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Contents
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Generic Features of Tertiary Chromatin Structure
Various studies have attempted to visualize tertiary chromatin structure, but virtually all of them have examined the structures in cells that were subjected to various extraction procedures and/or chemical fixation. A prevailing concern about these studies has, therefore, been whether the structures detected are artifacts of the preparation procedure. To address this concern, we and others have examined tertiary chromatin structures in live cells. Although the resolution limit of the light microscope (approximately 0.2 μm) does not permit visualization of the folding of the 10- or even 30-nm chromatin fibers, live cell imaging completely eliminates the question of fixation artifacts. We achieved live cell imaging of tertiary structures by using large tandem arrays of transcription factor binding sites integrated into a chromosome and then visualized by a green fluorescent protein (GFP) tag. When transcriptionally activated, these tandem arrays unfolded from a single punctum (about 0.5 μm in diameter) into a series of adjacent puncta that we referred to as “beads.” These beaded structures rapidly refolded into a single bead when transcription was inhibited. Although tandem arrays occur naturally—for example, the ribosomal gene cluster—most normal chromatin is composed of different genes with different promoters that are often widely interspersed with non-coding DNA. Thus, despite the advantage of imaging live cells, it is not clear whether the results from tandem array systems can be extrapolated to most chromatin. Consequently, we identified completely natural systems in which to investigate tertiary chromatin structure. As a start, we hypothesized that the tandem arrays would be good models for regions of natural chromatin where a high percentage of genes were active. We identified two such regions. One was the MHCII cluster on human chromosome 6 in which at least 13 genes spanning a nearly 750-kb domain are simultaneously activated by interferon. The other was a nearly 400-kb region on human chromosome 22, which we identified by a bioinformatics analysis of microarray data. We found that this region was much more transcriptionally active in Jurkat cells than in Raji cells. At present there is no live cell imaging technique to visualize tertiary chromatin structure in these transcriptionally active domains, so we resorted to a fixation and hybridization procedure known as DNA fluorescent in situ hybridization (FISH). To validate that this DNA FISH procedure did not disrupt the tertiary structures observed by light microscopy, we compared DNA FISH images of the tandem array with live cell images and showed that the two yielded comparable structures. With confidence that tertiary structure would be preserved, we then used DNA FISH to examine these structures in the transcriptionally active domains on human chromosomes 6 and 22. Remarkably, we found structures very similar to those detected in the live cell tandem arrays. The prominent feature was again a series of adjacent puncta or beads approximately 0.5 μm in diameter that refolded into a single bead when transcription was inhibited. Thus, despite vast differences in gene density and levels of transcriptional activation, these disparate chromosomal domains shared common beaded features of tertiary chromatin structure. Consequently, we propose that beads may be generic features of higher-order chromatin organization. The presence of beads in light microscope images indicates that higher-order chromatin must be clustered into largely distinct subdomains. The ability of these beads to split into a series of adjacent beads indicates that the folding of chromatin within a cluster or bead must be sufficiently organized to readily enable this bifurcation process. These results, therefore, impose constraints on how tertiary chromatin must be organized. The determination of the actual folding patterns of a 30-nm chromatin fiber within a bead requires higher resolution imaging than afforded by light microscopy. We are now pursuing methods for correlative fluorescence and X-ray microscopy that will permit three-dimensional reconstruction of cryo-preserved intact nuclei at 20-nm resolution and, thereby, enable us to assess 30-nm chromatin fiber organization within the beaded structures detected in our GFP-tagged tandem array.
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processes involving DNA, including replication, transcription, and repair, must
occur within the context of chromatin. However, this packaging of the DNA molecule
is understood with certainty at only its lowest level of organization, namely
the wrapping of DNA around nucleosomes yielding a fiber 10 nm in diameter. This
nucleosomal fiber is further wound in some way—the details are still controversial—to
yield a thicker fiber of approximately 30 nm. Yet these first two levels of
packaging are just the beginning, as much more compaction is required to fit
our genome into the nucleus. These unknown higher levels of chromatin folding
are referred to as higher-order or “tertiary” chromatin structure.
Our recent work has suggested that generic, conserved features are within such
tertiary chromatin structure.