Position effect takes precedence over target sequence in determination of adenine methylation patterns in the nuclear genome of a eukaryote, Tetrahymena thermophila

Abstract

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N 6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5'-NAT-3', but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation.

Methylation of adenine in the nuclear DNA of Tetrahymena is internucleosomal and independent of histone H1

“…In transformation experiments in Tetrahymena the position effect took precedence over the methylation state of the transforming DNA, i.e. methylation was lost from a chromosomal site inserted in the rDNA (13). Thus the evidence suggests that adenine methylation is governed by chromatin structure, rather than the reverse.…”

“…The position effect for methylation of nuclear DNA in Tetrahymena suggested that methylation may be related to chromatin structure (13). Early experiments with MNase digestion of bulk chromatin suggested that methyladenine was preferentially located in the linker DNA of Tetrahymena (14).…”

“…Sequence of the target site is not sufficient for DNA methylation in Tetrahymena. In transformation experiments a site that is uniformly methylated on the chromosome was not methylated when a fragment containing the site was inserted into the rDNA minichromosome (13). This was true regardless of the methylation state of the DNA at the time it was introduced into the cell.…”

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena

“…However, in the AT-rich Tetrahymena genome (overall AT > 75%) ( 25 , 54 ), only a very small portion of adenine and the AT-motif are methylated (( 17 ) and this study). Indeed, it has been reported that 6mA methylation is lost when a DNA fragment containing a methylated site is moved to a different genomic locus ( 65 , 66 ), suggesting that the genomic context and chromatin environment are crucial for 6mA occurrence.…”

DNA Methylation in Plants

“…About 0.8% of adenine residues are found as m 6 A in DNA of the transcriptionally active macronuclei of Tetrahymena (Gorovsky et al 1973;Kirnos et al 1980). A methylation site is 5 -NAT-3 (Bromberg et al 1982), and about 3% methylation sites are GATC (Harrison et al 1986;Karrer and Van Nuland 1998).…”