Large-scale chromatin fibers of living cells display a discontinuous functional organization

We investigated the chromatin organization of living cells with a combinati on of recently developed approaches for histone and DNA labeling. Nucleosom al DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusio n protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucl eolar regions chromatin was organized into large-scale fibers of 2 to 8 mum in length and 300 to 500 nm in diameter. Within the nuclear interior we ob served similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection pro cedures revealed major differences in the chromatin organization observed, Chromatin organization revealed by the distribution of histone H2B-GFP was directly compared with the functional organization of chromatin by Cy3-dUTP labeling of DNA replicating at a specific time, DNA regions replicating at a specific time display characteristic physical and functional properties. Analysis of Cy3-labeled foci revealed that they are associated with all th ree forms of chromatin organization (fibrillar, focal and diffuse). In part icular, Cy3-labeled foci appeared as discontinuous regions of large-scale f ibers, These results demonstrate that large-scale chromatin fibers have dis continuous functional characteristics.