Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold

Mammalian sperm DNA is the most tightly compacted eukaryotic DNA, being at least sixfold more highly condensed than the DNA in mitotic chromosomes. To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin. This differs markedly from the bulkier DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. The overall organization of mammalian sperm DNA, however, resembles that of somatic cells in that both the linear arrays of sperm chromatin and the 30-nm solenoid filaments of somatic cell chromatin are organized into loop domains attached at their bases to a nuclear matrix. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored when the nuclear matrix is disrupted during decondensation. In somatic cells, proper function of DNA is dependent upon the structural organization of the DNA by the nuclear matrix, and the structural organization of sperm DNA is likely to be just as vital to the proper functioning of the spermatozoa.

DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells.

https://www.cell.com/cell/pdf/0092-8674(85)90117-5.pdf

Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases.

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

https://rupress.org/jcb/article-pdf/110/3/569/1059214/569.pdf

Core filaments of the nuclear matrix.

Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.

https://science.sciencemag.org/content/sci/259/5099/1326.full.pdf

Higher level organization of individual gene transcription and RNA splicing

In the chicken oviduct, it has been well documented that steroid hormones stimulate the transcription of specific genes such as the ovalbumin gene. In addition to the presence of specific hormone receptors in the tissue, gene expression seems to require that target genes exist in large DNase I sensitive chromosomal domains. This structure appears necessary but not sufficient for transcriptional activation. In search of still other levels of control, we have investigated the interactions of genes with the nuclear matrix, a structure which has been implicated in DNA synthesis, transcription and RNA processing. Here we have isolated nuclear matrix and used a nondegradative method to fractionate nuclear DNA based on its preferential association with the matrix. The preparation was digested with a restriction enzyme and both matrix-bound and released DNAs were recovered. We found that only actively expressed genes were associated with the matrix. Furthermore, within a 100-kilobase (kb) DNase I sensitive chromosomal domain, only the transcribed regions were associated with the matrix. This association was shown to be reversible when hormone was withdrawn. Our results suggest that the nuclear matrix is the site of nuclear transcription and may represent another potential level of control for regulation of gene expression in the eukaryotic cell.

Elena M. Ciejek

Papers

Actively transcribed genes are associated with the nuclear matrix