Visualizing nuclear proteins together with transcribed and inactive genes in structurally preserved cells.
Recent data support the idea that the mammalian nucleus is organized in a functionally significant way. Immunocytochemistry has revealed the existence of diverse nuclear "bodies" and compartments. Fluorescence in situ hybridization (FISH) has shown that chromosomes change their spatial relationships during dynamic cell cycle progression and that nuclear organization can change during development and differentiation when patterns of gene expression are established or modified. To determine the relationship between nuclear organization and genome function is an important goal for biology. This article describes an immunoFISH technique, which is a useful tool for investigating the functional organization of the nucleus. It combines immunocytochemistry with FISH to allow associations between proteins, DNA, and RNA to be visualized in a single-step analysis using confocal microscopy. Immunocytochemistry and FISH were thought incompatible since cell preparation flattens nuclei and the harsh DNA denaturation treatment required for FISH destroys proteins. The immunoFISH technique successfully overcomes these problems and can reveal interactions between nuclear components not readily detectable using other experimental approaches. The interactions of single-copy, endogenous loci with nuclear proteins or bodies can be seen, as can spatial compartmentalization of these loci, in cells preserved in three dimensions, representative of the situation in vivo. Allelic differences in transcription can be related to nuclear location and protein interactions of the individual alleles since genes, RNA and proteins can be visualized together. Chromosome behavior can be followed through mitosis to investigate centromere activity or vector segregation efficiency, for example. Visual data obtained using the immunoFISH technique have provided insight into the functional significance of nuclear organization and its role in cell biology.
Brown
Karen
K
MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. karen.brown@csc.mrc.ac.uk
D004268:DNA-Binding Proteins; D020869:Gene Expression Profiling; D051740:Ikaros Transcription Factor; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051740:Ikaros Transcription Factor