Chromosomal DNA Packaging Research Articles

Characterization, expression, and antimicrobial activity of histones from Japanese flounder Paralichthys olivaceus

Histone proteins are not only structurally important for chromosomal DNA packaging but also involved in the regulation of gene expression and the immune response of host against pathogens. Japanese flounder (Paralichthys olivaceus) as one of the most important marine flatfish, suffered from widespread outbreaks of diseases, and its immunological functioning remained to be elucidated. In the present study, we reported the expression patterns of four histones (H1, H2A, H3, and H3.3) and functional characterization of the histone H3.3 from flounder. Quantitative real time RT-PCR (RT-qPCR) analysis showed that expression of the four histones occurred in multiple tissues, but their levels of expression were relatively high in immune organs, and inducible in response to pathogens infection. Infection with extracellular and intracellular bacterial pathogens and viral pathogen regulated the expression of histones in a manner that depended on tissue type, pathogen, and infection stage. Specifically, H1 expression was highly induced by intracellular viral pathogens; H2AX and H3 expressions were highly induced by intracellular bacterial pathogen; dissimilarly, H3.3 expression was slightly induced by extracellular bacterial pathogen, but was inhibited by intracellular bacterial and viral pathogens. To further investigate H3.3 function, recombinant H3.3 (rH3.3) was obtained, and in vitro experiments showed rH3.3 possessed the capability of binding to both Gram-negative and Gram-positive bacteria and inhibiting the growth of some target bacteria. Consistently, In vivo results showed that overexpression of H3.3 promoted the host defense against invading pathogenic microorganism and regulated the expressions of several cytokines. These results suggested that flounder histones exhibit different expression patterns in response to the infection of different microbial pathogens, and H3.3 serves as an immune-related protein and plays an important role in antimicrobial immunity of Japanese flounder. Taken together, this study is the first report about the expression profile of different histones upon different kind of pathogens and anti-infectious immunity of H3.3 in teleost, which offered new insights into the immunological function of histones in teleost.

Downregulation of Nuclear Protein H2B Induces Salicylic Acid Mediated Defense Against PVX Infection in Nicotiana benthamiana.

Histone H2B protein is not only structurally important for chromosomal DNA packaging but is also involved in the regulation of gene expression, including the immune response of plants against pathogens. In this study, we show that the potato virus X (PVX) infection resulted in the reduced expression of H2B at both the mRNA and protein level in Nicotiana benthamiana. Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) was then used to down-regulate the expression of H2B in N. benthamiana and tests showed that the titre of TRV was similar in these plants to that in control treated plants. When these H2B-silenced plants were inoculated with PVX, the virus spread more slowly through the plant and there was a lower titre of PVX compared to non-silenced plants. Abnormal leaf development and stem necrosis were observed in the H2B-silenced plants, which were alleviated in H2B-silenced NahG transgenic plants suggesting the involvement of salicylic acid (SA) in the production of these symptoms. Indeed, quantitative reverse transcription (qRT)-PCR and liquid chromatography tandem mass spectroscopy (LC-MS) results showed that endogenous SA is increased in H2B-silenced N. benthamiana. Thus, downregulation of H2B induced the accumulation of endogenous SA, which was correlated with stem necrosis and a decreased accumulation of PVX in N. benthamiana.

Differential Ability of Five DNA Glycosylases to Recognize and Repair Damage on Nucleosomal DNA.

Damage to genomic DNA leads to mutagenesis and disease. Repair of single base damage is initiated by DNA glycosylases, the first enzymes in the base excision repair pathway. Although eukaryotic packaging of chromosomal DNA in nucleosomes is known to decrease DNA glycosylase efficiency, the impact on individual glycosylases is unclear. Here, we present a model system in which we examine the repair of site-specific base damage in well-characterized nucleosome core particles by five different DNA glycosylases. We find that DNA glycosylase efficiency on nucleosome substrates depends not only on the geometric orientation of the damaged base but also on its identity, as well as on the size, structure, and mechanism of the glycosylase. We show via molecular modeling that inhibition of glycosylase activity is largely due to steric obstruction by the nucleosome core.

Applications of Magnetic Tweezers to Studies of NAPs.

Nucleoid-associated proteins (NAPs) are important factors in shaping bacterial nucleoid and regulating global gene expression. A great deal of insights into NAPs can be obtained through studies using single DNA molecule, which has been made possible owing to recent rapid development of single-DNA manipulation techniques. These studies provide information on modes of binding to DNA, which shed light on the mechanism underlying the regulatory function of NAPs. In addition, how NAPs organize DNA and thus their contribution to chromosomal DNA packaging can be determined. In this chapter, we introduce transverse magnetic tweezers that allows for convenient manipulation of long DNA molecules, and its applications in studies of NAPs as exemplified by the E. coli H-NS protein. We describe how transverse magnetic tweezers is a powerful tool that can be used to characterize the DNA binding and organization modes of NAPs and how such information leads to better understanding of its roles in DNA packaging of bacterial nucleoid and transcription regulation.

Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties.

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.

Impact of DNA Twist Accumulation on Progressive Helical Wrapping of Torsionally Constrained DNA

DNA wrapping is an important mechanism for chromosomal DNA packaging in cells and viruses. Previous studies of DNA wrapping have been performed mostly on torsionally unconstrained DNA, while in vivo DNA is often under torsional constraint. In this study, we extend a previously proposed theoretical model for wrapping of torsionally unconstrained DNA to a new model including the contribution of DNA twist energy, which influences DNA wrapping drastically. In particular, due to accumulation of twist energy during DNA wrapping, it predicts a finite amount of DNA that can be wrapped on a helical spool. The predictions of the new model are tested by single-molecule study of DNA wrapping under torsional constraint using magnetic tweezers. The theoretical predictions and the experimental results are consistent with each other and their implications are discussed.

Human mitotic chromosome structure: what happened to the 30-nm fibre?

EMBO J 31 7, 1644–1653 February172012 The long-standing view of chromosome packaging is that 10-nm beads-on-a string chromatin fibres fold into higher-order 30-nm fibres, which further twist and coil to form highly condensed chromosomes. After four decades of intense pursuit of the structure and properties of 30-nm chromatin fibres, work of Nishino et al (2012) in this issue of The EMBO Journal demonstrates that regular 30-nm fibres are absent from human mitotic chromosomes. The emerging view is that chromosome-level condensation can be achieved through packaging of 10-nm fibres in a fractal manner.

Multiple Binding Modes and DNA Organization by Non-Specific Binding of the E. Coli Nucleoid Associated Protein IHF

The integration host factor (IHF) protein is an abundant E.Coli nucleoid protein. It is most well known as an essential cofactor in the process of phage Δ site-specific recombination where it binds to highly specific DNA sequences with a dissociation coefficient of less than 0.1 nM. Surprisingly, the intra-cellular concentration of IHF is in the range of 10 - 100 uM, extraordinarily larger than the need by its site-specific function. This raises an important question regarding the roles of non-specific binding of IHF that are yet to be understood. Here, we report several novel findings that IHF can bind to DNA with multiple distinct modes under physiological buffer conditions, and can organize DNA into highly condensed complex structures. Our findings have broad implications and highlight a role of IHF as an essential chromosomal DNA packaging protein.

Gene silencing H-NS paralogue StpA forms a rigid protein filament along DNA that blocks DNA accessibility

Nucleoid-associated proteins are bacterial proteins that are responsible for chromosomal DNA compaction and global gene regulation. One such protein is Escherichia coli Histone-like nucleoid structuring protein (H-NS) which functions as a global gene silencer. Whereas the DNA-binding mechanism of H-NS is well-characterized, its paralogue, StpA which is also able to silence genes is less understood. Here we show that StpA is similar to H-NS in that it is able to form a rigid filament along DNA. In contrast to H-NS, the StpA filament interacts with a naked DNA segment to cause DNA bridging which results in simultaneous stiffening and bridging of DNA. DNA accessibility is effectively blocked after the formation of StpA filament on DNA, suggesting rigid filament formation is the important step in promoting gene silencing. We also show that >1 mM magnesium promotes higher order DNA condensation, suggesting StpA may also play a role in chromosomal DNA packaging.

Computational analysis suggests a highly bendable, fragile structure for nucleosomal DNA

Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone affinity for DNA depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown. Here, we show relationships between nucleosome positioning and two structural characteristics of DNA conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional stability was correlated with characteristics of DNA. This result explains how histone positioning is partially determined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging that controls cellular dynamics.

Nano-Positioning-System Provides Structural Insights into ATP-Dependent Nucleosome Remodeling

The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. In order to allow central nuclear processes such as DNA replication, recombination, repair and transcription, cells have evolved so-called chromatin remodeling complexes, which use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes.Chd1 (Chromodomain-helicase-DNA-binding protein 1) is a single-subunit chromatin remodeler consisting of three domains, namely the characteristic tandem-chromodomain, the helicase domain and the DNA-binding domain. It has been shown to assemble, slide and space nucleosomes in vitro and associate with actively transcribed regions of the genome in vivo, but the molecular mechanism of Chd1 remodeling is still not well understood.Single-molecule (sm) techniques such as sm-FRET allow the direct and real-time investigation of conformational changes within macromolecular complexes and hence are perfectly suited to study the mechanism of molecular processes such as chromatin remodeling.Here, we apply sm-FRET and the recently developed Nano-Positioning-System (NPS) [1] to determine the conformation of Chd1 bound to a mononucleosome. Briefly, we determine the distances between several known positions on the nucleosome to unknown positions on different domains of Chd1. The NPS computes the 3-dimensional position probability densities of the sites on Chd1 relative to the nucleosome. By combining our position probability densities with data from SAXS and x-ray crystallography on the structure of the different Chd1 domains, a structure of Chd1 bound to a mononucleosome can be modeled.[1] Muschielok et al., Nature Methods, 5, 965 (2008).

The Biology of Chromatin Remodeling Complexes

The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome composition in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. Here, we address many aspects of remodeler biology: their targeting, mechanism, regulation, shared and unique properties, and specialization for particular biological processes. We also address roles for remodelers in development, cancer, and human syndromes.

HP1beta and HP1gamma, but not HP1alpha, decorate the entire XY body during human male meiosis.

During meiosis in male mammals, the X and Y chromosomes become heterochromatic and transcriptionally silent, and form the XY body. Although the HP1 proteins are known to be involved in the packaging of chromosomal DNA into repressive heterochromatin domains, their involvement in facultative heterochromatinization has not been precisely determined. Here, we analyse, for the first time in humans, the subcellular distribution of the heterochromatin protein HP1alpha, HP1beta and HP1gamma isoforms, in male pachytene spermatocytes, and the XY body facultative heterochromatin in particular. Our results demonstrate that HP1beta and HP1gamma, but not the HP1alpha isoforms, decorate the entire XY body in half the pachytene nuclei observed. In some nuclei, the XY body appears to be only partially labelled. In these cases, the HP1beta and HP1gamma signals are adjacent to the Yq12 constitutive heterochromatin and signal appears to originate in this region before spreading over the entire XY body. This distribution suggests that HP1beta and HP1gamma proteins, which are components of the constitutive heterochromatin, may also be involved in the facultative heterochromatinization of the XY body. Nevertheless, their absence from the early pachytene substage, even though the XY body is already condensed, suggests that these proteins are not involved in the initiation of this process.

Prokaryotic and eukaryotic chromosomes: what's the difference?

It is widely held that the profound differences in cellular architecture between prokaryotes and eukaryotes, in particular the housing of eukaryotic chromosomes within a nuclear membrane, also extends to the properties of their chromosomes. When chromosomal multiplicity, ploidy, linearity, transcriptional silencing, partitioning, and packaging are considered, no consistent association is found between any of these properties and the presence or absence of a nuclear membrane. Some of the perceived differences can be attributed to cytological limitations imposed by the small size of bacterial nucleoids and the arbitrary choice of representative organisms for comparison. We suggest that the criterion of nucleosome-based packaging of chromosomal DNA may be more useful than the prokaryote/eukaryote dichotomy for inferring the broadest phylogenetic relationships among organisms.

M31, a murine homolog of Drosophila HP1, is concentrated in the XY body during spermatogenesis

The formation of the sex vesicle, or XY body, during male meiosis and pairing of the sex chromosomes are thought to be essential for successful spermatogenesis. Despite its cytological discovery a century ago, the mechanism of XY body formation, particularly heterochromatinization of the sex chromosomes, has remained unclear. The HP1 class of chromobox genes are thought to encode proteins involved in the packaging of chromosomal DNA into repressive heterochromatin domains, as seen, for example, in position-effect variegation. Study of the distribution of a murine HP1-like chromodomain protein, M31, during spermatogenesis revealed spreading from the tip of the XY body in mid-stage pachytene spermatocytes to include the whole of the XY body in late-pachytene spermatocytes. We also demonstrate that the formation of the XY body during spermatogenic progression in neonatal mice coincides with the expression of a novel nuclear isoform of M31, M31^p21. These results support the view that a common mechanistic basis exists for heterochromatin-induced repression, homeotic gene silencing, and sex-chromosome inactivation during mammalian spermatogenesis.

Packaging of transducing DNA by bacteriophage P1.

P1 transduces bacterial chromosomal markers with widely differing frequencies. We use quantitative Southern hybridisations here to show that, despite this, most markers are packaged at similar levels. Exceptions are a group of markers near 2 min and another at 90 min which seem to be packaged at levels two- to threefold higher. We thus conclude that certain marker frequency variations in transduction can be explained by differences in packaging level, but that most cannot. The limited range in packaging levels suggests that P1 can initiate the packaging of chromosomal DNA from many sites. This idea is supported by our failure to find any chromosomal sequences with homology to the phage pac site and by the occurrence of hybridising bands which seem to suggest sequential packaging from a large number of specific sites. We eliminate the possibility that chromosomal DNA packaging is the result of endonucleolytic cutting by the P1 res enzyme.

Transduction of bacteriophage resistance in Streptococcus pyogenes with coinfection lysates of the virulent phage A25ts1-2 and a temperate bacteriophage 2511

Two new mutants of Streptococcus pyogenes designated KS107 and KS136 were shown to allow adsorption of a virulent bacteriophage KV1, but replication of viral DNA did not occur in these mutants. The novel observation was made that coinfection of donor ceils with phages A25ts1-2 and 2511 enhanced transduction of phage resistance and of antibiotic resistance. Mixed donor lysates did not give enhanced transduction. Antiserum to A25ts1-2 blocked transduction, suggesting that the presence of 2511 promoted packaging of chromosomal DNA into A25ts1-2 particles. Phage 2511 was isolated by induction of a clinical strain, but it behaved as a lytic phage in laboratory strains. Phage KV1 was isolated from sewage and was found to be closely related to A25ts1-2, whereas 2511 was clearly different. The phage resistance gene of KS107 was not linked to any of three antibiotic resistance genes by cotransduction tests.