Irina Solovei*, @, Antonio Cavallo 1, Lothar Schermelleh*, Françoise Jaunin 2, Catia Scasselati 2, Dusan Cmarko 2, Christoph Cremer 1, Stanislav Fakan 2, and Thomas Cremer*
*Department of Biology II, Ludwig-Maximilians University of Munich,
Germany
1 Kirchhoff Institute for Applied Physics, University
of Heidelberg, Heidelberg, Germany
2 Centre of Electron Microscopy, University of Lausanne,
Lausanne, Switzerland
@ To whom correspondence and reprint requests should be
addressed.
Fax: (+49) 89 21806719.
E-mail: irina.solovei@lrz.uni-muenchen.de
3D-FISH has become a major tool for studying the higher order chromatin
organization in the cell nucleus. It is not clear, however, to what extent
chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects
the order in living cells. To study this question, we compared higher order
chromatin arrangements in living cells with those found after the 3D-FISH
procedure. For in vivo studies we employed replication labeling
of DNA with Cy3-conjugated nucleotides and/or chromatin labeling by GFP-tagged
histone 2B. At the light microscope level, we compared the intranuclear
distribution of
H2B-GFP-tagged chromatin and the positions of replication-labeled
chromatin domains in the same individual cells in vivo, after fixation
with 4% paraformaldehyde, and after 3D-FISH. Light microscope data demonstrate
a high degree of preservation of the spatial arrangement of ~1-Mb chromatin
domains. Subsequent electron microscope investigations of chromatin structure
showed strong alterations in the ultrastructure of the nucleus caused mainly
by the heat denaturation step. Through this step chromatin acquires the
appearance of a net with mesh size of 50-200 nm roughly corresponding to
the average displacement of the chromatin domains observed at light microscope
level. We conclude that 3D-FISH is a useful tool to study chromosome territory
structure and arrangements down to the level of ~1-Mb
chromatin domain positions. However, important ultrastructural details
of the chromatin architecture are destroyed by the heat denaturation step,
thus putting a limit to the usefulness of 3D-FISH analyses at nanometer
scales.
1. Mahy NL, Perry PE, Gilchrist S, Baldock RA, and Bickmore WA, "Spatial Organization of Active and Inactive Genes and Noncoding DNA within Chromosome Territories".
2. Tanabe H, Mueller S, Neusser M, von Hase J, Calcagno E, Cremer M, Solovei I, Cremer C, and Cremer T, "Evolutionary Conservation of Chromosome Territory Arrangements in Cell Nuclei from Higher Primates".
3. Frenster JH, Nakatsu SL, and Masek MA, "Ultrastructural Probes of DNA Templates within Human Bone Marrow and Lymph Node Cells".
4. Frenster JH, "Uni-Polar Clustering of Lymphocyte DNA Templates Toward Neoplastic Target Cells Within Hodgkin's Disease Lymph Nodes".
5. Frenster JH, Papalian MM, Masek MA, and Frenster JA, "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease".
6. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bone Marrow Cells".
7. Frenster JH, "Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".
8. Frenster JH, "Activation of DNA Transcription within Repressed Chromatin".
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