The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus

Abstract

The nucleus is an intricately structured integration of many functional domains whose complex spatial organization is maintained by a nonchromatin scaffolding, the nuclear matrix. We report here a method for preparing the nuclear matrix with improved preservation of ultrastructure. After the removal of soluble proteins, the structures of the nucleus were extensively cross-linked with formaldehyde. Surprisingly, the chromatin could be efficiently removed by DNase I digestion leaving a well preserved nuclear matrix. The nuclear matrix uncovered by this procedure consisted of highly structured fibers, connected to the nuclear lamina and built on an underlying network of branched 10-nm core filaments. The relative ease with which chromatin and the nuclear matrix could be separated despite extensive prior cross-linking suggests that there are few attachment points between the two structures other than the connections at the bases of chromatin loops. This is an important clue for understanding chromatin organization in the nucleus.

Figure 1

DAPI staining shows that DNA is released from formaldehyde treated nuclei by DNase I digestion. CaSki cells were grown on coverslips and processed as described. (Upper) Cells were permeabilized with 0.5% Triton X-100 in CSK buffer before formaldehyde cross-linking and DAPI staining. (Left) Phase contrast image. (Right) Fluorescent image. (Lower) Cells were treated in the same way and then digested with 400 μg/ml DNase I for 50 min at 32°C before staining. The micrograph exposures and magnifications are identical to those in the Upper panels. Almost all detectable DNA was removed by the DNase I digestion despite the prior, extensive cross-linking of the structure.

Figure 2

Histones are released from formaldehyde-treated nuclei by DNase I digestion. CaSki cells were labeled with [³H]thymidine before fractionation, reversal of cross-links, and electrophoretic analysis as described. Histones, because of their very high lysine content, strongly labeled. Most of the histones detectable in this experiment were released from the nucleus by DNase I digestion (lane 1). Few were released by postdigestion washes (lane 2). Little histone was retained with the cross-link stabilized nuclear matrix (lane 3). The positions of histones H1 and H4 on the gel are marked with histones H2A, H2B, and H3 running between them.

Figure 3

The ultrastructure of the nuclear matrix revealed by resinless section electron microscopy. The nuclear matrix of a CaSki cell was prepared by the cross-link stabilized nuclear matrix preparation procedure and visualized by resinless section electron microscopy (–29). (A) The nuclear matrix consisted of two parts, the nuclear lamina (L) and a network of intricately structured fibers connected to the lamina and well distributed through the nuclear volume. The matrices of nucleoli (Nu) remained and were connected to the fibers of the internal nuclear matrix. Three remnant nucleoli may be seen in this section. Few intermediate filaments were connected to the outside of the lamina. (B) Seen at higher magnification the highly structured fibers of the internal nuclear matrix seemed to be built on an underlying structure of 10-nm filaments that are occasionally branched. These were seen most clearly when, for short stretches, they were free of covering material (arrowheads). The irregular fibers, with granules well integrated into their structure, may be built on this filamentous core structure. [Bars = 1 μM (A) and 100 nm (B).]

Figure 4

The nuclear matrix retains the RNP network of the unextracted nucleus with good structural preservation. CaSki cells were prepared for conventional thin section microscopy before (A) and after (B) the removal of chromatin by the cross-linking/DNase I procedure. Thin sections were selectively stained for RNA by the EDTA regressive staining procedure (14) to visualize the RNP network that is an important part of the nuclear matrix. The nuclear lamina (L) forms the periphery of both nucleus (A) and nuclear matrix (B). The removal of chromatin after formaldehyde cross-linking did not substantially alter the structure or spatial distribution of the nuclear RNP network. (C) At higher magnification interchromatin granule clusters, sites of RNA-splicing factor concentration, could be seen with good preservation in the RNP network of the cross-link stabilized nuclear matrix. The CaSki nuclear matrix in this panel was counterstained with the B4A11 antibody which recognizes an RNA splicing factor and with a colloidal gold-conjugated second antibody. This comparison of RNP network ultrastructure in the nucleus and cross-link stabilized nuclear matrix showed good preservation of architecture. The RNP network was not substantially altered by the removal of chromatin if the structure was first extensively cross-linked. [Bars = 500 nm (A and B) and 200 nm (C).]