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.Nucleic acid metabolism is spatially organized in the cell nucleus. The application of powerful microscopy techniques has revealed an increasingly intricate domain organization within the nucleus (reviewed in ref. 1). Individual catalytic processes and the machinery they require are structurally constrained to spatial domains. The very intricate spatial organization of the nucleus presents an important research problem. Our goals are to identify the structure(s) maintaining the complex architecture of the nucleus and to characterize the molecular interactions that constrain components to specific locations.Much of the domain organization of the nucleus remains after the experimental removal of chromatin (2-9). This suggests that chromatin itself is not the fundamental structure organizing the nucleus. In fact, chromatin may itself be architecturally organized in loop domains attached at their bases to an underlying structure (10, 11).There is a second nucleic acid-containing structure distributed throughout the nucleus, an ribonucleoprotein (RNP)-containing network of fibrils and granules selectively stained by the EDTA-regressive method (12)(13)(14). This structure, identified in intact nuclei, corresponds to the nuclear matrix remaining after biochemical fractionation (15-17). The isolated nuclear matrix retains most nuclear RNA (15, 18), RNP proteins (17, 19), and may even require intact RNA for structural integrity (19,20). It is this nuclear matrix to which chromatin loops are anchored (11,21,22).Biochemical studies of the nuclear matrix and detailed ultrastructural studies of its architecture required the development of techniques to remove the larger mass chromatin while leaving the nuclear matrix undisturbed. Two very different and somewhat harsh fractionation protocols have uncovered a network of highly branched 10-nm filaments connected to the nuclear lamina and well distributed through the nuclear volume (18,23). These may form the core structure upon which the RNP-contain...

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

“…Nevertheless, it is possible that the majority of NMP spots which were present consistently on three 2-D gel patterns (0, 2, 4 h after etoposide treatment) may not be related to apoptosis. The relationship between the DNA fragmentation by in situ DNAend labeling and DNA ladder on agarose gels has been reported for apoptotic bone marrow cells and erythroid cells [27], [28]. Our results are in agreement with previous findings, that the characteristic morphology change appeared earlier than DNA fragmentation.…”

Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression

“…In contrast, HERV-K has a diffuse pattern throughout the nucleus and the cytoplasm ( Figure 4A). The localization of L1 proteins is reminiscent of that of the nuclear lamina (Nickerson et al, 1997;see Gruenbaum et al, 2003 for review). A wealth of evidence indicates the essential role of lamin proteins, and the nuclear lamina structure which they form, in control of fundamental functions, including the organization and positioning of active and nonexpressed chromatin, gene regulation, nuclear architecture and differentiation (reviewed by Goldman et al, 2002;Gruenbaum et al, 2003Gruenbaum et al, , 2005.…”

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

“…In situ nuclear matrix was prepared essentially as previously reported [24,25]. Briefly, HeLa cells grown on coverslips were washed twice with ice-cold PBS followed by incubation with CSK buffer [10mM Pipes (pH 6.8)/100mM NaCl/30mM sucrose/3mM MgCl2/1mM EGTA/0.5% (v/v) Triton X-100], supplemented with complete protease inhibitor mixture to remove cytosolic soluble proteins.…”

“…We obtained nuclear and nuclear matrix protein extracts and in situ nuclear matrix preparations by a sequential high salt extraction procedure previously described [22][23][24][25]. To verify the authenticity of the nuclear matrix preparations, we analyzed the distribution of the nuclear matrix proteins lamin B1 and actin as well as the distribution of the soluble transcription factor Sp3.…”