Shortly after Nan et al.57 and Jones et al.58 reported the role of DNA methylation and modification of chromatin in transcriptional silencing, Ng et al.62 found that cells deficient in MeCP2 (e.g., HeLa cells) were capable of repressing transcription as determined by reporter constructs. This finding suggested that MeCP2 was probably not the sole connection between the methylation of DNA and tran-scriptional silencing.
In 2001, Eric Selker and Hisashi Tamaru63 reported that dim-5, a gene that encodes a histone methyltransferase for methylation of lysines of histone tails of chromatin in the fungus, Neurospora crassa, was required for DNA methylation. They had accidentally generated a mutation in a previously unknown gene required for DNA methylation. In a series of clever experiments, they mapped the fungal mutant gene to a region homologous to histone methyltransferases and demonstrated through biochemical tests on a recombinant form of the gene that the expressed protein methylated histone H3. In characterizing the mutant gene they found a single nucleotide change (C to G) that generated a stop codon in the middle of a distinctive ~130 amino acid sequence motif called the SET domain,64 which identified the gene (in Drosophila) as required for heterochromatin formation, and possibly was one of the chromatin-associated SET methyltransfera-ses. Substitution of lysine at position 9 with either leucine or arginine in histone H3 caused the loss of DNA methylation in vivo, from which they inferred that histone methylation controlled DNA methylation.
The study of Jun-Ichi Nakayama and colleagues65 provided additional evidence that lysine 9 of histone 3 (H3 Lys9) was preferentially methylated at heterochromatin regions of fission yeast (Schizosaccharomyces pombe) and that modifications of histone tails were linked to heterochromatin assembly. They proposed that histone deacetylases and histone methyltransferases cooperate to establish a ''histone code'' that would result in self-propagating heterochromatin assembly. On the basis of conservation of certain transacting proteins that affect silencing (Clr4/SUV39H1 and Swi6/HP1), and the presence of H3 Lys9-methyl
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