Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA

Abstract

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Figure 1

Gel electrophoresis from different steps of nucleoskeleton preparations from stimulated human lymphocytes. (a) DNA was purified from agarose-embedded cells without cutting with enzymes (controls, 1), after cutting with EcoRI and HaeIII (2), and after cutting and removal of chromatin (3). Note the presence of nucleosomal bands in lane 2. Samples of DNA (1 μg in 1 and 2 and 0.5 μg in 3) were subjected to electrophoresis on a 1.7% agarose gel (M1: λ/EcoRI, HindIII; M2: pUCBM21/HpaII, DraI, HindIII). (b) Extracted fraction of chromatin after gel electrophoresis of cells cut with EcoRI and HaeIII. The two lanes displayed were loaded with roughly equal numbers of beads. Electrophoresis was performed on a 0.8% agarose gel that was subsequently treated with RNAse A and proteinase K (see Materials and Methods) before staining with ethidium bromide. (c) DNA hybridization of the blotted extracted fraction of chromatin using a human total genomic probe.

Figure 2

Unstimulated human lymphocytes, control cells, and nucleoskeleton preparations hybridized with the centromeric alpha-satellite probe and the telomeric repeat probe. Nucleoskeleton preparations were produced by permeabilizing the agarose-encapsulated cells with 0.2% Triton X-100 and cutting of DNA with the restriction enzymes EcoRI and HaeIII. Afterwards, the cells were placed into the slots of an agarose gel, electroeluted, and used for in situ hybridization. All figures are projections of stacks of optical sections. (a) Control cell, centromeric probe; (b) nucleoskeleton preparation, centromeric probe; (c), control cell, telomeric probe; (d) nucleoskeleton preparation, telomeric probe. Bar, 2 μm.

Figure 3

Unstimulated human lymphocytes, control cells, and nucleoskeleton preparations hybridized with the A (part of the transcribed unit) and D_HH (part of the intergenic spacer) fragments of the ribosomal DNA. (a) Control cell, A fragment; (b) nucleoskeleton preparation, A fragment; (c) control cell, D_HH fragment; (d) nucleoskeleton preparation, D_HH fragment. Bar, 2 μm.

Figure 4

Stimulated human lymphocytes, control cells, and nucleoskeleton preparations hybridized with the A (part of the transcribed unit) and D_HH (part of the intergenic spacer) fragments of the ribosomal DNA. (a) Control cell, A fragment; (b) nucleoskeleton preparation, A fragment; (c) control cell, D_HH fragment; (d) nucleoskeleton preparation, D_HH fragment. Bar, 2 μm.

Figure 5

Stimulated human lymphocytes, control cells, and nucleoskeleton preparations hybridized simultaneously with centromeric alpha-satellite probe and ribosomal DNA probe, or with telomeric repeat probe and ribosomal DNA probe. (a) Control cell, centromeric probe detected with FITC-labeled avidin and A fragment detected with rhodamine-labeled antibodies; (b) nucleoskeleton preparation, hybridization protocol as in a; (c) control cell, D_HH fragment detected with FITC-labeled avidin, telomeric probe detected with rhodamine-labeled antibodies; (d) nucleoskeleton preparation, hybridization protocol as in c. Bar, 2 μm.

Figure 6

Blot hybridization of chromatin electroeluted from beads. (a) Hybridization with the centromeric alpha-satellite probe; (1 and 2) unstimulated lymphocytes, (3 and 4) stimulated lymphocytes. (b) Hybridization with the telomeric probe; (1 and 2) unstimulated lymphocytes, (3 and 4) stimulated lymphocytes. The very faint signal represents most likely nonattached extratelomeric TTAGGG repeats. (c) Unstimulated lymphocytes; (1 and 2) hybridization with the A fragment (part of the transcribed unit of rDNA), (3 and 4) hybridization with the D_HH fragment (part of the intergenic spacer of rDNA). (d) Stimulated lymphocytes; (1 and 2) hybridization with the A fragment, (3 and 4) hybridization with the D_HH fragment. The insert shows dot blots of equal amounts of human placenta-DNA hybridized with the A (left) and D_HH (right) fragments. To be able to compare the amount of eluted A and D_HH fragment in unstimulated as well as stimulated lymphocytes, the signal intensities in the blots c and d were normalized against these dot blots. This results in relative values for signal intensities A:D_HH = 1.62:1 in unstimulated lymphocytes and A:D_HH = 0.97:1 in stimulated lymphocytes.