Dnmt2-dependent methylomes lack defined DNA methylation patterns

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms

Emerging Role of HMGB1 in the Pathogenesis of Schistosomiasis Liver Fibrosis

In chronic schistosomiasis, liver fibrosis is linked to portal hypertension, which is a condition associated with high mortality and morbidity. High mobility group box 1 (HMGB1) was originally described as a nuclear protein that functions as a structural co-factor in transcriptional regulation. However, HMGB1 can also be secreted into the extracellular milieu under appropriate signal stimulation. Extracellular HMGB1 acts as a multifunctional cytokine that contributes to infection, injury, inflammation, and immune responses by binding to specific cell-surface receptors. HMGB1 is involved in fibrotic diseases. From a clinical perspective, HMGB1 inhibition may represent a promising therapeutic approach for treating tissue fibrosis. In this study, we demonstrate elevated levels of HMGB1 in the sera in experimental mice or in patients with schistosomiasis. Using immunohistochemistry, we demonstrated that HMGB1 trafficking in the hepatocytes of mice suffering from acute schistosomiasis was inhibited by Glycyrrhizin, a well-known HMGB1 direct inhibitor, as well as by DIC, a novel and potential anti-HMGB1 compound. HMGB1 inhibition led to significant downregulation of IL-6, IL4, IL-5, IL-13, IL-17A, which are involved in the exacerbation of the immune response and liver fibrogenesis. Importantly, infected mice that were treated with DIC or GZR to inhibit HMGB1 pro-inflammatory activity showed a significant increase in survival and a reduction of over 50% in the area of liver fibrosis. Taken together, our findings indicate that HMGB1 is a key mediator of schistosomotic granuloma formation and liver fibrosis and may represent an outstanding target for the treatment of schistosomiasis

CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated.We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis

Chronic viral hepatitis induced by hepatitis C but not hepatitis B virus infection correlates with increased liver angiogenesis.

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections lead to cirrhosis and increase the risk for the development of hepatocellular carcinoma (HCC). Angiogenesis is an essential step in oncogenesis and contributes to tumor progression in adult organs; however, to what extent angiogenesis occurs in the liver during chronic viral hepatitis has not been studied. Ninety-nine matched patients affected by chronic hepatitis due to either HBV or HCV were studied together with 13 controls (5 patients were affected by familial hyperbilirubinemia with normal liver histology; 6 patients with stage II primary biliary cirrhosis; and 2 patients with pseudo inflammatory tumor). Microvessel density was assessed in liver biopsies by immunostaining using two different antibodies against endothelial cell antigens, QB-END/10 and Factor VIII. In addition, the liver homogenates and sera of HCV- or HBV-positive patients and controls were tested for their capacity to stimulate the migration and proliferation of freshly isolated human endothelial cells in vitro. Evidence of angiogenesis was significantly more frequent in HCV-positive patients compared with HBV-infected subjects or controls (74\% vs. 39\% vs. 8\%) (chi2 = 20.78; P < .0001) (HCV+ vs. HBV+ vs. controls). The degree of microvessel density was also higher in HCV- than in HBV-positive patients or controls (chi2 = 12.28; P < .005). In addition, HCV-positive sera and liver homogenates stimulated a higher migration and proliferation of human endothelial cells in vitro compared with HBV-positive or control sera and liver homogenates. These observations indicate that angiogenesis is particularly linked to HCV infection, suggesting a possible contribution to HCV-related liver oncogenesis

Binding isotherm of HMGB1 to fluorescently labeled linear DNA.

<p>A) FAM-labeled 20-bp dsDNA at a 50 nM concentration was titrated with increasing HMGB1 (black circles) or HMGB1ΔC (red circles) concentrations, and the fluorescence polarization (P) of the fluorescent probe was measured after a 15-min incubation at 25 °C. (a) The binding stoichiometry of HMGB1 or HMGB1ΔC to FAM-labeled dsDNA was calculated. Increasing protein concentrations were added to a solution containing a mixture of 2 μM unlabeled dsDNA and 50 nM FAM-labeled dsDNA; thus, the [Protein]/[DNA] ratio varied from 0 to 15. The polarization values were measured by exciting the probe at 490 nm and reading the FAM-emission fluorescence at 520 nm after a 15-min incubation at 25 °C.</p

Binding of HMGB1 protein to linear dsDNA monitored by fluorescence spectroscopy.

<p>A) Interaction between HMGB1 (black circles) or HMGB1ΔC (red circles) with 20-bp DNA was analyzed by the quenching of the Trp emission fluorescence. Both proteins were kept at 2 μM, and the DNA concentration was varied from 0 to 2 μM. Trp emission spectra were collected after a 15-min incubation at 25 °C. B) Interaction between HMGB1 or HMGB1ΔC with 20-bp DNA, as analyzed by bis-ANS displacement. The protein and bis-ANS concentrations were 0.5 μM and 10 μM, respectively, whereas the DNA concentration varied from 0 to 1.2 μM. The emission spectra of bis-ANS were acquired after a 15-min incubation time at 25 °C. Normalized spectrum areas were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#pone-0079572-g004" target="_blank">Figure 4</a>. Control experiments were performed similarly but in the absence of protein.</p

Thermal denaturation of the HMGB1 protein.

<p>A) The Trp fluorescence emission spectra of HMGB1 (black circles) and HMGB1ΔC (red circles) at each temperature were acquired and converted into CM and α according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn1" target="_blank">Equations 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn2" target="_blank">2</a>, respectively. The curves were adjusted by sigmoidal fitting, and the T_m was obtained directly from the fitting. B) The CD signal at 222 nm for the HMGB1 and HMGB1ΔC spectra at each temperature was converted into the loss of secondary structure content. The buffer contained 10 mM Tris.HCl at pH 7.2, 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA and 5% of glycerol.</p

Influence of low pH on the HMGB1 structure.

<p>A) HMGB1 (black circles) and HMGB1ΔC (red circles) at 5 μM concentration were incubated at different pH values (in citrate/citric acid buffer), and the CM variation (ΔCM) was calculated. Because of the small change in ΔCM, even in a very acidic pH, both proteins were also incubated with Gdn.HCl at pH 2.3 and 5.5 M (black triangle for HMGB1 and red triangle for HMGB1ΔC). B) The secondary structure content of 5 μM HMGB1 at neutral pH (black straight lines) and pH 2.3 (black medium-dashed lines) and of HMGB1ΔC at neutral pH (red straight lines) and pH 2.3 (red medium-dashed lines) was monitored by CD at 20 °C. Spectra were converted to molar ellipticity, as described in the Material & Methods section. C) The interaction of bis-ANS and the proteins was assessed by exciting 10 μM probe in a solution containing 5 μM HMGB1 (black circles) or HMGB1ΔC (red circles) at different pH values after a 1-h incubation at 25 °C. For comparison, HMGB1 and HMGB1ΔC were incubated at pH 2.3 in the presence of 5.5 M Gdn.HCl (closed triangles). Normalized spectrum areas were obtained by dividing the spectrum area value of each pH point by the area value at neutral pH.</p

Denaturation of HMGB1 and HMGB1ΔC as a function of increasing Gdn.HCl concentration.

<p>A) The CM of HMGB1 (black circles) and HMGB1ΔC (red circles) at 5 μM was obtained for each [Gdn.HCl] using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn1" target="_blank">Equation 1</a>, as described in the Material and Methods Section. B) Trp fluorescence spectra were obtained and converted to degree of denaturation (α) according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#eqn2" target="_blank">Equation 2</a>. The resistance to unfolding can be analyzed by G_1/2, which reflects the concentration necessary to unfold 50% of the protein population and is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079572#pone-0079572-t001" target="_blank">Table 1</a>. </p

Schematic representation of HMGB1-mediated DNA bending.

<p>A 20-bp oligonucleotide labeled with FAM (green star, F) and TAMRA (orange star, T) fluorophores in the presence of HMGB1 or HMGB1∆C undergoes bending at different angles, measured by the distance between these two fluorophores. Bending angle values were obtained using the two-kinked model. The difference observed in size and color intensity of the fluorophores molecules is proportional to their emission quenching. The acidic tail of HMGB1 and its interaction with other part of the molecule are represented by green and dashed lines, respectively.</p