Immunolocalization of Histone Modifications as a Tool to Visualize Chromatin Dynamics in Plants

Abstract

The nuclear DNA of eukaryotic organisms is associated with several proteins. These proteins together with the DNA form chromatin. In all eukaryotes, DNA is folded around a core of histones to form nucleosomes. Chromatin was previously believed to serve mainly to organize and compact the genetic material. However, in recent years, chromatin has been regarded has a highly dynamic structure responsible for controlling gene expression. Therefore, chromatin is highly flexible, to make genetic information accessible when needed, and the degree of compaction has to be tightly regulated. DNA staining, using 4',6-diamidino-2-phenylindole (DAPI), provided the basis for a cytological distinction between weakly stained euchromatin, now known to be gene rich, and brightly stained heterochromatin, which usually contains various repetitive sequences. In many organisms, heterochromatic DNA is hypermethylated and this is used as an additional mechanism to regulate transcription. Histones can be modified by acetylation, phosphorylation, methylation or ubiquitination. These mechanisms provide signals to which other factors can bind and additionally alter the biochemical properties of chromatin (i.e., “histone code”). In general, acetylation of histones is correlated with active genes, whereas methylation of histones at different positions results in changes in gene expression. The methylation of lysine 9 in histone 3 (H3K9) is associated with heterochromatin formation while methylation of lysine 4 of histone 3 (H3K4) methylation is related to gene activation and positioned in euchromatin. DNA and histone modifications recruit various non-histone proteins to specific chromosomal regions and eventually create a defined nuclear structure that is able to affect gene expression.