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

Abstract

Linker histones (e.g. H1, H5, H1 degrees ) 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.

A specialized histone H1 variant is required for adaptive responses to complex abiotic stress and related DNA methylation in Arabidopsis

“…Notably, the proportions of relative enrichment between the stress-inducible and main variants changed in favor of the former in genic compared with typical heterochromatin regions. Interestingly, we observed differences in the distribution of intensity profiles along chromosomes between the main and stress-inducible variants that were consistent with patterns of staining with H1 variant-specific antibodies (Ascenzi and Gantt, 1999b) and imply some underlying differences in chromatin localization preferences. To elucidate the possible reasons for these differences, we analyzed correlations between the distribution of H1 variants and the known distribution profiles of H3K4me3 and H3K9me2 methylation marks, the functionally opposed epigenetic signatures associated with transcriptionally active and repressive chromatin states, respectively.…”

“…A dash indicates that the parameters were not measured. (2) 6.2 6 0.6 (2) 16.2 6 1.3 (4) 6.8 6 0.5 (7) 73.9 6 1.9 (2) 91.1 6 3.4 (2) 63.7 6 2.0 (4) 86.1 6 2.4 (7) H1.2 21.3 6 1.8 (10) 21.1 6 1.3 (7) 22.3 6 4.0 (4) 16.8 6 1.2 (9) 43.0 6 1.5 (10) 77.9 6 2.0 (7) 29.5 6 2.2 (4) 72.3 6 2.0 (9) H1.3 ---3.4 6 0.3 (11) ---97.1 6 3.4 (11) H2B 25.7 6 4.4 (6) 23.5 6 5.2 (6) 13.1 6 2.6 (10) 14.8 6 1.9 (4) 64.8 6 5.0 (6) 35.3 6 3.4 (6) 38.5 6 2.7 (10) 23.1 6 1.1 (4) Stress H1.1 10.3 6 0.8 (11) 8.5 6 0.5 (8) 15.2 6 0.8 (10) 8.4 6 0.5 (5) 72.4 6 2.0 (11) 86.0 6 1.9 (8) 71.1 6 1.4 (10) 86.3 6 2.1 (5) H1.2 15.6 6 1.0 (10) 15.1 6 0.6 (5) 14.8 6 2.2 (7) 19.4 6 1.3 (6) 73.3 6 1.7 (10) 80.2 6 1.2 (5) 23.1 6 1.2 (7) 74.4 6 2.0 (6) H1.3 2 6 0.3 (10) 1.3 6 0.2 (6) 1.0 6 0.1 (5) 3.0 6 0.2 (13) 99.7 6 6.7 (10) 100.0 6 10.0 (6) 99.8 6 10.7 (5) 100.0 6 2.6 (13) H2B 24.9 6 4.2 (10) 12.2 6 0.8 (6) 29.4 6 5.1 (9) 23.6 6 3.1 (5) 48.6 6 3.7 (10) 33.3 6 0.8 (6) 47.3 6 4.0 (9) 47.4 6 2.7 (5) chromatin in a diffuse pattern distinct from 49,6-diamino-2-phenylindole staining (Ascenzi and Gantt, 1999b). The relative abundance of the main H1 variants was higher on heterochromatic compared with euchromatic transposons (Fig.…”

“…Importantly, immunostaining of whole nuclei with specific antibodies showed that the distribution of H1.3 differed from that of H1.1 and H1.2 (which was identical), suggesting that H1.3 binds to different genomic regions compared with the main H1 variants (Ascenzi and Gantt, 1999b). Arabidopsis, wild tomato, and tobacco plants with down-regulation of the stress-inducible linker histone variant do not show defects in development and global chromatin organization under normal conditions (Ascenzi and Gantt, 1999a;Scippa et al, 2000Scippa et al, , 2004Przewloka et al, 2002), suggesting that this group of linker histones has no major role in the basal functions of plant development or in chromatin structure.…”

Telomeric nucleosomes: Forgotten players at chromosome ends

“…Subsequently, velocity sedimentation analysis and isolation of telomere nucleoproteins revealed the presence of H1 on rat telomeres [85] but at a lower H1/nucleosome ratio than bulk chromatin. A reduced H1 stoichiometry was found also in A. thaliana [86] and in human adult fibroblasts, where H1 at telomeres is nearly fourfold less than on inactive chromatin [87]. The reduced H1 content at telomeres is in agreement with their short nucleosome spacing, since there is a linear relationship between H1 stoichiometry and nucleosome repeat length [88].…”

Influence of change in the proportion of H1 histone variants on microsporogenesis and development of male gametophyte in transgenic plants of tobacco (Nicotiana tabacum L.)

“…Their induction caused by a shortage of water was studied in tomato (Wei and OConnel 1996) and in Arabidopsis (Ascenzi and Gantt 1997). The rise in their levels in Arabidopsis indicates the existence of various intranuclear mechanisms for separating the major H1 histone variants, which suggests their role in regulating specific sets of genes (Ascenzi and Gantt 1999). Thus, the function of particular variants of the H1 histone in the normal physiology of plants and organisms has not yet been fully explained.…”