Subnuclear distribution of the entire complement of linker histone variants in Arabidopsis thaliana

Linker histones (e.g. H1, H5, H1 degrees) are thought to exert control on c hromatin function by restricting nucleosomal dynamics. All higher eukaryote s possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complem ent of linker histone structural variants and therefore is an ideal model f or investigating functional differentiation among linker histones. Histones H1-1 and H1-2 are relatively similar proteins that are expressed in a wide variety of tissues and make up the majority of linker histone while H1-3 i s a highly divergent minor variant protein that is induced by drought stres s. We are interested in determining whether the in vivo distribution of eac h of these proteins also differs. To this end, we have produced subtype-spe cific antibodies and have localized each of the three proteins at the intra nuclear and DNA sequence levels by indirect immunofluorescence and immunopr ecipitation, respectively. Antibodies against linker histones H1-1 and H1-2 decorate nuclei in patterns very similar to 4',6-diamidino-2-phenylindole (DAPI) staining, but different than the staining pattern of total histones. In contrast, antibodies made against two regions of H1-3 bind to chromatin in a diffuse pattern distinct from the DAPI-staining pattern. We also desc ribe a technique to determine the localization of plant linker histone vari ants along regions of chromatin, employing in vivo chemical DNA-protein cro ss-linking to preserve native associations followed by immunoprecipitation with subtype-specific antibodies. We use this technique to demonstrate that , in contrast to the major linker histones, H1-3 does not bind the repetiti ve sequences pAL1 and 5S rDNA. In addition, we show that linker histones ar e bound to the compacted nucleosomal arrays at the telomere but with reduce d stoichiometry. Taken together, our results suggest that plants, as has be en shown for animals, possess a variant linker histone that is differential ly localized.