Nuclear envelope organization in papillary thyroid carcinoma

Papillary thyroid carcinomas (PTCs) have characteristic nuclear shape changes compared to follicular-type thyroid epithelium. We tested the hypothesis that the altered nuclear shape results from altered distribution or expression of the major structural proteins of the nuclear envelope. Lamin A, lamin B1, lamin C, lamin B receptor (LBR), lamina-associated polypeptide 2 (LAP2), emerin, and nuclear pores were examined. PTC's with typical nuclear features by H&E were compared to non-neoplastic thyroid and follicular neoplasms using confocal microscopy, and semi-quantitative immunoblotting. Lamin A/C, lamin B1, LAP2, emerin, and nuclear pores all extend throughout the grooves and intranuclear inclusions of PTC. Their distribution and fluorescent intensity is not predictably altered relative to nuclear envelope irregularities. By immunoblotting, the abundance (per cell) and electrophoretic mobilities of lamin A, lamin B1, lamin C, emerin, and LAP2 proteins do not distinguish PTC, normal thyroid, or follicular neoplasms. These results do not support previously published predictions that lamin A/C expression is related to a loss of proliferative activity. At least three LAP2 isoforms are identified in normal and neoplastic thyroid. LBR is sparse or undetectable in all the thyroid samples. The results suggest that the irregular nuclear shape of PTC is not determined by these nuclear envelope structural proteins per se. We review the structure of the nuclear envelope, the major factors that determine nuclear shape, and the possible functional consequences of its alteration in PTC.     

Nuclear visions enhanced: chromatin structure, organization and dynamics

The EMBO Workshop on 'Chromatin Structure, Organization and Dynamics' took place in April 2011 in Prague, Czech Republic. Scientists from the life sciences, chemistry and biophysics presented their latest data on the generation of three-dimensional and, eventually, four-dimensional models of the genome, working to correlate changes in the organization of chromatin with the functional state of the genome.     

Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies

The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.     

The epigenetics of nuclear envelope organization and disease

Mammalian chromosomes and some specific genes have non-random positions within the nucleus that are tissue-specific and heritable. Work in many organisms has shown that genes at the nuclear periphery tend to be inactive and altering their partitioning to the interior results in their activation. Proteins of the nuclear envelope can recruit chromatin with specific epigenetic marks and can also recruit silencing factors that add new epigenetic modifications to chromatin sequestered at the periphery. Together these findings indicate that the nuclear envelope is a significant epigenetic regulator. The importance of this function is emphasized by observations of aberrant distribution of peripheral heterochromatin in several human diseases linked to mutations in NE proteins. These debilitating inherited diseases range from muscular dystrophies to the premature aging progeroid syndromes and the heterochromatin changes are just one early clue for understanding the molecular details of how they work. The architecture of the nuclear envelope provides a unique environment for epigenetic regulation and as such a great deal of research will be required before we can ascertain the full range of its contributions to epigenetics.     

Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos

Chicken sperm chromatin initiated an assembly of interphase-like nuclei in a cell-free cytoplasmic preparation from 1-6 h old Drosophila melanogaster embryos. The formation of these interphase-like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti-Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell-free extract.     

Nuclear organization: Uniting replication foci,chromatin domains and chromosome structure

In higher eukaryotes, ‘replication factories’ coordinate DNA synthesis within local clusters of chromatin domains. Recent experiments^(1,2) have confirmed the complexity of these clusters and established that the organization of sites labelled during S phase persists throughout the cell cycle. This implies that domain clusters are critical elements of an hierarchy that is fundamental to both nuclear and chromosome structure.     

Nuclear envelope structure.

The past 18 months have seen significant advances in our knowledge of the constituents of the nuclear envelope, their interactions during interphase and the mechanisms involved in their mitotic dynamics. Although most of the new data are in general agreement with, and contribute detail to, our traditional image of the nuclear envelope, a few observations appear to mark the beginning of new and important directions in research.     

Nuclear envelope dynamics.

The nuclear envelope (NE) provides a semi permeable barrier between the nucleus and cytoplasm and plays a central role in the regulation of macromolecular trafficking between these two compartments. In addition to this transport function, the NE is a key determinant of interphase nuclear architecture. Defects in NE proteins such as A-type lamins and the inner nuclear membrane protein, emerin, result in several human diseases that include cardiac and skeletal myopathies as well as lipodystrophy. Certain disease-linked A-type lamin defects cause profound changes in nuclear organization such as loss of peripheral heterochromatin and redistribution of other nuclear envelope components. While clearly essential in maintenance of nuclear integrity, the NE is a highly dynamic organelle. In interphase it is constantly remodeled to accommodate nuclear growth. During mitosis it must be completely dispersed so that the condensed chromosomes may gain access to the mitotic spindle. Upon completion of mitosis, dispersed NE components are reutilized in the assembly of nuclei within each daughter cell. These complex NE rearrangements are under precise temporal and spatial control and involve interactions with microtubules, chromatin, and a variety of cell-cycle regulatory molecules.     

Supranucleosomal organization of chromatin

A systematic study of the effect of different ionic conditions on the morphology of the 25–30 nm chromatin fiber from chicken erythrocytes has revealed that, as the ionic strength is increased, knobby fibers with a clear superbead structure are formed in the presence of either Mg⁺⁺ or Na⁺, or both. A further increase in ionic strength results in smooth chromatin fibers due to a tight packing of superbeads. Cross-linking such fibers with formaldehyde and reversal of the ionic conditions, demonstrates the superbead structures underlying the smooth fibers observed at high ionic concentrations. The average size of the superbeads is 34 nm along the length of the fibers, in agreement with the value found in embedded sea cucumber chromatin. A second class of superbeads has an average length of 25 nm and probably corresponds to partially disrupted structures.     

Nucleomeric organization of chromatin

Chromatin in the nuclei fixed in tissue and in the nuclei isolated by low ionic strength solutions in the presence of Mg2+ is represented by globular (nucleomeric) fibrils, 20-25 nm in diameter. The staphylococcal or endogenous nuclear nuclease splits the chromatin fibrils resulting in fragments corresponding to nucleomers and their multimers. Upon removal of firmly bound Mg2+ the nucleomers unfold to form chains consisting of 4-6-8 nucleosomes. Mild hydrolysis of nuclear chromatin by staphylococcal nuclease results in a split-off of mono-, di- and trimers of nucleomers sedimenting in a sucrose density gradient in the presence of EDTA as particles with the sedimentation coefficients of 37, 47 and 55S, respectively. The sedimentation coefficient for the mononucleomer in a sucrose density gradient with MgCl2 is 45S. Determination of the length of DNA fragments of chromatin split-off by staphylococcal nuclease showed that the nucleomer consists of 8 nucleosomes, while the dimer and trimer of the nucleomer consists of 14-16 and 21-24 nucleosomes, respectively. The nucleomeric monomer undergoes structural transition from the compact (45S) to the "loose" state (37S) after removal of Mg2+. This transition is completely reversible, when the nucleomer contains histone H1. The removal of the latter or dialysis of the nucleomer against EDTA in low ionic strength solutions results in a complete unfolding of the nucleomer into a nucleosomal chain fragment. A model for the nucleomer fibril structure in which the helical organization of the nucleosomal chain in the nucleomer (2 turns with 4 nucleosomes in each) is alternated with the impaired helical bonds between the nucleomers is discussed. The functional significance of the nucleomeric organization of chromatin may be an additional restriction of the site-specific recognition of DNA in chromatin with the possibility of local (at the level of one nucleomer) changes in chromatin conformation excluding this restriction.     

Ribosomal chromatin organization.

Mammalian cells contain approximately 400 copies of the ribosomal RNA genes organized as tandem, head-to-tail repeats spread among 6-8 chromosomes. Only a subset of the genes is transcribed at any given time. Experimental evidence suggests that, in a specific cell type, only a fraction of the genes exists in a conformation that can be transcribed. An increasing body of study indicates that eukaryotic ribosomal RNA genes exist in either a heterochromatic nucleosomal state or in open euchromatic states in which they can be, or are, transcribed. This review will attempt to summarize our current understanding of the structure and organization of ribosomal chromatin.     

Nuclear DNA contents, rDNAs, chromatin organization, and karyotype evolution inVicia sect,faba

Summary Automated karyotype analyses, nuclear DNA contents, and sequences of rDNA internal transcribed spacers of the nine species inVicia sect.faba are reported. As karyomorphological parameters are used to evaluate the karyotype evolution, so the determination of the heterochromatin by Feulgen absorption at different thresholds of optical density provided further evidence on the chromatin organization withinVicia sect.faba. The comparison of sequences of rDNA spacers has enabled the definition of the phylogenetic relationships between the analyzed species.     

Revisiting higher-order and large-scale chromatin organization

The past several years has seen increasing appreciation for plasticity of higher-level chromatin folding. Four distinct '30nm' chromatin fiber structures have been identified, while new in situ imaging approaches have questioned the universality of 30nm chromatin fibers as building blocks for chromosome folding in vivo. 3C-based approaches have provided a non-microscopic, genomic approach to investigating chromosome folding while uncovering a plethora of long-distance cis interactions difficult to accommodate in traditional hierarchical chromatin folding models. Recent microscopy based studies have suggested complex topologies co-existing within linear interphase chromosome structures. These results call for a reappraisal of traditional models of higher-level chromatin folding.