Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH).
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 approximately 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 approximately 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.
Solovei
Irina
I
Department of Biology II, Ludwig-Maximilians University of Munich, Germany. irina.solovei@lrz.uni-muenchen.de
Cavallo
Antonio
A
Schermelleh
Lothar
L
Jaunin
Françoise
F
Scasselati
Catia
C
Cmarko
Dusan
D
Cremer
Christoph
C
Fakan
Stanislav
S
Cremer
Thomas
T
D002232:Carbocyanines / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D002478:Cells, Cultured; D002843:Chromatin / Q000648:ultrastructure*; D004261:DNA Replication; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D005347:Fibroblasts / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006657:Histones / Q000032:analysis / Q000235:genetics; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007202:Indicators and Reagents / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014407:Tumor Cells, Cultured
C088941:3'-deoxy-5-(cyanine dye 3)uridine 5'-trisphosphate; D002232:Carbocyanines; D002843:Chromatin; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D006657:Histones; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins