Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH)

Irina Solovei, Antonio Cavallo, Lothar Schermelleh, Françoise Jaunin, Catia Scasselati, Dusan Cmarko, Christoph Cremer, Stanislav Fakan, Thomas Cremer

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)10-23
Number of pages14
JournalExperimental Cell Research
Volume276
Issue number1
DOIs
Publication statusPublished - 2002

Keywords

  • 3D-FISH
  • H2B-GFP
  • Nucleus ultrastructure
  • Replication labeling in vivo

ASJC Scopus subject areas

  • Cell Biology

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