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

Abstract

We investigated the chromatin organization of living cells with a combination of recently developed approaches for histone and DNA labeling. Nucleosomal DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusion protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucleolar regions chromatin was organized into large-scale fibers of 2 to 8 microm in length and 300 to 500 nm in diameter. Within the nuclear interior we observed similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection procedures 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 three forms of chromatin organization (fibrillar, focal and diffuse). In particular, Cy3-labeled foci appeared as discontinuous regions of large-scale fibers. These results demonstrate that large-scale chromatin fibers have discontinuous functional characteristics.

Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones

“…9g and h). Similar nuclear arrangements of SC35, nucleoli, H4K8ac, and transcriptionally active and heterochromatic domains, as observed here in chicken HD12 cells, have been observed in a great variety of different vertebrate cell types when cultivated as monolayers (see for instance (Moen et al 1995;Sadoni et al 1999;Sadoni et al 2001)). .…”

“…Nuclear organization of the splicing factor SC35 and of active isoforms of RNA Pol II As chromatin-depleted regions are often enriched in splicing factors and represent splicing speckles in monolayer cells (Moen et al 1995;Sadoni et al 2001), which are associated with GC-and gene-rich DNA (Shopland et al 2003), we wondered whether the central DNA-depleted zone in RS nuclei might also be enriched in splicing factors. As the essential splicing factor SC35 is typically enriched in splicing speckles, we performed immunostaining of RS with a specific antibody against SC35.…”

Three-dimensional Organization of pKi-67: A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold

“…Bar ϭ 0.5 m. has been described both as a solenoid and as a zigzag conformation (Bednar et al 1998). On the other hand, the 250-300-nm cord is similar to a level of chromatin folding found in both late-replicating heterochromatic homogeneously staining regions (HSRs), which are several hundreds of mega-base pairs in size , and in living cells (Sadoni et al 2001). …”

Characterization of nuclear compartments identified by ectopic markers in mammalian cells with distinctly different karyotype

“…GC-rich DNA was found to be evenly distributed throughout chromosome territories, whereas AT-rich sequences were found statistically more often in the territory interior (Tajbakhsh et al 2000). The comparison of DAPI staining and H2B-GFP labeled chromatin showed that these two methods recognize different portions of the chromatin (Sadoni et al 2001). However, DAPI-stained AT-rich regions concentrate around the nucleoli and at the nuclear periphery (Saccone et al 2002) and are known to coincide with parts of heterochromatic regions (Minc et al 1999).…”