No. 10] Proc. Japan Acad ., 78, Ser. B (2002) 293 DNA methylation and Lamarckian inheritance By Hiroshi SANo Research and Education Center for Genetic Information, Nara Institute of Science and Technology Ikoma, Nara 630-0192 (Communicated by Yasuyuki YAMADA,M. J. A., Dec. 12, 2002) Abstract: Jean Baptiste de Lamarck (1744-1829) maintained that characteristics that were acquired during an organism's lifetime are passed on to its offspring. This theory, known as Lamarckian inheritance, was later completely discredited. However, recent progress in epigenetics research suggests it needs to be reexamined in consideration of DNA methylation. In this article, I summarize our observations, which support Lamarckian inheritance. Initial experiments indicate that (1) artificially induced demethy- lation of rice genomic DNA results in heritable dwarfism, and (2) cold stress induces extensive demethy- lation in somatic cells of the maize root. Based on these results, I propose the hypothesis that traits that are acquired during plant growth are sometimes inherited by their progeny through persistent alteration of the DNA methylation status. Key words: Acquired traits; DNA methylation; epigenetics; Lamarckian inheritance; vernalization. Lamarckian inheritance. In 1809, Lamarck pro- In plants, however, it has long been known that epi- posed that evolution is governed by two laws. First, genetically acquired traits can be sexually transmit- organs are improved with repeated use and weakened by ted.5~ The best known example is flax (Linum usitatis- disuse. Second, such environmentally determined simum). Plants treated with fertilizer exhibit a more acquisition or loss of traits may be passed on to the off- branched pattern of growth and broader leaves than spring. l~ This theory was the first statement of inheri- untreated plants.6~ Other characteristics are alteration in tance of acquired characteristics, and became known as the plant weight at maturity,6~ the number of hairs on the Lamarckism or Lamarckian inheritance. After intensive seed-capsule septa7~ and the mobilities of isozymes of discussion, however, the theory was discredited by peroxidase and acid phosphatase.8 These traits are in most geneticists after the 1930s. fact seed-transmissible.6~ In Nicotiana rustica, heritable Nevertheless, recent studies have raised the possi- differences have also been observed after a single gen- bility that Lamarckian inheritance may play a partial role eration of growth in specific environments.9~ One of the in development and evolution. This idea is based on the causes of such epigenetic inheritance is postulated to be concept of "epigenetics", which is defined as change in the alteration of chromatin structure.10~ Consequently, gene expression without base sequence alteration.2~ the idea has been proposed that an environmental stim- This typically occurs during somatic cell differentiation in ulus can induce heritable chromatin modification as an animal cells, in which the clonal expansion of a single cell adaptive response, and therefore that inherited epige- leads to a diversity of cell types.3~ In other words, alter- netic changes in the structure of chromatin can cause a ation of the genetic system within an organism is trans- type of Lamarckian inheritance.5~ missible from cell to cell, this being called epimutation. DNA methylation. DNA of higher eukaryotes con- Such cellular inheritance is common during ontogeny, tains 5-methylcytosine (m5C), comprising up to 30% of but is usually erased when germ cells are formed.4~ This the total cytosines. In vertebrates, m5C is located generally precludes inheritance of the epimutation by the almost exclusively in CpG, while in plants it occurs in next generation, and therefore cannot be considered to both CpG and CpNpG," and also in non-symmetrical necessarily support the concept of Lamarckian inheri- cytosines.12~ The distribution within the genome is non- tance. random, and varies depending on the tissue and the 294 H. SANO [Vol. 78(B), Table I. Characteristics of azadC-treated and untreated rice cultivated in the field developmental stage. The physiological function of m5C major groups.20~ One prefers hemi-methylated DNA, i.e. is essentially to silence gene expression, which is DNA with methylated cytosines on only one strand, and important for host DNA defense against incorporation of is possibly associated with the DNA replication complex parasitic DNA. 13)Intensive studies on animal cells have functioning in maintenance of methylation patterns. revealed distinct examples, including inactivation of The other methylates DNA on both strands, and estab- the X-chromosome, imprinting of genes and silencing of lishes methylation patterns during embryonic develop- parasitic DNA such as retroelements and trans- ment. These properties of two distinct enzymes make the posons.14~ In plants, mobilization of transposons is also DNA methylation patterns to be generally maintained reported to be repressed by DNA methylation.l5~ through cell division, yet to be variable under certain cir- The molecular mechanism of gene silencing is cumstances. It is therefore believed that one of the fac- thought to involve changes in DNA structure caused by tors responsible for epimutation is indeed methylation of methylation of cytosines. Two possibilities have been pro- DNA.13~ posed. One is direct repression due to methylated pro- Background observations. DNA methylation in moter regions blocking binding of transcriptional dwarf plants. To determine whether or not plant machinery, and the other is indirect repression due to growth is affected by DNA methylation status, the rela- altered nucleosome structures affecting the chromatin tionship between DNA methylation and dwarfism in conformation.l3> Recent studies have indicated that the maize (Zea mat's) was initially examined.21~ The latter occurs frequently and plays an essential role in amount of m5C in the DNA of a single-gene dwarf controlling global gene expression.l6~ For example, vari- mutant, d5, was found to be about 8% lower than that of ous disorders including ICF (immunodeficiency, cen- its tall, near-isogenic counterpart. To establish whether tromeric region instability and facial anomalies) have this DNA undermethylation was at all correlated with been found to be the result of abnormal chromosome dwarfism, germinated seeds of a common tall strain (Z. structure caused by defective DNA methylation.17~ It is mat's, cultivar Honey Bantum) were treated for 16 h also known that chromosomal DNA is locally hyperme- with 5-azacytidine (azaC), a powerful inhibitor of DNA thylated and globally hypomethylated in cancer cells.'8 methylation in vivo. After the chemical had been The controlling system of the on-off switch for removed by repeated washing, seedlings were planted DNA methylation, however, is still largely unclear. and the growth was measured. At maturity, the total While a set of DNA methyltransferases has been identi- stem length of treated plants was 28% less than that of fied in various organisms, including plants, the presence untreated controls. Genomic DNA, isolated from azaC- of DNA demethylases is controversiaL19~ Eukaryotic treated plants, showed about an 8% reduction in m5C DNA methyltransferases can be classified into two content. No such effects were observed when seeds were No. 10] DNA methylation and Larnarck jail inheritance 2.5 Table 11. Frequency of mr'C in total cytosine residues in rice DNA''".,I Fig. 1. Induction of heritable dwarfism in rice plants. Seeds of rice cultivars Akil.akomaclo (A and 13) or Yamadanishiki (C) were imbibed for ,3 days, treated with 0.1 mM azadC for 16 h, and cul- tivated under standard field conditions. Self-poll sated offspring were successively obtained each year and tested for their traits. Fig. 2. 1lenome-wide demethylatiou in maize root tissues upon (A) Seedlings of M., generation (right) and control wild-type chilling.2~' The amount of in'C in leaf blades (shaded) and root (left) of Akital:omachi. (13) Mature M, (right) and control wild- tissues (closed) was quantified by HPLC. DNA samples were tvpe (left) of Akitakonlachi. (C) Mature M., (right) and control extracted from 1:3-dap-old seedlings grown at 23 °C (day 0); from wild-lype (left) of Yanladanishilci. seedlings that were further cold Irealed at 4 °C for 8 duvs (day 8); and from seedlings returned to 23 °C for-'3 days ((lay 11) or 10 treated with other nucleotide derivatives, such as days ((ay 18) after cold treatment. Note that cold-triggered cleoiethylation proceeded even after samples werc returned to 3'azido3'deoxythyrnidine and arabinosyl cytidine, indi- the higher temperature. The ratio of m'C to total (, is expressed cating that the observed phenotypic and genornic alter- as a percentage (Ys). ations were azaC-specific. The results suggested that DNA methylation is indeed involved in the expression of genes regulating plant, growth. (65%). The M., offspring of self-fertilized dwarf M, In,ductio a of heritable dwarfism and demethyla- plants were also dwarf, while those from tall M, plants tion. Because of its agronomic importance and the were only tall (Table 1). The heading time of these dwarf accessibility of good genetic materials, we further con- plants was 11 to 15 days earlier than with the controls. tinued the project with rice (Oryza saliva) and analyzed The dwarf and early heading properties were stably the properties of azaC-induced dwarf plants in detail .'2r A transmitted up to at least the M, generation (Fig. 1), single exposure