title: Chromatin structure and DNA methylation
Creators: name: Davis, Terence
Publisher: name: University of Glasgow
Year: 1984

description: The DNA of eukaryotes has been shown to contain the minor base 5-methylcytosine. This arises by the enzymic modification of cytosine already incorporated into DNA. The distribution of methylation in chromatin is not random but there are distinct methylated and unmethylated domains, one such unmethylated domain consists of transcriptionally active DNA. The work undertaken during the tenure of this award has been to examine the effects of chromatin structure and chromatin constituents upon DNA methylation, with the aim of finding a specific inhibitor of methylation which may be responsible for the undermethylation of transcribing regions of chromatin. When the distribution of methyl groups in chromatin is examined, it is found that nucleosomal core DNA is enriched in these groups when compared to linker DNA. This DNA is also enriched in the bases cytosine and guanine. When nuclei from log phase cells are methylated in vitro with the endogenous methylase, the methyl groups are added predominantly to core DNA. As DNA synthesis is not continuing in vitro, and most of the methylation has already occurred in vivo, the methylation in these nuclei is "delayed methylation" Evidence is presented that this methylation is occurring in the nuclear matrix, and the methylation pattern observed simply reflects the pattern in vivo. But when mouse ascites methylase is added to these nuclei, the methyl groups go on to linker DNA, showing that this DNA is more susceptible to methylation than core DNA. These two observations suggest that the methylation pattern found in vivo is not a function of chromatin structure, and it is thought that nucleosomes bind to DNA enriched in cytosine and guanine, and that the enrichment of methylcytosine is a consequence of this. This idea is reinforced by several lines of evidence; i) Histones are very effective inhibitors of DNA methylation in vitro when added to a methylase assay. ii) Chromatin is a poor acceptor of methyl groups compared to DNA. Core particles, which have a higher histone to DNA ratio, are not as good acceptors as chromatin. iii) When native mouse DNA and histones are reconstituted into chromatin, the nucleosomes are again found on methyl rich and cytosine and guanine rich DNA. When transcriptionally competent chromatin is examined, the DNA is found to be deficient in 5-niethylcytosine compared to total chromatin. When nuclei from log phase cells are incubated in vitro the methylation occurs predominantly in the fraction of chromatin containing transcribing regions. As this fraction is also thought to contain DNA associated with the nuclear matrix it is not certain which DNA is being methylated. As the methylation in isolated nuclei is not affected by extraction with 0.2 M NaCl, which removes all of the soluble DNA methylase, this methylation is a product of a bound DNA methylase which is thought to be in the nuclear matrix. Adding soluble DNA methylase to nuclei in vitro results in an elevated methylation of DNA in this fraction, and again the methyl groups are being added to transcribing (matrix) chromatin. When the effects of nuclear non-histone proteins on DNA methylation in vitro are examined no inhibition is observed by either high mobility group proteins or nuclear non-histone proteins in general. These results suggest that there are no specific proteins in transcribing regions which inhibit DNA methylation in vivo.