During interphase, chromosomes are arranged into territories within a highly organized nuclear space containing several compartments. It is becoming clear that this complex nuclear arrangement is important for gene regulation and therefore expression. The study of chromosome organization in interphase requires high-resolution imaging methods that at the same time allow for flexible labelling strategies and preserve nuclear structure. Tokuyasu cryosections of cells or tissues provide a simple, high-resolution platform for performing immunolabelling and fluorescence in situ hybridization (FISH) on well-preserved samples. Here we show how FISH performed on thin cryosections (cryo-FISH) can be used for the study of chromosome organization at high resolution and in a quantitative manner. We have measured chromosome intermingling, volume and radial position, in resting and activated human lymphocytes, and observed chromosome-specific differences between the two cellular states. These differences are in part related to the nuclear expansion that occurs during activation, but are also likely to be tied to their different transcriptional profiles. Extrapolation of our dataset to the whole genome suggests that activated cells contain a lower amount of chromatin involved in intermingling than resting cells.
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Relative and absolute intermingling volumes in resting and PHA-activated human lymphocytes obtained after cryo-FISH (DOC 26 kb).
Relative and absolute chromosome volumes in resting and PHA-activated human lymphocytes obtained after cryo-FISH (DOC 27 kb).
CT radial positions in resting and PHA-activated human lymphocytes obtained after cryo-FISH (DOC 27 kb).
Example of the empirical thresholding criteria. A cryo-FISH of chromosome 5 (A) was segmented using different threshold levels of empirically find a mask that best fits the fluorescent signal from the chromosome paint (B). The mask of the ynder-thresholded image includes patches away from the main body of the CT that are likely to be background (arrowhead), whilst the mask of the over-thresholded image excludes areas of fluorescence that are clearly above the background level (arrowhead). Bar, 0.5 μm. (TIFF 1.81 mb)
Frequency of section radii upon random sectioning. A, Schematic representation of how to obtain the frequency (f) of a class of section sizes from their radii (ri) and the 3D nuclear radius (R), using the formula shown on the right (zi is the axial position of the section of radius ri). B, Relative frequency of the different section sizes obtained from the sectioning of a nucleus of radius 1. (TIFF 887 kb)
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Branco, M.R., Branco, T., Ramirez, F. et al. Changes in chromosome organization during PHA-activation of resting human lymphocytes measured by cryo-FISH. Chromosome Res 16, 413 (2008). https://doi.org/10.1007/s10577-008-1230-x
- chromosome territory
- lymphocyte activation
- radial position