DNA Methylation is Reduced in DNasel-Sensitive Regions of Plant Chromatin

Abstract

Pea, barley, and com chromatin was isolated and subjected to limited digestion with DNasel. The preferentially degraded, small molecular weight fraction, presumably consisting of sequences in an active state of expression, was isolated and its 5-methylcytosine content determined. The DNasel-sensitive chromatin fraction from all three plant species investigated contained a markedly reduced level of DNA methylation compared to total DNA.The DNA of higher animals and plants contains the modified base m5C4 in the sequence CG and, in plants, CNG (N = any base). Only a fraction of the cytosines occurring in these sequences are methylated (13), and the distribution of cytosine methylation is not random. High levels of m5C are found in unexpressed regions ofgenomes, such as centromeric and heterochromatic areas of animal chromosomes (20), several types of satellite DNA (6), and inactive mammalian Xchromosomes (14). A number of studies have demonstrated that certain genes are undermethylated in tissues where they are actively expressed, but highly methylated in tissues (or different developmental stages of the same tissue) where the genes are silent (8). Thus, DNA methylation is postulated to be a determinant of gene expression.Chromatin containing actively expressed genes (or genes that are poised for expression) is maintained in a structure that renders it more susceptible to nuclease attack, and presumably more accessible to transcription factors, than chromatin containing inactive genes (30). This nuclease-sensitive fraction of animal chromatin has been shown to have a reduced level of methylation (I1, 21). In addition, the nuclease-sensitive fraction of animal chromatin is enriched in HMG-proteins and depleted in histone HI (4,31 'Abbreviation: m5C, 5-methylcytosine. 451Plant genomes are methylated to a far greater extent than animal genomes. Up to 40% of the cytosine residues are modified in certain plant species, compared to 4 to 6% in mammals (1). The role, however, of DNA methylation in the plant genome is not well understood. The relationship between plant DNA methylation and gene expression has been primarily examined for specific T-DNA (2, 12, 15, 23) and transposon insertions (9,25). In all of these cases, expression has been found to correlate with reduced methylation. In addition, the methylation state ofa maize seed storage protein gene was recently found to correlate with its expression at different developmental stages (7). Although a relationship between chromatin structure and gene activity has been found for a number of plant genes (17 and references therein), and HMG-like proteins have been implicated in the determination of plant chromatin structure (26), little is known about the relationship between DNA methylation and plant chromatin structure. In a study of T-DNA expression in tobacco cells, Reid et al. (24) found a correlation between nuclease-sensitive chromatin, gene expression, and reduced methylation of the T-DNA insertion. A similar correlation was found for pea rRNA genes (29). H...

Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing

“…Subsequent studies, however, found a close correlation between chromatin structure and transcriptional activity, and characteristic dierences in chromatin structure among dierent tissues (Baluska 1990). DNase I accessibility studies showed that DNase I-sensitive regions of plant chromatin showed markedly reduced DNA methylation (Klaas and Amasino 1989), and demonstrated a generally open chromatin structure for transgenes (Weising et al 1990) and endogenous genes (Conconi and Ryan 1993) that was independent of the transcriptional activity of the genes.…”

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

“…[ [33][34][35][36] The latter is not a preferred option due to the potential of contamination from 5-methylcytosine derived from RNA that maybe present in the DNA sample which is also released by acid hydrolysis.…”

The role of chromatin remodeling in transgene silencing and plant development

“…DNasel-sensitive regions are undermethylated relative to total plant DNA (Klaas and Amasino, 1989) but methylation patterns do not necessarily change with modifications of chromatin accessibility. Appearance of DNaseI-hypersensitive sites in the maize Adhl gene (Nick et al, 1986) and a pea rbcS gene (Goerz et al, 1988) after induction was not accompanied by changes in DNA methylation.…”