Remodeling of nuclear architecture by the thiodioxoxpiperazine metabolite chaetocin.

Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored.

Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.

Fluorescence in situ hybridization (FISH) of specific DNA probes has become a widely used technique mostly for chromosome analysis and for studies of the chromosomal location of specific DNA segments in metaphase preparations as well as in interphase nuclei. FISH on 3D-preserved nuclei (3D-FISH) in combination with 3D-microscopy and image reconstruction is an efficient tool to analyze the spatial arrangement of targeted DNA sequences in the nucleus. Recent developments of a "new generation" of confocal microscopes that allow the distinct visualization of at least five different fluorochromes within one experiment opened the way for multicolor 3D-FISH experiments. Thus, numerous differently labeled nuclear targets can be delineated simultaneously and their spatial interrelationships can be analyzed on the level of individual nuclei.In this chapter, we provide protocols for the preparation of complex DNA-probe sets suitable for 3D-FISH with up to six different fluorochromes, for 3D-FISH on cultured mammalian cells (growing in suspension or adherently) as well as on tissue sections, and for 3D immuno-FISH.In comparison with FISH on metaphase chromosomes and conventional interphase cytogenetics, FISH on 3D-preserved nuclei requires special demands with regard to probe quality, fixation, and pretreatment steps of cells in order to achieve the two goals, namely the best possible preservation of the nuclear structure and at the same time an efficient probe accessibility.

Histone lysine methylation patterns in human cell types are arranged in distinct three-dimensional nuclear zones.

The impact of histone lysine methylation as an essential epigenetic mechanism for gene regulation has been demonstrated by numerous studies where it was functionally and structurally linked to euchromatin and heterochromatin. Most of these data have been obtained by biochemical and two-dimensional (2D)-microscopic techniques providing little information about the global nuclear arrangement of histone modifications. We investigated the 3D architecture and spatial interrelationships of different histone lysine methylation sites (tri-H3K4, mono-H4K20, mono-H3K9, tri-H3K27, tri-H4K20 and tri-H3K9) in various human cell types. Immunofluorescence and confocal microscopy were used together with a quantitative evaluation of 3D images, to reveal spatial relations of specific methylation sites with either centromeres, nascent RNA or with each other. A close association with centromeres was found only for histone methylation sites previously linked to constitutively repressed chromatin. Differences observed in these sites in relation to the cell cycle emphasize the potential relevance of the dynamic properties of heterochromatin for nuclear functions. Nascent RNA was found associated, though to a different degree, with all histone methylation sites, supporting the increasing evidence that transcription occurs across a wide range of the human genome. Finally we demonstrated by simultaneous visualization of different histone lysine methylation sites that methylation patterns are organized in distinct nuclear zones with little apparent intermingling.