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

The nuclear lamina, a protein meshwork lining the nucleoplasmic surface of the inner nuclear membrane, is thought to provide a framework for organizing nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromatin. In several higher eukaryotic cells, the lamina appears to be a polymer comprised mainly of one to three immunologically related polypeptides of relative molecular mass (Mr) 60,000-75,000 (60-70K) termed lamins. Three lamins (A, B, and C) are typically present in mammalian somatic cells. Previous studies on nuclear envelopes of rat liver and Xenopus oocytes suggested that the lamina has a fibrillar or filamentous substructure. Interestingly, protein sequences recently deduced for human lamins A and C from complementary DNA clones indicate that both of these polypeptides contain a region of approximately 350 amino acids very similar in sequence to the coiled-coil alpha-helical rod domain that characterizes all intermediate-type filament (IF) proteins. Here we analyse the supramolecular organization of the native nuclear lamina and the structure and assembly properties of purified lamins, and show that the lamins constitute a previously unrecognized class of IF polypeptides.

The nuclear lamina is a meshwork of intermediate-type filaments.

https://www.cell.com/cell/pdf/0092-8674(86)90761-0.pdf

Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.

https://rupress.org/jcb/article-pdf/100/5/1706/1051652/1706.pdf

Topoisomerase II is a structural component of mitotic chromosome scaffolds

MECHANISMS OF NUCLEOCYTOPLASMIC TRANSPORT 343 The Pore Complex as a Molecular Seive 344 Localization Sequences in Nuclear Proteins 345 Functions of Nuclear Localization Signals 347 Regulated Protein Import into Nuclei . . . . . . . . . .. . . . . . . ...... . . . . . . . . . . . . . 348 Export of RNA from the Nucleus 349 Cell-free Nuclear Transport Systems 351 The Nuclear Pore Complex as a Macromolecular Translocation Machine 352

Functional organization of the nuclear envelope

Non-histone proteins and long-range organization of HeLa interphase DNA.

Evidence for two levels of DNA folding in histone-depleted HeLa interphase nuclei☆☆☆

SUMMARY The protein compositions of purified metaphase chromosomes, nuclei and their residual scaffold and matrix structures, are reported. The protein pattern of nuclei on sodium dodecyl sulphate/polyacrylamide gels is considerably more complex and rich in non-histone proteins than that of chromosomes. Nuclei contain about three to four times more non-histone proteins relative to their histones than chromosomes. Besides the protein components of the peripheral lamina, several protein bands are specific or at least highly enriched in nuclei. Conversely, two proteins X0 (33×10 3 M r ) and X1 (37×10 3 M r ) are highly enriched in the pattern of metaphase chromosomes. We have compared morphologically the previously defined nuclear matrices type I and II. The type I nuclear matrix is composed of the known lamina proteins, which form the peripheral lamina structure, and a complex series of proteins that form the internal network of the matrix as observed by electron microscopy. This internal network is stabilized similarly to the metaphase scaffolding by metalloprotein interaction. Both the scaffolding and the internal network of the matrix dissociate if thiols or certain metal chelators are used in the extraction buffer. Under these conditions the resulting nuclear structure, called matrix type II, appears empty in the electron microscope, with the exception of some residual nucleolar material. This latter material can be extracted from the internal network by exhaustive treatment of the nuclei with RNase before extraction with high salt. Immunoblotting and activity studies show RNA polymerase II to be tightly bound to the type I, but not to the type II matrix, or to the scaffolding structure. No polymerase II enzyme was detected in isolated metaphase chromosomes. Another nuclear enzyme, poly(ADP-ribose) polymerase is not bound to either of the residual nuclear matrices or to the scaffolding structures. The association of RNA polymerase with the internal network of the nuclear matrix is consistent with the idea that transcription occurs in close association with this structure.

/pdf/interphase-nuclear-matrix-and-metaphase-scaffolding-34hy0b1aay.pdf

Interphase nuclear matrix and metaphase scaffolding structures.

https://portlandpress.com/biochemsoctrans/article-pdf/9/2/6P/831439/bst009006pa.pdf

Chromosome structural studies

. Recent evidence suggests that metallo-protein interactions are important in maintaining one level of organization of the DNA in histone-depleted chromosomes and nuclei. When treated with the metal chelators, 1,10-phenanthroline, neocuproine, or thiols, but not with EDTA, histone-depleted chromosomes and nuclei unfold their compacted DNA as evidenced by a reduction in their sedimentation coefficient. For nuclei, the s value is reduced by such chelation from a fast form I (18000s) to a slow form II (8500s). With chromosomes, a similar shift from a fast form I (5500s) to a slow form II (1500s) is observed. It is unlikely that this relaxation of the DNA is due to the possible degradative processes of 1,10-phenanthroline known to occur under some conditions.

Jane S. Lebkowski

Papers

Non-histone proteins and long-range organization of HeLa interphase DNA.

Evidence for two levels of DNA folding in histone-depleted HeLa interphase nuclei☆☆☆

Interphase nuclear matrix and metaphase scaffolding structures.

Chromosome structural studies

Evidence for a structural role of copper in histone-depleted chromosomes and nuclei