% of the total cytosines. In vertebrates, m5C is located almost exclusively in CpG, whereas in plants it occurs in both CpG and CpNpG (1Gruenbaum Y. Naveh-Many T. Cedar H. Razin A. Nature. 1981; 292: 860-862Crossref PubMed Scopus (593) Google Scholar). The distribution within the genome is non-random and varies depending on the tissue and the developmental stages. The physiological function of m5C is essentially to silence gene expression, which is important for host DNA defenses against incorporation of "parasitic" DNA (2Yoder J.A. Walsh C.P. Bestor T.H. Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1501) Google Scholar, 3Colot V. Rossignol J.L. Bioessays. 1999; 21: 402-411Crossref PubMed Scopus (271) Google Scholar). Two systems are involved: one directly blocks transcriptional machinery attached to promoter regions of genes by altering DNA structure and the other indirectly interferes with transcription by influencing nucleosome conformation and stability (4Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1541) Google Scholar). Recent studies indicated the latter to occur frequently, as shown by various disorders due to abnormal chromosome structures that are caused by defective DNA methylation (5Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (869) Google Scholar). One hallmark of cancer cells is local hypermethylation and global hypomethylation of chromosomal DNA (6Smith S.S. J. Mol. Biol. 2000; 302: 1-7Crossref PubMed Scopus (42) Google Scholar). Abnormal methylation in the promoter regions of regulatory genes may indeed result in cancer development (7Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1513) Google Scholar). Using antisense inhibition of DNMT1, a maintenance type DNA methyltransferase, about 10% of all genes in cultured mouse cells were found to be activated (8Jackson-Grusby L. Beard C. Possemat R. Tudoe M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (562) Google Scholar). These observations confirmed that DNA methylation functions as a global repressor of gene expression (9Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5330) Google Scholar). The reverse case, i.e. global demethylation has also been inferred to be critical during embryogenesis in mammals (9Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5330) Google Scholar, 10Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2395) Google Scholar). What controls the on-off switch for DNA methylation, however, is still largely unclear. While a set of DNA methyltransferases has been identified in various organisms, including plants, the presence of DNA demethylases is controversial (7Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1513) Google Scholar). Among several candidate proteins, 5-methylcytosine DNA glycosylase was shown to induce genome-wide demethylation upon transfection into mouse myoblasts (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar). Whatever the mechanism may be, reprogramming of DNA methylation appears to be fundamental in normal development (10Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2395) Google Scholar). Epigenetic inheritance is defined as change in gene expression without base sequence alteration (12Riggs A.D. Porter T.N. Russo V.E.A. Martienssen R.A. Riggs A.D. Epigenetic Mechanisms of Gene Regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 29-45Google Scholar). This typically occurs during somatic cell differentiation, in which the clonal expansion of a single cell leads to a diversity of cell types (13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar). Such a cellular inheritance is common during ontogeny but is usually erased before gametes are produced (14Reik W. Walter J. Nat. Rev. Genet. 2001; 2: 21-32Crossref PubMed Scopus (1806) Google Scholar). In plants, however, it has long been known that epigenetically acquired traits can be sexually transmitted, as exemplified by flax (Linum) (15Smith J.M. J. Theor. Biol. 1990; 143: 41-53Crossref PubMed Scopus (72) Google Scholar). In this case, epigenetic changes were induced by external factors such as nutrients and temperature (16Durrant A. Heredity. 1962; 17: 27-61Crossref Scopus (148) Google Scholar, 17Cullis C.A. CRC Crit. Rev. Plant Sci. 1983; 1: 117-131Crossref Scopus (42) Google Scholar). Consequently, the idea has been proposed that an environmental stimulus can induce heritable chromatin modifications as an adaptive response (18Jablonka E. Lamb M.J. J. Theor. Biol. 1989; 139: 69-83Crossref PubMed Scopus (186) Google Scholar). It is established that some clonal epigenetic changes are mediated through DNA methylation (6Smith S.S. J. Mol. Biol. 2000; 302: 1-7Crossref PubMed Scopus (42) Google Scholar,13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar), but evidence is limited for involvement of the latter in inheritance of acquired characteristics (19Martienssen R.A. Colot V. Science. 2001; 293: 1070-1074Crossref PubMed Scopus (401) Google Scholar). We have previously shown that a single exposure of germinated rice seeds to the DNA demethylating agent, 5-azadeoxycytidine, induced dwarfism at maturity (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). Genomic DNA isolated from dwarf plants showed a 16% reduction in the m5C content in comparison with DNA from untreated plants. Both hypomethylation and dwarfism were transmitted to progeny for at least three generations (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). Thus, the acquired phenotype due to acquired changes in DNA methylation was heritable. However, whether or not such an epigenetic inheritance occurs under natural condition remained to be solved. In this report we provide support for the conclusion that levels of DNA methylation change in the cores of nucleosomes in maize root tissues in response to cold stress or environmental cues. This finding allowed us to speculate that DNA methylation may function as a common switch of gene expression and that naturally induced changes in DNA methylation may result in heritable epigenetic modification of gene expression. DISCUSSIONNucleosomes are organized into precise positions on DNA by signals in the sequence (31Davey C. Pennings S. Allan J. J. Mol. Biol. 1997; 267: 276-288Crossref PubMed Scopus (82) Google Scholar). One such signal has been proposed to be m5C, but the experimental evidence is controversial. For example, opposite results as to preferential binding of histone H1 to methylated DNA have been reported (32McArthur M. Thomas J.O. EMBO J. 1996; 15: 1705-1714Crossref PubMed Scopus (62) Google Scholar, 33Campoy F.J. Meehan R.R. McKay S. Nixon J. Bird A. J. Biol. Chem. 1995; 270: 26473-26481Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and currently the interaction between histones and methylated DNA is in general not considered to be specific. However, under certain circumstances, nucleosome assembly appears to depend on CpG methylation (31Davey C. Pennings S. Allan J. J. Mol. Biol. 1997; 267: 276-288Crossref PubMed Scopus (82) Google Scholar, 34Godde J.S. Kass S.U. Hirst M.C. Wolffe A.P. J. Biol. Chem. 1996; 271: 24325-24328Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This suggests a dynamic interaction between methylated DNA and proteins that constitute chromatin. Methylation of histones was shown to be essential for triggering DNA methylation in filamentous fungi, and it was suggested that propagation of DNA methylation patterns is dependent on feed-back loops between modification of chromatin proteins and DNA (35Tamaru H. Selker E.U. Nature. 2001; 414: 277-283Crossref PubMed Scopus (849) Google Scholar). Our result showing periodic oscillation of DNA methylation within the nucleosomal structure is consistent with this view and further suggests a positive role for methylation in determining the chromatin infrastructure. Our findings also substantiate occurrence of demethylation upon environmental stress.During differentiation of mammalian cells, genome-wide loss of DNA methylation occurs, for which active or passive mechanisms have been proposed (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar, 36Zhu B. Benjamin D. Zheng Y. Angliker H. Thiry S. Siegmann M. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5031-5036Crossref PubMed Scopus (69) Google Scholar). In active demethylation, m5C is enzymatically removed from DNA, and in passive demethylation, newly replicated DNA is not methylated (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar). Our results with maize indicate that genome-wide demethylation occurs, possibly through active demethylation, because chilled tissues immediately ceased DNA replication (28Steward N. Kusano T. Sano H. Nucleic Acids Res. 2000; 28: 3250-3259Crossref PubMed Scopus (123) Google Scholar). A rapid and active decrease in global DNA methylation was also observed during seed germination of Silene latifolia (37Zluvova J. Janousek B. Vyskot B. J. Exp. Bot. 2001; 52: 2265-2273Crossref PubMed Scopus (81) Google Scholar). Active demethylation is catalyzed by 5-methylcytosine DNA glycosidase (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar, 36Zhu B. Benjamin D. Zheng Y. Angliker H. Thiry S. Siegmann M. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5031-5036Crossref PubMed Scopus (69) Google Scholar), although such activity has not yet been found in plants. A specific feature of our findings was that demethylation predominantly occurred at the nucleosome core regions as revealed by direct methylation mapping. A question arises, then, as to how such a differential demethylation pattern is formed. Currently we have no explanation for this, but some clues for speculation are available. For example, nucleosome cores are proposed to be located outside the 30-nm fiber, which is composed of a unit of six nucleosomes. Linker regions are connected by histone H1 inside the fiber. This structure makes 5-methylcytosine DNA glycosylase more accessible to the core than to the linker. Alternatively, 5-methylcytosine DNA glycosylase may be selectively recruited to the core by specific protein(s) such as, for example, 5-methylcytosine binding proteins. Another distinct finding of the direct methylation mapping is ubiquitous demethylation at CpG, CpNpG and other sites, suggesting no restriction to only one strand of DNA. This means that, for remethylation, de novo methyltransferase activity is necessary. If this enzyme is absent in quiescent cells, for example, in root tissues, the methylated status can not be restored. This could account for the apparently progressive demethylation in cold-chased seedlings.While the average methylation level of total genomic DNA in untreated root tissues was ∼18%, that of ZmMI1 was 38%, 2-fold higher. This indicates a heterogeneous distribution of m5C in the genome, consistent with the histochemical observations, and suggests ZmMI1 to be located in heterochromatin. However, because demethylation occurred throughout the genome, including theZmMI1 sequence, the periodic methylation pattern found in the latter may be common to other parts of genome. If so, the occurrence of demethylation mainly in the core regions of nucleosomes could induce an alteration of chromatin structure and thus influence gene expression. Increase in ZmMI1 transcripts substantiates this hypothesis. The advantage of this feature is that expression of many genes could be simultaneously controlled. Indeed, it was estimated that plants may express as many as several hundred genes upon cold stress (38Thomashow M.F. Meyerowitz E.M. Somerville C.R. Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 807-834Google Scholar). It is attractive to speculate that plants have developed the methylation/demethylation system to simultaneously regulate a vast number of genes at once, rather than to individually regulate each gene. Alternatively, because ∼70% of the maize genome is composed of retroposon-like sequences (39Bennetzen J.L. Plant Mol. Biol. 2000; 42: 251-269Crossref PubMed Scopus (605) Google Scholar) and because demethylation has been shown to reactivate transposons (40Dennis E.S. Brettell R.I. Philos. Trans. R. Soc. Lond-Biol. Sci. 1990; 362: 217-229Google Scholar), the global demethylation possibly transcriptionally activates such sequences to translocate, resulting in secondary activation/inactivation of other genes (41Miura A. Yonebayashi S. Watanabe K. Toyama T. Shimada H. Kakutani T. Nature. 2001; 411: 212-214Crossref PubMed Scopus (465) Google Scholar). However, the reason why such demethylation occurs only in root tissues is not clear. One explanation might be that above-ground parts are equipped with a more complete self-defense system than those underground, as exemplified by the presence and absence of maintenance-type DNA methyltransferase in the respective tissues (28Steward N. Kusano T. Sano H. Nucleic Acids Res. 2000; 28: 3250-3259Crossref PubMed Scopus (123) Google Scholar).Heredity of epigenetic modification, or acquired traits, in response to environmental conditions has long been speculated, and DNA methylation has been proposed as one of its promoters (13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar). In animal cells, however, DNA methylation patterns are strictly regulated in somatic cells so that upon gametogenesis those that are acquired are usually completely erased and not transmitted to the progeny (14Reik W. Walter J. Nat. Rev. Genet. 2001; 2: 21-32Crossref PubMed Scopus (1806) Google Scholar). In plants, the distinction between somatic and germ cells is less obvious in comparison with animals, making possible transmission of acquired DNA methylation patterns. We have experimentally shown this by treatment of germinated rice seeds with 5-azadeoxycytidine, a chemical that powerfully induces demethylation of DNA in vivo (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). At maturity, plants exhibited a global demethylation and altered phenotypes, including dwarfism. The acquired traits and demethylation patterns were inherited for up to at least six generations. 2N. Steward, M. Ito, Y. Yamaguchi, N. Koizumi, and H. Sano, unpublished observation. Thus alteration of DNA methylation can induce changed expression of some genes, resulting in a new phenotype, both of which are heritable (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar).Our above-mentioned experiments suggested that the acquired trait due to an acquired methylation pattern is sexually transmittable, but evidence of methylation patterns changing depending on the environment was missing. The present study showed that this can indeed occur. However, maize roots are differentiated tissues consisting of somatic cells and they do not form germ cells. In this context, the demethylation pattern would not be transmittable to progeny. The fact that no obvious demethylation was observed in stem mesocotyl tissues, which contain cells with the potentiality to develop into germ cells, suggests that these latter are well protected against environmental stresses in terms of DNA methylation. However, if a DNA methylation pattern did change, it could result in heritable epimutations. If such mutations were advantageous for survival, they might persist for generations. An example is the flower morphology change inLinaria vulgaris, considered to be the result of hypermethylation of the Lcyc gene which occurred 250 years ago (42Cubas P. Vincent C. Coen E. Nature. 1999; 401: 157-161Crossref PubMed Scopus (901) Google Scholar). Also, in plants, vegetative reproduction is not rare as seen with tuber propagation, making it possible to directly transmit altered methylation patterns to the next generation. We therefore speculate that Lamarckian inheritance does exist, being mediated through DNA methylation. Nucleosomes are organized into precise positions on DNA by signals in the sequence (31Davey C. Pennings S. Allan J. J. Mol. Biol. 1997; 267: 276-288Crossref PubMed Scopus (82) Google Scholar). One such signal has been proposed to be m5C, but the experimental evidence is controversial. For example, opposite results as to preferential binding of histone H1 to methylated DNA have been reported (32McArthur M. Thomas J.O. EMBO J. 1996; 15: 1705-1714Crossref PubMed Scopus (62) Google Scholar, 33Campoy F.J. Meehan R.R. McKay S. Nixon J. Bird A. J. Biol. Chem. 1995; 270: 26473-26481Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and currently the interaction between histones and methylated DNA is in general not considered to be specific. However, under certain circumstances, nucleosome assembly appears to depend on CpG methylation (31Davey C. Pennings S. Allan J. J. Mol. Biol. 1997; 267: 276-288Crossref PubMed Scopus (82) Google Scholar, 34Godde J.S. Kass S.U. Hirst M.C. Wolffe A.P. J. Biol. Chem. 1996; 271: 24325-24328Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This suggests a dynamic interaction between methylated DNA and proteins that constitute chromatin. Methylation of histones was shown to be essential for triggering DNA methylation in filamentous fungi, and it was suggested that propagation of DNA methylation patterns is dependent on feed-back loops between modification of chromatin proteins and DNA (35Tamaru H. Selker E.U. Nature. 2001; 414: 277-283Crossref PubMed Scopus (849) Google Scholar). Our result showing periodic oscillation of DNA methylation within the nucleosomal structure is consistent with this view and further suggests a positive role for methylation in determining the chromatin infrastructure. Our findings also substantiate occurrence of demethylation upon environmental stress. During differentiation of mammalian cells, genome-wide loss of DNA methylation occurs, for which active or passive mechanisms have been proposed (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar, 36Zhu B. Benjamin D. Zheng Y. Angliker H. Thiry S. Siegmann M. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5031-5036Crossref PubMed Scopus (69) Google Scholar). In active demethylation, m5C is enzymatically removed from DNA, and in passive demethylation, newly replicated DNA is not methylated (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar). Our results with maize indicate that genome-wide demethylation occurs, possibly through active demethylation, because chilled tissues immediately ceased DNA replication (28Steward N. Kusano T. Sano H. Nucleic Acids Res. 2000; 28: 3250-3259Crossref PubMed Scopus (123) Google Scholar). A rapid and active decrease in global DNA methylation was also observed during seed germination of Silene latifolia (37Zluvova J. Janousek B. Vyskot B. J. Exp. Bot. 2001; 52: 2265-2273Crossref PubMed Scopus (81) Google Scholar). Active demethylation is catalyzed by 5-methylcytosine DNA glycosidase (11Jost J.P. Oakeley E.J. Zhu B. Benjamin D. Thiry S. Siegmann M. Jost Y.-C. Nucleic Acids Res. 2001; 29: 4452-4461Crossref PubMed Scopus (88) Google Scholar, 36Zhu B. Benjamin D. Zheng Y. Angliker H. Thiry S. Siegmann M. Jost J.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5031-5036Crossref PubMed Scopus (69) Google Scholar), although such activity has not yet been found in plants. A specific feature of our findings was that demethylation predominantly occurred at the nucleosome core regions as revealed by direct methylation mapping. A question arises, then, as to how such a differential demethylation pattern is formed. Currently we have no explanation for this, but some clues for speculation are available. For example, nucleosome cores are proposed to be located outside the 30-nm fiber, which is composed of a unit of six nucleosomes. Linker regions are connected by histone H1 inside the fiber. This structure makes 5-methylcytosine DNA glycosylase more accessible to the core than to the linker. Alternatively, 5-methylcytosine DNA glycosylase may be selectively recruited to the core by specific protein(s) such as, for example, 5-methylcytosine binding proteins. Another distinct finding of the direct methylation mapping is ubiquitous demethylation at CpG, CpNpG and other sites, suggesting no restriction to only one strand of DNA. This means that, for remethylation, de novo methyltransferase activity is necessary. If this enzyme is absent in quiescent cells, for example, in root tissues, the methylated status can not be restored. This could account for the apparently progressive demethylation in cold-chased seedlings. While the average methylation level of total genomic DNA in untreated root tissues was ∼18%, that of ZmMI1 was 38%, 2-fold higher. This indicates a heterogeneous distribution of m5C in the genome, consistent with the histochemical observations, and suggests ZmMI1 to be located in heterochromatin. However, because demethylation occurred throughout the genome, including theZmMI1 sequence, the periodic methylation pattern found in the latter may be common to other parts of genome. If so, the occurrence of demethylation mainly in the core regions of nucleosomes could induce an alteration of chromatin structure and thus influence gene expression. Increase in ZmMI1 transcripts substantiates this hypothesis. The advantage of this feature is that expression of many genes could be simultaneously controlled. Indeed, it was estimated that plants may express as many as several hundred genes upon cold stress (38Thomashow M.F. Meyerowitz E.M. Somerville C.R. Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 807-834Google Scholar). It is attractive to speculate that plants have developed the methylation/demethylation system to simultaneously regulate a vast number of genes at once, rather than to individually regulate each gene. Alternatively, because ∼70% of the maize genome is composed of retroposon-like sequences (39Bennetzen J.L. Plant Mol. Biol. 2000; 42: 251-269Crossref PubMed Scopus (605) Google Scholar) and because demethylation has been shown to reactivate transposons (40Dennis E.S. Brettell R.I. Philos. Trans. R. Soc. Lond-Biol. Sci. 1990; 362: 217-229Google Scholar), the global demethylation possibly transcriptionally activates such sequences to translocate, resulting in secondary activation/inactivation of other genes (41Miura A. Yonebayashi S. Watanabe K. Toyama T. Shimada H. Kakutani T. Nature. 2001; 411: 212-214Crossref PubMed Scopus (465) Google Scholar). However, the reason why such demethylation occurs only in root tissues is not clear. One explanation might be that above-ground parts are equipped with a more complete self-defense system than those underground, as exemplified by the presence and absence of maintenance-type DNA methyltransferase in the respective tissues (28Steward N. Kusano T. Sano H. Nucleic Acids Res. 2000; 28: 3250-3259Crossref PubMed Scopus (123) Google Scholar). Heredity of epigenetic modification, or acquired traits, in response to environmental conditions has long been speculated, and DNA methylation has been proposed as one of its promoters (13Holliday R. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 452-468Crossref Scopus (30) Google Scholar). In animal cells, however, DNA methylation patterns are strictly regulated in somatic cells so that upon gametogenesis those that are acquired are usually completely erased and not transmitted to the progeny (14Reik W. Walter J. Nat. Rev. Genet. 2001; 2: 21-32Crossref PubMed Scopus (1806) Google Scholar). In plants, the distinction between somatic and germ cells is less obvious in comparison with animals, making possible transmission of acquired DNA methylation patterns. We have experimentally shown this by treatment of germinated rice seeds with 5-azadeoxycytidine, a chemical that powerfully induces demethylation of DNA in vivo (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). At maturity, plants exhibited a global demethylation and altered phenotypes, including dwarfism. The acquired traits and demethylation patterns were inherited for up to at least six generations. 2N. Steward, M. Ito, Y. Yamaguchi, N. Koizumi, and H. Sano, unpublished observation. Thus alteration of DNA methylation can induce changed expression of some genes, resulting in a new phenotype, both of which are heritable (20Sano H. Kamada I. Youssefian S. Katsumi M. Wabilko H. Mol. Gen. Genet. 1990; 220: 441-447Crossref Scopus (114) Google Scholar). Our above-mentioned experiments suggested that the acquired trait due to an acquired methylation pattern is sexually transmittable, but evidence of methylation patterns changing depending on the environment was missing. The present study showed that this can indeed occur. However, maize roots are differentiated tissues consisting of somatic cells and they do not form germ cells. In this context, the demethylation pattern would not be transmittable to progeny. The fact that no obvious demethylation was observed in stem mesocotyl tissues, which contain cells with the potentiality to develop into germ cells, suggests that these latter are well protected against environmental stresses in terms of DNA methylation. However, if a DNA methylation pattern did change, it could result in heritable epimutations. If such mutations were advantageous for survival, they might persist for generations. An example is the flower morphology change inLinaria vulgaris, considered to be the result of hypermethylation of the Lcyc gene which occurred 250 years ago (42Cubas P. Vincent C. Coen E. Nature. 1999; 401: 157-161Crossref PubMed Scopus (901) Google Scholar). Also, in plants, vegetative reproduction is not rare as seen with tuber propagation, making it possible to directly transmit altered methylation patterns to the next generation. We therefore speculate that Lamarckian inheritance does exist, being mediated through DNA methylation. We thank Prof. A. B. Pardee and Dr. A. Goodmann for helpful discussions and comments on the manuscript.
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