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Histones H2A and H2B form part of the same nucleosomal structure as H3 and H4. Stable HeLa cell lines expressing histones H2B, H3, and H4 tagged with green fluorescent protein (GFP) were established; the tagged molecules were assembled into nucleosomes. Although H2B-GFP was distributed like DNA, H3-GFP and H4-GFP were concentrated in euchromatin during interphase and in R-bands in mitotic chromosomes. These differences probably result from an unregulated production of tagged histones and differences in exchange. In both single cells and heterokaryons, photobleaching revealed that H2B-GFP exchanged more rapidly than H3-GFP and H4-GFP. About 3% of H2B exchanged within minutes, whereas approximately 40% did so slowly (t(1/2) approximately 130 min). The rapidly exchanging fraction disappeared in 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and so may represent H2B in transcriptionally active chromatin. The slowly exchanging fraction was probably associated with chromatin domains surrounding active units. H3-GFP and H4-GFP were assembled into chromatin when DNA was replicated, and then >80% remained bound permanently. These results reveal that the inner core of the nucleosome is very stable, whereas H2B on the surface of active nucleosomes exchanges continually.

Type

Journal article

Journal

J Cell Biol

Publication Date

25/06/2001

Volume

153

Pages

1341 - 1353

Keywords

Cell Line, Cell Nucleus, Chromatin, Chromosomes, DNA, Dichlororibofuranosylbenzimidazole, Green Fluorescent Proteins, HeLa Cells, Histones, Humans, Kinetics, Luminescent Proteins, Macromolecular Substances, Microscopy, Fluorescence, Nucleosomes, Photochemistry, Protein Binding, Recombinant Fusion Proteins, S Phase, Transfection