DNA is wrapped by the nuclear aggregates of polyamines: the imaging evidence

In the cell nucleus, putrescine, spermidine, and spermine self-assemble with phosphate ions to generate three forms of compounds, named nuclear aggregates of polyamines (NAPs), which may interact with DNA. In an in vitro setting mimicking the cell nucleus milieu, this molecular aggregation occurs within well-defined ratios. Structural and functional analogies exist between the in vitro NAPs (ivNAPs) and their extractive homologues. The present Article reports images of ivNAPs at different resolution levels. Independent of the DNA template, ivNAPs become hierarchically stacked to produce ultimately macroscopic filamentous structures. The ivNAP-DNA complexes arranged in long and repetitive structures that displayed the self-similar features of natural fractals when dehydrated onto glass slides. Atomic force microscopy showed that ivNAPs have a cyclic structure and dispose around the DNA in a tube-like arrangement. Overall, the images indicate that these aggregates envelope the genomic DNA, thus proving that NAPs play a crucial role in DNA compaction and functioning.     

Nuclear aggregates of polyamines are supramolecular structures that play a crucial role in genomic DNA protection and conformation

In a previous study we showed that natural polyamines interact in the nuclear environment with phosphate groups to form molecular aggregates [nuclear aggregates of polyamines (NAPs)] with estimated molecular mass values of 8000, 4800 and 1000 Da. NAPs were found to interact with genomic DNA, influence its conformation and interfere with the action of nucleases. In the present work, we demonstrated that NAPs protect naked genomic DNA from DNase I, whereas natural polyamines (spermine, spermidine and putrescine) fail to do so. In the context of DNA protection, NAPs induced noticeable changes in DNA conformation, which were revealed by temperature-dependent modifications of DNA electrophoretic properties. In addition, we presented, for NAPs, a structural model of polyamine aggregation into macropolycyclic compounds. We believe that NAPs are the sole biological forms by which polyamines efficiently protect genomic DNA against DNase I, while maintaining its dynamic structure.     

The nuclear matrix: a heuristic model for investigating genomic organization and function in the cell nucleus.

Despite significant advances in deciphering the molecular events underlying genomic function, our understanding of these integrated processes inside the functioning cell nucleus has, until recently, met with only very limited success. A major conundrum has been the "layers of complexity" characteristic of all cell structure and function. To understand how the cell nucleus functions, we must also understand how the cell nucleus is put together and functions as a whole. The value of this neo-holistic approach is demonstrated by the enormous progress made in recent years in identifying a wide variety of nuclear functions associated with the nuclear matrix. In this article we summarize basic properties of in situ nuclear structure, isolated nuclear matrix systems, nuclear matrix-associated functions, and DNA replication in particular. Emphasis is placed on identifying current problems and directions of research in this field and illustrating the intrinsic heuristic value of this global approach to genomic organization and function.     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPalpha and TEBPbeta. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPalpha in attaching telomeres in the nucleus and in recruiting TEBPbeta to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPbeta in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPbeta.     

NapM,a new nucleoid‐associated protein,broadly regulates gene expression and affects mycobacterial resistance to anti‐tuberculosis drugs

Nucleoid‐associated proteins (NAPs) play important roles in the global organization of bacterial chromosomes. However, potential NAPs and their functions are barely characterized in mycobacteria. In this study, NapM, an alkaline protein, functions as a new NAP. NapM is conserved in all of the sequenced mycobacterial genomes, and can recognize DNA in a length‐dependent but sequence‐independent manner. It prefers AT‐rich DNA and binds to the major groove. NapM possesses a clear DNA‐bridging function, and can protect DNA from DNase I digestion. NapM globally regulates the expression of more than 150 genes and the resistance of Mycobacterium smegmatis to two anti‐tuberculosis drugs, namely, rifampicin and ethambutol. An ABC transporter operon was found to be specifically responsible for the napM‐dependent ethambutol resistance of M. smegmatis. NapM also presents a similar regulation of anti‐tuberculosis drug resistance in M. tuberculosis. These results suggest that NapM is a new member of the mycobacterial NAP family. Our findings expand the range of identified NAPs and improve the understanding on the relationship between NAPs with antibiotic resistance in mycobacteria.     

Mechanotransduction from the ECM to the genome: Are the pieces now in place?

A multitude of biochemical signaling processes have been characterized that affect gene expression and cellular activity. However, living cells often need to integrate biochemical signals with mechanical information from their microenvironment as they respond. In fact, the signals received by shape alone can dictate cell fate. This mechanotrasduction of information is powerful, eliciting proliferation, differentiation, or apoptosis in a manner dependent upon the extent of physical deformation. The cells internal "prestressed" structure and its "hardwired" interaction with the extra-cellular matrix (ECM) appear to confer this ability to filter biochemical signals and decide between divergent cell functions influenced by the nature of signals from the mechanical environment. In some instances mechanical signaling through the tissue microenvironment has been shown to be dominant over genomic defects, imparting a normal phenotype on cells that otherwise have transforming genetic lesions. This mechanical control of phenotype is postulated to have a central role in embryogenesis, tissue physiology as well as the pathology of a wide variety of diseases, including cancer. We will briefly review studies showing physical continuity between the external cellular microenvironment and the interior of the cell nucleus. Newly characterized structures, termed nuclear envelope lamina spanning complexes (NELSC), and their interactions will be described as part of a model for mechanical transduction of extracellular cues from the ECM to the genome.     

Nuclear aggregates of polyamines

Nuclear aggregates of polyamines (NAPs) are cyclic supramolecular compounds made of polyamines and phosphate groups. Three different aggregates, s-NAP, m-NAP and l-NAP, with a molecular weight of 1035, 5175 and 9552 Da, respectively, are described. These molecules interact with genomic DNA. In consequence of this interaction, NAPs not only protect DNA from nucleases with extraordinarily greater efficiency than single polyamines (spermine, spermidine and putrescine), but also induce noticeable changes in DNA condensation status, as shown by temperature-dependent modifications of DNA electrophoretic properties. The biochemical characterization of these compounds has allowed the definition of a structural model for each NAP. According to this model, five s-NAPs assemble together to form a m-NAP unit. We hypothesize that the complexation of s-NAP into m-NAP favours the transition to Z-DNA through the progressive widening of DNA strands and the exposure of bases. We propose that NAPs, by wrapping the DNA helixes, form supramolecular tunnel-like structures that confer efficient protection without affecting DNA elasticity.     

Compartmentalization of regulatory proteins in the cell nucleus

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.     

Till disassembly do us part: a happy marriage of nuclear envelope and chromatin

A characteristic feature of eukaryotic cells is the presence of nuclear envelope (NE) which separates genomic DNA from cytoplasm. NE is composed of inner nuclear membrane (INM), which interacts with chromatin, and outer nuclear membrane, which is connected to endoplasmic reticulum. Nuclear pore complexes are inserted into NE to form transport channels between nucleus and cytoplasm. In metazoan cells, an intermediate filament-based meshwork called as nuclear lamina exists between INM and chromatin. Sophisticated collaboration of these molecular machineries is necessary for the structure and functions of NE. Recent research advances have revealed that NE dynamically communicates with chromatin and cytoskeleton to control multiple nuclear functions. In this mini review, I briefly summarize the basic concepts and current topics of functional relationships between NE and chromatin.     

The skeletal function of non‐genic nuclear DNA: new evidence from ancient cell chimaeras

DNA can be divided functionally into three categories: (1) genes — which code for proteins or specify non-messenger RNAs; (2) semons — short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g. loops or domains) or selfish genetic elements; (3) secondary DNA — which does not function by means of specific sequences. Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton. The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity. This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes. Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function. Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei. Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether junk or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes. New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated independently by secondary symbiogenesis (i.e., the merger of two radically different eukaryote cells) several hundred million years ago. In both groups the nucleus and plasma membrane of the former algal symbiont persist as the nucleomorphs and periplastid membrane, respectively. The fact that nucleomorphs have undergone a 200- to 1000-fold reduction in genome size and have virtually no secondary DNA shows that selection against non-functional nuclear DNA is strong enough to eliminate it very efficiently; therefore, the large amounts of secondary DNA in the former host nuclei of these chimaeras, and in nuclei generally, must be being maintained by positive selection. The divergent selection for secondary DNA in the nucleus and against it in nucleomorphs is readily explicable by the skeletal DNA hypothesis, given the different spectrum of gene functions that it encodes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Recruitment of alkaloid-specific homospermidine synthase (HSS) from ubiquitous deoxyhypusine synthase: Does Crotalaria possess a functional HSS that still has DHS activity?

Quinolizidine alkaloids are the most prominent group of alkaloids occurring in legumes, except for many members of the tribe Crotalarieae that accumulate pyrrolizidine alkaloids (PAs). To study the evolution of PA biosynthesis as a typical pathway of plant secondary metabolism in this tribe, we have searched for a cDNA coding for homospermidine synthase (HSS), the enzyme catalyzing the first specific step in this biosynthesis. HSS was shown to have been recruited from deoxyhypusine synthase (DHS) by independent gene duplication in several different angiosperm lineages during evolution. Except for a cDNA sequence coding for the DHS of Crotalaria retusa, no data is available concerning the origin of PA biosynthesis within this tribe of the Fabaceae. In addition to several pseudogenes, we have identified one functional DHS in C. scassellatii and two in C. juncea. Despite C. juncea plants under study being devoid of PAs, we have found that the two sequences of C. juncea are different with respect to their genomic organization, their tissue-specific expression, and their biochemical activities. Supported by the branching pattern of a maximum likelihood analysis of these sequences, they have been classified as "class 1" and "class 2" DHS. It remains open whether the duplicated DHS belonging to class 2 is involved in the biosynthesis of PAs.     

Immunocytochemical demonstration of polyamines in nucleoli and nuclei

Although biochemical studies have shown that polyamines (PAs) occur in the nucleus, only few studies have examined the intranuclear distribution of these organic cations. By immunocytochemistry, we have previously demonstrated that PAs are located in ribosomes. We now show that PAs also are present in both nucleoli and nuclei of a variety of cell types. Detection of nucleolar and nuclear PAs required novel pretreatment procedures involving protease and/or DNase digestion of specimens prior to immunoreaction. Double fluorescence staining confirmed the localizations. This suggests that PAs may be important to the formation of ribosomes in nucleoli, as well as adds support to biochemical studies suggesting that PAs are involved in many biological events in the nucleus. Further biochemical studies will be needed to substantiate this hypothesis.     

Interactions between oestrogen and 1α,25(OH)<Subscript>2</Subscript>-vitamin D<Subscript>3</Subscript> signalling and their roles in spermatogenesis and spermatozoa functions

Oestrogens and 1α,25(OH)₂-vitamin D₃ (1,25-D₃) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D₃ are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D₃ in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.