Genome-wide DNA methylation profiling of CD8+ T cells shows a distinct epigenetic signature to CD4+ T cells in multiple sclerosis patients

Background Multiple sclerosis (MS) is thought to be a T cell-mediated autoimmune disorder. MS pathogenesis is likely due to a genetic predisposition triggered by a variety of environmental factors. Epigenetics, particularly DNA methylation, provide a logical interface for environmental factors to influence the genome. In this study we aim to identify DNA methylation changes associated with MS in CD8+ T cells in 30 relapsing remitting MS patients and 28 healthy blood donors using Illumina 450K methylation arrays. Findings Seventy-nine differentially methylated CpGs were associated with MS. The methylation profile of CD8+ T cells was distinctive from our previously published data on CD4+ T cells in the same cohort. Most notably, there was no major CpG effect at the MS risk gene HLA-DRB1 locus in the CD8+ T cells. Conclusion CD8+ T cells and CD4+ T cells have distinct DNA methylation profiles. This case–control study highlights the importance of distinctive cell subtypes when investigating epigenetic changes in MS and other complex diseases.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

CNT loading into cationic cholesterol suspensions show improved DNA binding and serum stability and ability to internalize into cancer cells

Methods which disperse single-walled carbon nanotubes (SWNTs) in water as `debundled', while maintaining their unique physical properties are highly useful. We present here a family of cationic cholesterol compounds (Chol(+)) {Cholest-5en-3 beta-oxyethyl pyridinium bromide (Chol-PB+), Cholest-5en-3 beta-oxyethyl N-methyl pyrrolidinium bromide (Chol-MPB+), Cholest-5en-3 beta-oxyethyl N-methyl morpholinium bromide (Chol-MMB+) and Cholest-5en-3 beta-oxyethyl diazabicyclo octanium bromide (Chol-DOB+)}. Each of these could be easily dispersed in water. The resulting cationic cholesterol (Chol(+)) suspensions solubilized single-walled carbon nanotubes (SWCNTs) by the non-specific physical adsorption of Chol(+) to form stable, transparent, dark aqueous suspensions at room temperature. Electron microscopy reveals the existence of highly segregated CNTs in these samples. Zeta potential measurements showed an increase in potential of cationic cholesterol aggregates on addition of CNTs. The CNT-Chol(+) suspensions were capable of forming stable complexes with genes (DNA) efficiently. The release of double-helical DNA from such CNT-Chol(+) complexes could be induced upon the addition of anionic micellar solution of SDS. Furthermore, the CNT-based DNA complexes containing cationic cholesterol aggregates showed higher stability in fetal bovine serum media at physiological conditions. Confocal studies confirm that CNT-Chol(+) formulations adhere to HeLa cell surfaces and get internalized more efficiently than the cationic cholesterol suspensions alone (devoid of any CNTs). These cationic cholesterol-CNT suspensions therefore appear to be a promising system for further use in biological applications.

Role of Specific Cations and Water Entropy on the Stability of Branched DNA Motif Structures

DNA three-way junctions (TWJs) are important intermediates in various cellular processes and are the simplest of a family of branched nucleic acids being considered as scaffolds for biomolecular nanotechnology. Branched nucleic acids are stabilized by divalent cations such as Mg2+, presumably due to condensation and neutralization of the negatively charged DNA backbone. However, electrostatic screening effects point to more complex solvation dynamics and a large role of interfacial waters in thermodynamic stability. Here, we report extensive computer simulations in explicit water and salt on a model TWJ and use free energy calculations to quantify the role of ionic character and strength on stability. We find that enthalpic stabilization of the first and second hydration shells by Mg2+ accounts for 1/3 and all of the free energy gain in 50% and pure MgCl2 solutions, respectively. The more distorted DNA molecule is actually destabilized in pure MgCl2 compared to pure NaCl. Notably, the first shell, interfacial waters have very low translational and rotational entropy (i.e., mobility) compared to the bulk, an entropic loss that is overcompensated by increased enthalpy from additional electrostatic interactions with Mg2+. In contrast, the second hydration shell has anomalously high entropy as it is trapped between an immobile and bulklike layer. The nonmonotonic entropic signature and long-range perturbations of the hydration shells to Mg2+ may have implications in the molecular recognition of these motifs. For example, we find that low salt stabilizes the parallel configuration of the three-way junction, whereas at normal salt we find antiparallel configurations deduced from the NMR. We use the 2PT analysis to follow the thermodynamics of this transition and find that the free energy barrier is dominated by entropic effects that result from the decreased surface area of the antiparallel form which has a smaller number of low entropy waters in the first monolayer.

Effect of a natural mutation in the 5 ` untranslated region on the translational control of p53 mRNA

Tumor-suppressor protein p53, the `guardian of the genome', is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, Delta N-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5' untranslated region (5' UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5' UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5' UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5' UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2-M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5' UTR. Taken together, the results demonstrate for the first time that SNP in the 5' UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene.

Mycobacterium tuberculosis DinG Is a Structure-specific Helicase That Unwinds G4 DNA IMPLICATIONS FOR TARGETING G4 DNA AS A NOVEL THERAPEUTIC APPROACH

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' -> 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' -> 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

Structure, stability and elasticity of DNA nanotubes

DNA nanotubes are tubular structures composed of DNA crossover molecules. We present a bottom up approach for the construction and characterization of these structures. Various possible topologies of nanotubes are constructed such as 6-helix, 8-helix and tri-tubes with different sequences and lengths. We have used fully atomistic molecular dynamics simulations to study the structure, stability and elasticity of these structures. Several nanosecond long MD simulations give the microscopic details about DNA nanotubes. Based on the structural analysis of simulation data, we show that 6-helix nanotubes are stable and maintain their tubular structure; while 8-helix nanotubes are flattened to stabilize themselves. We also comment on the sequence dependence and the effect of overhangs. These structures are approximately four times more rigid having a stretch modulus of similar to 4000 pN compared to the stretch modulus of 1000 pN of a DNA double helix molecule of the same length and sequence. The stretch moduli of these nanotubes are also three times larger than those of PX/JX crossover DNA molecules which have stretch moduli in the range of 1500-2000 pN. The calculated persistence length is in the range of a few microns which is close to the reported experimental results on certain classes of DNA nanotubes.