c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Bacterial Second Messenger Cyclic di-AMP Modulates the Competence State in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger cyclic di-AMP (c-di-AMP) is an emerging player in the stress responses of many pathogens. In S. pneumoniae, c-di-AMP is produced by a diadenylate cyclase, CdaA, and cleaved by phosphodiesterases Pde1 and Pde2. c-di-AMP binds a transporter of K+ (Trk) family protein, CabP, which subsequently halts K+ uptake via the transporter TrkH. Recently, it was reported that Pde1 and Pde2 are essential for pneumococcal virulence in mouse models of disease. To elucidate c-di-AMP-mediated transcription that may lead to changes in pathogenesis, we compared the transcriptomes of wild-type (WT) and Δpde1 Δpde2 strains by transcriptome sequencing (RNA-Seq) analysis. Notably, we found that many competence-associated genes are significantly upregulated in the Δpde1 Δpde2 strain compared to the WT. These genes play a role in DNA uptake, recombination, and autolysis. Competence is induced by a quorum-sensing mechanism initiated by the secreted factor competence-stimulating peptide (CSP). Surprisingly, the Δpde1 Δpde2 strain exhibited reduced transformation efficiency compared to WT bacteria, which was c-di-AMP dependent. Transformation efficiency was also directly related to the [K+] in the medium, suggesting a link between c-di-AMP function and the pneumococcal competence state. We found that a strain that possesses a V76G variation in CdaA produced less c-di-AMP and was highly susceptible to CSP. Deletion of cabP and trkH restored the growth of these bacteria in medium with CSP. Overall, our study demonstrates a novel role for c-di-AMP in the competence program of S. pneumoniaeIMPORTANCE Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.

Uridine adenosine tetraphosphate and purinergic signaling in cardiovascular system: An update.

Uridine adenosine tetraphosphate (Up4A), biosynthesized by activation of vascular endothelial growth factor receptor (VEGFR) 2, was initially identified as a potent endothelium-derived vasoconstrictor in perfused rat kidney. Subsequently, the effect of Up4A on vascular tone regulation was intensively investigated in arteries isolated from different vascular beds in rodents including rat pulmonary arteries, aortas, mesenteric and renal arteries as well as mouse aortas, in which Up4A produces vascular contraction. In contrast, Up4A produces vascular relaxation in porcine coronary small arteries and rat aortas. Intravenous infusion of Up4A into conscious rats or mice decreases blood pressure, and intravenous bolus injection of Up4A into anesthetized mice increases coronary blood flow, indicating an overall vasodilator influence in vivo. Although Up4A is the first dinucleotide described that contains both purine and pyrimidine moieties, its cardiovascular effects are exerted mainly through activation of purinergic receptors. These effects not only encompass regulation of vascular tone, but also endothelial angiogenesis, smooth muscle cell proliferation and migration, and vascular calcification. Furthermore, this review discusses a potential role for Up4A in cardiovascular pathophysiology, as plasma levels of Up4A are elevated in juvenile hypertensive patients and Up4A-mediated vascular purinergic signaling changes in cardiovascular disease such as hypertension, diabetes, atherosclerosis and myocardial infarction. Better understanding the vascular effect of the novel dinucleotide Up4A and the purinergic signaling mechanisms mediating its effects will enhance its potential as target for treatment of cardiovascular disease.

Inhibition of Enterococcus faecalis Growth and Biofilm Formation by Molecule Targeting Cyclic di-AMP Synthetase Activity.

INTRODUCTION: Enterococcus faecalis is correlated with oral diseases including recurrent root canal treatment failure because of its biofilm formation ability and various virulence factors. Cyclic di-AMP (c-di-AMP) is an omnipresent second messenger involved in many crucial cellular physiological processes, including biofilm formation. ST056083 is a small molecule working as an inhibitor of the c-di-AMP synthetase DNA integrity scanning protein (DisA) in vitro. In this study, the impact of ST056083 on E. faecalis DisA activity, bacterial growth, and biofilm formation was tested. METHODS: The binding affinity between the protein and ligand was evaluated using the Amber score, and the binding mode was analyzed and visualized using UCSF Chimera (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco, San Francisco, CA). The effect of ST056083 on E. faecalis DisA was evaluated using the coralyne assay. The effect of ST056083 on E. faecalis biofilm formation was determined by the biofilm quantification assay, scanning electron microscopic examination, and 3-dimensional confocal laser scanning microscopic assay. The effect of ST056083 on E. faecalis exopolysaccharide synthesis was measured by the anthrone-sulfuric method. RESULTS: We expressed and purified E. faecalis DisA in vitro and confirmed the inhibitory effect of ST056083 on its biological activity. In addition, we showed the inhibitory effect of ST056083 on E. faecalis growth, biofilm formation, and exopolysaccharide synthesis. CONCLUSIONS: Our findings enhance the understanding of the physiological role of c-di-AMP in E. faecalis and represent a preliminary study on the ST056083 inhibitory effect and mechanism.

[c-di-AMP regulates bacterial biofilm formation].

Bacterial biofilm plays an important role in persistent microbial infection. Delineation of the formation and development of bacterial biofilm would provide a promising strategy to treat recalcitrant infection. c-di-AMP (Cyclic diadenosine monophosphate) is a recently identified second messenger of bacteria and involved in plethora of bacterial activities, including cell growth, cell wall homeostasis, biofilm formation and microbial pathogenicity. Here we review the recent literature pertinent to the role and molecular mechanisms of c-di-AMP in regulating biofilm formation of bacteria. The potential application of c-di-AMP and its related proteins in the development of novel antimicrobial therapeutics has also been discussed.

Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria.

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the ß-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.

A New Second Messenger: Bacterial c-di-AMP.

Nucleotide-based second messengers transduce signals originating from both outside and inside the cell to adaptive responses accordingly. c-di-AMP is a newly established second messenger employed by many organisms. We summarize recent advances in bacterial c-di-AMP-mediated signaling, especially the interaction between c-di-AMP signaling and the host.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures.

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.

Constrictor prostanoids and uridine adenosine tetraphosphate: vascular mediators and therapeutic targets in hypertension and diabetes.

Vascular dysfunction plays a pivotal role in the development of systemic complications associated with arterial hypertension and diabetes. The endothelium, or more specifically, various factors derived from endothelial cells tightly regulate vascular function, including vascular tone. In physiological conditions, there is a balance between endothelium-derived factors, that is, relaxing factors (endothelium-derived relaxing factors; EDRFs) and contracting factors (endothelium-derived contracting factors; EDCFs), which mediate vascular homeostasis. However, in disease states, such as diabetes and arterial hypertension, there is an imbalance between EDRF and EDCF, with a reduction of EDRF signalling and an increase of EDCF signalling. Among EDCFs, COX-derived vasoconstrictor prostanoids play an important role in the development of vascular dysfunction associated with hypertension and diabetes. Moreover, uridine adenosine tetraphosphate (Up4 A), identified as an EDCF in 2005, also modulates vascular function. However, the role of Up4 A in hypertension- and diabetes-associated vascular dysfunction is unclear. In the present review, we focused on experimental and clinical evidence that implicate these two EDCFs (vasoconstrictor prostanoids and Up4 A) in vascular dysfunction associated with hypertension and diabetes.

Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library.

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.

The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication.

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-ß, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.

Cyclic di-nucleotide signaling enters the eukaryote domain.

Cyclic (c-di-GMP) is the prevalent intracellular signaling intermediate in bacteria. It triggers a spectrum of responses that cause bacteria to shift from a swarming motile phase to sessile biofilm formation. However, additional functions for c-di-GMP and roles for related molecules, such as c-di-AMP and c-AMP-GMP continue to be uncovered. The first usage of cyclic-di-nucleotide (c-di-NMP) signaling in the eukaryote domain emerged only recently. In dictyostelid social amoebas, c-di-GMP is a secreted signal that induces motile amoebas to differentiate into sessile stalk cells. In humans, c-di-NMPs, which are either produced endogenously in response to foreign DNA or by invading bacterial pathogens, trigger the innate immune system by activating the expression of interferon genes. STING, the human c-di-NMP receptor, is conserved throughout metazoa and their closest unicellular relatives, suggesting protist origins for human c-di-NMP signaling. Compared to the limited number of conserved protein domains that detect the second messengers cAMP and cGMP, the domains that detect the c-di-NMPs are surprisingly varied.

Uridine adenosine tetraphosphate (Up4A) is a strong inductor of smooth muscle cell migration via activation of the P2Y2 receptor and cross-communication to the PDGF receptor.

The recently discovered dinucleotide uridine adenosine tetraphosphate (Up(4)A) was found in human plasma and characterized as endothelium-derived vasoconstrictive factor (EDCF). A further study revealed a positive correlation between Up(4)A and vascular smooth muscle cell (VSMC) proliferation. Due to the dominant role of migration in the formation of atherosclerotic lesions our aim was to investigate the migration stimulating potential of Up(4)A. Indeed, we found a strong chemoattractant effect of Up(4)A on VSMC by using a modified Boyden chamber. This migration dramatically depends on osteopontin secretion (OPN) revealed by the reduction of the migration signal down to 23% during simultaneous incubation with an OPN-blocking antibody. Due to inhibitory patterns using specific and unspecific purinoreceptor inhibitors, Up(4)A mediates it's migratory signal mainly via the P2Y(2). The signaling behind the receptor was investigated with luminex technique and revealed an activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. By use of the specific PDGF receptor (PDGFR) inhibitor AG1296 and siRNA technique against PDGFR-ß we found a strongly reduced migration signal after Up(4)A stimulation in the PDGFR-ß knockdown cells compared to control cells. In this study, we present substantiate data that Up(4)A exhibits migration stimulating potential probably involving the signaling cascade of MEK1 and ERK1/2 as well as the matrix protein OPN. We further suggest that the initiation of the migration process occurs predominant through direct activation of the P2Y(2) by Up(4)A and via transactivation of the PDGFR.

Uridine adenosine tetraphosphate activation of the purinergic receptor P2Y enhances in vitro vascular calcification.

Purinergic signaling has a crucial role in different vascular processes. The endothelial-derived vasoconstrictor uridine adenosine tetraphosphate (Up(4)A) is a potent activator of the purinoceptor P2Y and is released under pathological conditions. Here we sought to measure purinergic effects on vascular calcification and initially found that Up(4)A plasma concentrations are increased in patients with chronic kidney disease. Exploring this further we found that exogenous Up(4)A enhanced mineral deposition under calcifying conditions ex vivo in rat and mouse aortic rings and in vitro in rat vascular smooth muscle cells. The addition of Up(4)A increased the expression of different genes specific for osteochondrogenic vascular smooth muscle cells such as Cbfa1, while decreasing the expression of SM22α, a marker specific for vascular smooth muscle cells. The influence of different P2Y antagonists on Up(4)A-mediated process indicated that P2Y(2/6) receptors may be involved. Mechanisms downstream of P2Y signaling involved phosphorylation of the mitogen-activated kinases MEK and ERK1/2. Thus, Up(4)A activation of P2Y influences phenotypic transdifferentiation of vascular smooth muscle cells to osteochondrogenic cells, suggesting that purinergic signaling may be involved in vascular calcification.

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress.

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 µM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.

Role of 10-11bp periodicities of eukaryotic DNA sequence in nucleosome positioning.

10-11bp repeating patterns of the particular dinucleotides were suggested in positioning nucleosomes in eukaryotes. In order to assess the role, the 10-11bp periodicities of the dinucleotides were examined both in human well-positioned nucleosome DNA sequences and in promoter DNA sequences of eight species. Our results indicated that the periodical occurrence of the particular dinucleotides correlates to positioning nucleosome. The signals of 10-11bp periodicities are more pronounced in the nucleosome DNA sequences than in the linker DNA sequences. Near the transcription start site, the signals reveal a similar feature that the nucleosome organization exhibits. But, it seems that the species do not share the same dinucleotides patterns. Furthermore, the dinucleotides patterns are dominant at the specific region of genome, indicating their diverse roles in forming and organizing nucleosomes. Moreover, the 10-11bp periodicities signals near the translation start negatively correlate with gene expression, demonstrating a vital role in transcription regulation. Our study reveals some details about the roles of DNA sequence in positioning nucleosomes.

Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes.

BACKGROUND: The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown. RESULTS: When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3) or 62 (10.4 × 6) base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4. CONCLUSIONS: Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well.The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the nucleosome positioning, we hypothesize that Alu-nucleosomes, especially, those that form tightly positioned runs, could serve as "anchors" in organizing the chromatin in human cells.