Measuring Changes in Chromatin Using Micrococcal Nuclease

Abstract

This chapter documents a simple protocol to identify the nucleosome positioning of any given genes. The procedure includes partitioning 200-bp DNA fragments constituting the nucleosomal core region by micrococcal nuclease digestion and semiquantitative polymerase chain reaction amplification using multiple sets of primers covering arbitrary regions of approximately 150 bp in the gene. If the nuclease-digested 200-bp DNA is efficiently amplified, the region is inside the core. If the amplification is poor, the region spans the linker region. By a combination of this method with direct methylation mapping, the core region of a maize gene, ZmMI1, was shown to be less methylated than the linker region. The potential usefulness of the technique is discussed.

Dissecting Epigenetic Silencing Complexity in the Mouse Lung Cancer Suppressor Gene Cadm1

“…Overall, highest amplification efficiency was observed in normal lung, then in lung tumor, and followed by the lung cancer cell lines. Similar findings have been demonstrated in maize gene, ZmMI1 in which the correlation of methylation status with the nucleosomal structure was analyzed after MNase digestion of chromatin [42].…”

ABI3 mediates dehydration stress recovery response in Arabidopsis thaliana by regulating expression of downstream genes

“…Nucleosomes were mapped specifically for the regions where ABI3 recruitment has been observed from our ChIP-based experiment, i.e., covering the ABI3 binding sites. The primers were designed such that a combination of each primer set (i.e., forward and reverse primer) can amplify around 200 bp DNA fragment, a region that may correspond to at least one nucleosome [54]. A primer set that generated PCR signal intensity from mono-nucleosomal DNA template comparable to the intensity derived from di-nucleosomal and genomic DNA template, or higher, was considered to cover a region with well positioned nucleosome.…”

Epigenetic Inheritance in Rice Plants

“…Total DNA was extracted from seedling leaves by the CTAB method (Murray and Thompson, 1980) and subjected to direct methylation mapping. The DNA was digested with restriction enzymes (EcoRI and KpnI) and recovered by ethanol precipitation after phenol -chloroform extraction, and the bisulfite modification reaction was performed as described (Steward and Sano, 2004) with slight modifications. The primers for PCR were designed for predicted sequences after modification, in which C and m 5 C were assumed to be converted into Y (C or T) in the forward and into R (A or G) in the reverse direction.…”