PerspectiveMolecular Biology

Methylation Talk Between Histones and DNA

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Science  07 Dec 2001:
Vol. 294, Issue 5549, pp. 2113-2115
DOI: 10.1126/science.1066726

Biological phenomena are complex, but biologists, being human, crave simplicity. Hence the frisson of excitement, mixed with relief, with the union of two hitherto separate domains of study—in this case, the methylation of DNA and the methylation of histone proteins. DNA methylation is a mark on genomic DNA made by addition of methyl groups to cytosine bases, whereas histone methylation marks proteins that coat the DNA by addition of methyl groups to certain lysine residues. In their recent Nature paper, Tamaru and Selker (1) report that DNA methylation and histone methylation share a common pathway in the filamentous fungus Neurosporacrassa. Their discovery sets the stage for an experimental attack on one of the abiding mysteries of the genome: How do patterns of DNA methylation originate?

The problems inherent in managing large eukaryotic genomes can be eased by marking regions of DNA. Such marked DNA becomes structurally adapted so that it can perform certain activities. The most direct mark is one applied to the DNA itself, but this addition presumably must avoid adverse effects on the genome's stability and coding properties. The only marking system that is widespread among eukaryotes is methylation of DNA cytosine rings at position 5 to give the modified base, 5-methylcytosine (m5C). More elaborate marking can be achieved by coating the genome with immobile proteins that are specifically designed to carry covalent messages. Core histones—the proteins assembled into the beadlike nucleosomes around which the DNA is wrapped—can be thought of as information modules that acquire coded information based on addition or removal of chemical groups. Their amino-terminal tails protrude from the nucleosome beads and can be modified through the attachment (or removal) of acetyl, phosphate, or methyl groups (2).

The resulting “histone code” affects the accessibility of the packaged DNA to transcriptional activator proteins, and hence the ability to switch on gene expression. For example, the lysine residue K9 in histone H3—the ninth amino acid from the amino-terminal end of H3—is acetylated in active chromatin, but methylated in regions of the genome that are silenced. When methylated, this lysine (called H3K9) attracts HP1, a protein associated with condensed (silenced) “heterochromatic” regions of the genome. Thus, methylation of H3K9 appears to be read by HP1 as a signal to condense that region of the DNA (3-5). Like H3K9 methylation, DNA methylation too is associated with transcriptional silencing and can lead in animals to the recruitment of m5C binding proteins (6). The resemblances have led some to wonder if the two methyltransfer reactions might turn out to be connected. There would be a precedent of sorts, because mammalian proteins that bind to genomic m5C sites recruit proteins that deacetylate histone tails, leading to transcriptional shutdown (7, 8). In that case, though, the flow of “epigenetic” information is from DNA to histones. The Tamaru and Selker report is exciting because it suggests the reverse: that histone modification can influence the methylation of DNA.

In Neurospora, DNA methylation affects duplicated DNA sequences that were detected early in the organism's sexual cycle and subjected to active mutation (9, 10). As a result of this process, known as repeat-induced point mutation (RIP), the wild-type Neurospora genome contains a small fraction of methylated DNA, the majority of the DNA remaining nonmethylated. The victims of RIP are predominantly mobile genetic elements that normally move from place to place within the genome, unless eliminated by a defense mechanism of this kind. Our story begins with a search for mutant forms of Neurospora that lack DNA methylation. Several dim (defective in methylation) genes that encode DNA methyltransferase enzymes have already been identified. Tamaru and Selker (1) now show that a mutation in a newly discovered Neurospora gene, dim-5, abolishes methylation of all tested DNA sequences. Through genetic mapping and DNA sequence analysis, these investigators show that this gene encodes a protein containing a so-called SET domain. Intriguingly, the SET domain of chromosomal proteins found in mammals, insects, and yeast can be an enzyme that transfers methyl groups to H3K9 (11). By providing the Neurospora mutant with the wild-type dim-5 gene, the authors were able to correct the mutation and to restore methylation.

To look for a connection between dim-5, DNA methylation, and gene silencing, Tamaru and Selker exposed different strains of Neurospora to the toxic drug hygromycin. Strains engineered to carry a nonmethylated hygromycin-resistance gene (hph) grow in the presence of the drug, but strains with a methylated hph gene cannot grow because the gene is silenced. Interfering with dim-5 gene expression in strains with a methylated hph gene caused demethylation and reactivation of hph, thereby allowing the fungal hyphae to grow in the presence of hygromycin. Methylation and silencing clearly were undermined in the absence of the dim-5 gene, but still the question remained: Was methylation of histones in the region of dim-5 responsible for this effect? Early results already favored the involvement of histone methylation, because the original dim-5mutation was located at a highly conserved residue of the catalytic SET domain. Following up on this lead, Tamaru and Selker expressed the DIM-5 protein and exposed it to core histones in the presence of labeled S-adenosyl methionine. Only histone H3 became methylated, although on which residue was not clear. It is important to know the particular residue, because methylation of H3K9 correlates with gene silencing, but methylation of a different lysine H3K4 in the same histone signifies transcriptionally active chromatin (12). To answer this question, the investigators created strains of Neurospora in which K9 in H3 was replaced with amino acids (arginine and leucine) that cannot be methylated. Although these strains retained their natural H3 genes, they showed marked reactivation and demethylation of the hph gene in the presence of mutant histone H3. Therefore, the presence of H3 lacking a K9 residue that could be methylated mimics the effect of losing the H3 methyltransferase DIM-5 through mutation. The obvious deduction is that methylation of cytosine in Neurospora DNA depends upon prior methylation of K9 in histone H3.

Eukaryotic DNA methylation does not cover the genome like a featureless blanket, but conforms to a pattern of methylated and nonmethylated DNA sequence domains. An attractive feature of the new data is its potential to shed light on the origin of these DNA-methylation patterns, a subject about which we currently know very little. It is possible that DNA methyltransferases can only productively access DNA that is wrapped around nucleosomal histones carrying an H3K9 methylation signal (see the figure). This scenario is compatible with other known features of H3K9 behavior: its acetylation in transcriptionally active chromatin, its deacetylation under the influence of many gene-expression silencers (transcriptional repressors), and its methylation by SET domain proteins such as SU(VAR)3-9 and DIM-5. The notional “descent” from active to profoundly inactive chromatin culminates in DNA methylation. This would also agree with the prevailing evidence (at least in animals) that cytosine methylation is not a primary agent of gene silencing, but affects genes that have already been shut down in other ways.

Methyl groups stick together.

Modifications of the lysine residue K9 in histone protein H3. Depicted are the effects of deacetylation, histone methylation, and DNA methylation of H3K9 on gene transcription. The H3 amino-terminal tail protrudes from a nucleosome (green). H3K9 can be acetylated (pink) in transcribed chromatin, but is deacetylated under the influence of repressors of transcription. This permits methylation (orange) of H3K9 by a SET domain protein. Methylated H3K9 may directly or indirectly affect enzymes called DNA methyltransferases (DNMT), leading to methylation of cytosine on nearby DNA (blue). DNA methylation prompted by methylation of H3K9 on a local nucleosome has not yet been demonstrated. Interaction of methylated H3K9 with chromodomain proteins such as HP1 and interactions of methylated cytosine residues with methylated DNA binding proteins are not shown.

Linear progressions of this kind (see the figure) are seductive, but should be viewed with suspicion. There is as yet no evidence in Neurospora that the sequences that become methylated under the influence of methyl-H3K9 are in the same genomic location as those harboring the methylated histone. The effect on DNA modification might be remote, rather than localized to the methylated nucleosome. Extending the relationship between H3K9 methylation and DNA methylation to nonfungal eukaryotes is also premature. DNA methylation is not a fact of life for many eukaryotes, including other fungi (yeasts) and certain animals (the nematode worm Caenorhabditis elegans), so rigorous conservation of its function cannot be assumed. Cautionary notes aside, however, there is little doubt that the search for SET domain proteins that influence mammalian DNA methylation will now proceed at a frantic pace. Thanks to the awesome power of Neurospora genetics, there has never been a better time to probe the mysterious origins of DNA methylation.

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