Pulsed-field gel electrophoresis analysis of higher-order chromatin structures of Zea mays. Highly methylated DNA in the 50 kb chromatin structure

We have investigated the presence of higher-order chromatin structures in different maize tissues. Taking advantage of the pulsed-field gel electrophoresis technique to analyse large DNA fragments from intact nuclei and cells, we have determined the size distribution of the high-molecular-weight DNA fragments obtained from chromatin degradation by endogenous nucleases in isolated nuclei. Chromatin digestion leads to the appearance of stable DNA fragments of about 50 kb in all the tissues examined, suggesting the folding of DNA in higher-order chromatin domain structures. It has been reported that such chromatin domains are formed by loops of the 30 nm fibres anchored to the nuclear matrix by a complex set of proteins, including DNA topoisomerase II. Treatment of maize protoplasts with the calcium ionophore A23187 and the antitumour drug VM-26, which specifically inhibit the religation of the cleaved DNA in the topoisomerase II reaction, also produces the 50 kb structure. Analysis of the DNA contained in the 50 kb chromatin structure shows a higher degree of methylation than in bulk maize chromosomal DNA. The role of methylated DNA in the chromatin folding is discussed.