Linker histones (e.g. H1, H5, H1°) are thought to exert control on chromatin function by restricting nucleosomal dynamics. All higher eukaryotes possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complement of linker histone structural variants and therefore is an ideal model for 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 is a highly divergent minor variant protein that is induced by drought stress. We are interested in determining whether the in vivo distribution of each of these proteins also differs. To this end, we have produced subtype-specific antibodies and have localized each of the three proteins at the intranuclear and DNA sequence levels by indirect immunofluorescence and immunoprecipitation, 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 describe a technique to determine the localization of plant linker histone variants along regions of chromatin, employing in vivo chemical DNA-protein cross-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 repetitive sequences pAL1 and 5S rDNA. In addition, we show that linker histones are bound to the compacted nucleosomal arrays at the telomere but with reduced stoichiometry. Taken together, our results suggest that plants, as has been shown for animals, possess a variant linker histone that is differentially localized.
Bibliographical noteFunding Information:
ty of Minnesota, College of Biological Sciences Imaging Center staff for excellent support with microscopy and the PhosphorIma-ger, and Judith Berman and Maryam Gerami-Nejad, Department of Plant Biology, University of Minnesota, for the RAP1 antibody and technical advice on immunofluorescence, respectively. We also thank David Stollar, Department of Biochemistry, Tufts University, for the calf H4 antibody, and Steven Spiker, Department of Genetics, North Carolina State University, for the wheat HMGa antiserum and comments on this manuscript. The Arabidopsis Biological Resource Center provided some of the cDNAs used as probes in this work. This research was funded in part by a grant from the USDA (95-37301-1800).
Copyright 2007 Elsevier B.V., All rights reserved.