Spatial association of homologous pericentric regions in human lymphocyte nuclei during repair

Abstract

Spatial positioning of pericentric chromosome regions in human lymphocyte cell nuclei was investigated during repair after H(2)O(2)/L-histidine treatment. Fifteen to three-hundred minutes after treatment, these regions of chromosomes 1, 15, and X were labeled by fluorescence in situ hybridization. The relative locus distances (LL-distances), the relative distances to the nuclear center (LC-distances), and the locus-nuclear center-locus angles (LCL-angles) were measured in approximately 5000 nuclei after two-dimensional microscopy. Experimental frequency histograms were compared to control data from untreated stimulated and quiescent (G(0)) nuclei and to a theoretical two-dimensional projection from random points. Based on the frequency distributions of the LL-distances and the LCL-angles, an increase of closely associated labeled regions was found shortly after repair activation. For longer repair times this effect decreased. After 300 min the frequency distribution of the LL-distances was found to be compatible with the random distance distribution again. The LL-distance frequency histograms for quiescent nuclei did not significantly differ from the theoretical random distribution, although this was the case for the stimulated control of chromosomes 15 and X. It may be inferred that, concerning the distances, homologous pericentric regions appear not to be randomly distributed during S-phase, and are subjected to dynamic processes during replication and repair.

FIGURE 1

Example of a cell nucleus showing the interactively determined nuclear diameter (d_n), the locus distance (LL-distance), the locus-to-nuclear-center distance (LC-distance), and the locus-nuclear center-locus angle (LCL-angle) for quantitative evaluation. Bars (L₁; L₂) indicate the hybridization signals. C = center of the cell nucleus; d_n = diameter of the nucleus. Shading represents the total DNA stained with DAPI, whereas the solid spots indicated with L₁ and L₂ are the labeled centromeres.

FIGURE 2

Frequency histogram of projected distances of randomly distributed points in a three-dimensional ball relative to the unit diameter. The individual columns of the histogram represent all values in a range of 0.1 d_n each. The frequencies are calculated by numerical simulation (see Materials and Methods).

FIGURE 3

False color images of cell nuclei of stimulated human lymphocyte after thymidine treatment and BrdU incorporation. The DNA of the nuclei was counterstained with DAPI (a). Pericentric regions of chromosome X were labeled with rhodamine (b). Fluorescence labeling of the BrdU incorporation sites by FITC (c) indicated that from three-to-five nuclei were in S-phase for this image. In the merged image [(d) = (a)+(b)+(c)] the nuclei were classified as 1, G₀/G₁ cells, associated; 2 and 4, S-phase, associated X-centromeres; 3, S-phase, nonassociated X-centromeres; and 5, G₀/G₁-cells, nonassociated.

FIGURE 4

Frequency distributions of LL-distances relative to the diameter of the cell nucleus (d_n) for nuclei subjected to the indicated treatments and in the indicated repair states. The individual columns represent the frequency of values in a range of 0.1 d_n each.

FIGURE 5

Frequency distributions of LC-distances relative to the diameter of the cell nucleus (d_n) for nuclei subjected to the indicated treatments and in the indicated repair states. The individual columns represent the frequency of values in a range of 0.1 d_n each.

FIGURE 6

Frequency distributions of LCL-angles for nuclei subjected to the indicated treatments and in the indicated repair states. The individual columns represent the frequency of values in a range of 10° each.

FIGURE 7

Association of pericentric regions shown in optical sections through three-dimensionally preserved cell nuclei. The solid spots are the labeled centromeres #1, visible in a group of five cells. The typical height of the cell nuclei was 6.7 ± 0.5 μm as measured by confocal laser scanning microscopy. BrdU incorporation during S-phase is shown in shading. In this example, according to the 0.15 d_n criterion, two of the five nuclei showed associated centromeres (c and d) after measuring the distance in three dimensions by the LSM software 3.0 (Carl Zeiss).