The Role of Histone H4 Biotinylation in the Structure of Nucleosomes

Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation

NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing.

Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA

Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1β)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1β processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1β processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1β hyper-activation loop included a large number of IL-1β-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1β and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. Trial Registration: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov)

IL-1β processing by MMP-7.

<p>(<b>A</b>) Gene expression profiling identified <i>Mmp7</i> as one of the top up-regulated gene in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with bladder pathology (CFT073 infected mice, 7 days). Log₂ fold change of <i>Mmp7</i> expression levels in individual mice are shown relative to the H&E pathology score. <i>Mmp7</i> was not regulated in C57BL/6 WT mice or in <i>Il1b</i>^<i>-/-</i> or <i>Casp1</i>^<i>-/-</i> mice. (<b>B</b>) Strong epithelial MMP-7 staining in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with bladder pathology. MMP-7 staining was very low in C57BL/6 WT, <i>Il1b</i>^<i>-/-</i> and <i>Casp1</i>^<i>-/-</i> mice. Scale bars = 50 μm. (<b>C</b>) Phenotype of <i>Mmp7</i>^-/- mice, 7 days after infection with CFT073. Intact mucosal tissue structure with inflammatory cell infiltration. Low bacterial and neutrophil counts in urine compared to <i>Asc</i>^-/- mice (<i>n</i> = 5 mice per group, means ± SEMs, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, two-tailed unpaired <i>t</i>-test). Scale bar = 1 mm. IL-1β levels were elevated in the urine of <i>Asc</i>^-/- mice but not in <i>Mmp7</i>^-/- mice, as detected by ELISA. (<b>D</b>) Proteolytic cleavage of pro-IL-1β by MMP-7 <i>in vitro</i>, using purified enzyme and GST-tagged pro-IL-1β. The IL-1β fragments generated by proteolysis were 18 and 16 kDa, defined by Western blot using an antibody specific for the mature form of IL-1β. Recombinant mature IL-1β and GST-tagged pro-IL-1β were used as controls, as well as recombinant MMP-7. One representative experiment out of three, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.s007" target="_blank">S7A Fig</a>. (<b>E</b>) Bioassay for IL-1β activity, measuring the PGE₂ response of human bladder epithelial cells to the IL-1β fragments generated by MMP-7 proteolysis of GST-tagged pro-IL-1β. The cleaved products activated PGE₂ but MMP-7 and pro-IL-1β had no effect (means ± SEMs of two experiments, ** <i>P</i> < 0.01, two-tailed Mann Whitney test).</p

Hyper-activation of IL-1β dependent gene expression and bladder pathology in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice.

<p>Transcriptomic analysis of whole bladder RNA from infected mice (CFT073, 7 days), compared to uninfected controls of each genotype (cut off FC 1.41, <i>P</i> < 0.05). (<b>A</b>) Heatmap of regulated genes in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with the highest bladder pathology score, defined by neutrophil infiltration, loss of tissue structure and epithelial thickness in H&E stained bladder tissue sections. Scale FC -4 to 4, red = upregulated, blue = downregulated. A distinct gene set distinguished the <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with a high histopathology score from C57BL/6 WT mice or <i>Il1b</i>^<i>-/-</i> mice without pathology. (<b>B</b>) Top up-regulated genes in the pathology-associated gene set, compared to uninfected controls of each genotype. Means ± SEMs of 5 mice for <i>Asc</i>^<i>-/-</i> mice and 2 <i>Nlrp3</i>^<i>-/-</i> mice. (<b>C</b>) Analysis of IL-1β, inflammasome activators and effectors in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice, detecting massive over-expression compared to <i>Il1b</i>^<i>-/-</i> and WT mice. Red = upregulated, blue = suppressed. The data set included gene expression profiles from 7 <i>Asc</i>^<i>-/-</i> and 5 <i>Nlrp3</i>^<i>-/-</i> mice, and two each of the C57BL/6 WT and <i>Il1b</i>^<i>-/-</i> controls. Uninfected control RNA of each genotype were used to define significantly regulated genes (≥ 2 mice per genotype). Histopathology scores and group numbers for individual mice (see also Experiments 1, 2 and 3 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.s011" target="_blank">S1 Table</a>).</p

Regulation of <i>MMP7</i> expression by ASC and NLRP-3.

<p>(<b>A</b>) MMP-7, ASC and NLRP-3 responses to infection were visualized by confocal microscopy. An increase in MMP-7 and decrease in ASC staining were detected after infection of HTB-9 cells with CY-17 and CY-92 for 1 hour, compared to uninfected control cells. NLRP-3 staining was weakly affected. (<b>B</b>) Quantification of total fluorescence intensity (open pin-hole) after subtraction of the background staining in uninfected cells (PBS). Medians ± SEMs of 50 cells, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, compared to PBS control, two-tailed unpaired <i>t</i>-test (MMP-7 and NLRP-3) or two-tailed Mann Whitney test (ASC). One of three experiments is shown. (<b>C</b>) Western blot confirming the change in cellular content of MMP-7, ASC and NLRP-3, 1 hour after infection with the indicated strains. Fold change compared to PBS of normalized values (against GAPDH). One experiment out of 2 is shown. (<b>D</b>) Increase in MMP-7 expression in HTB-9 cells transfected with siRNAs specific for <i>ASC</i> or <i>NLRP3</i> and infected with CY-17 (4 hours, scale bars = 20 μm). (<b>E</b>) Western blot confirming the knock-down of ASC or NLRP-3 with siRNAs. A further reduction in ASC expression was detected after CY-17 infection (4 hours, quantified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.s008" target="_blank">S8B Fig</a>, one experiment out of 2 is shown.). (<b>F</b>) PCR amplification of a 259 bp fragment in the <i>MMP7</i> promoter (P1, -18/-276 relative to the transcription start site). (<b>G</b>) EMSA of the amplified fragment and nuclear extract from CY-17 infected HTB-9 cells (4 hours). Binding of ASC and NLRP-3 to P1 was identified as a band shift (arrow indicating protein-DNA complex). The band shift was inhibited by ASC- or NLRP-3-specific antibody. Free DNA formed a single low molecular weight band (arrow indicating free probe). The band shift was not affected by the IgG isotype control. One of three similar experiments is shown. (<b>H</b>) EMSA of the 259 bp <i>MMP7</i> promoter fragment P1 and recombinant ASC. Dose-dependent formation of an ASC-P1 complex is shown as a band shift (arrow indicating ASC-DNA complex), which was inhibited by 0.5 and 1.0 μg of anti-ASC antibodies. The band shift was not affected by negative control murine IgG control. One of three similar experiments is shown.</p

Acute cystitis immunotherapy, using an IL-1 receptor antagonist (IL-1RA) or an MMP inhibitor.

<p>(<b>A</b>) Overview of therapeutic regimen used to inhibit bladder pathology. <i>Asc</i>^-/- mice were pre-treated with Anakinra (IL-1RA), 30 min before infection and daily after infection with <i>E</i>. <i>coli</i> CFT073 (1 mg in 100 μl of PBS i.p. per mouse) and sacrificed 7 days after infection. Alternatively, <i>Asc</i>^-/- mice were pre-treated with the matrix metalloproteinase inhibitor (MMPI) Batimastat, 30 min before infection and daily after infection with <i>E</i>. <i>coli</i> CFT073 (0.5 mg in 100 μl of PBS i.p. per mouse, except day 3) (<i>n</i> = 7 per treatment group, total of two experiments). (<b>B</b>) Difference in gross bladder pathology between untreated controls and IL-1RA or MMPI treated mice. Two representative mice per group are shown. The IL-1RA therapy reduced macroscopic bladder pathology in <i>Asc</i>^<i>-/-</i> mice. Scale bar = 1 mm. The MMPI therapy showed a similar but less pronounced effect. (<b>C</b>) Edema, hyperemia and size of the bladders were used as scoring parameters for the pathology score. Pathology scores from individual mice are shown. The gross pathology score was reduced by the inhibitors (<i>P</i> < 0.001, for IL-1RA compared to untreated <i>Asc</i>^-/- mice and <i>P</i> = 0.002, for MMPI, compared to untreated <i>Asc</i>^-/- mice, means ± SEMs of two experiments, two-tailed Mann Whitney test). (<b>D</b>) Protection from bladder tissue pathology shown in H&E stained sections from treated versus control mice. Arrows indicate mucosal sloughing, edema and subepithelial abscesses in untreated mice. Inhibition of mucosal neutrophil aggregate formation in bladder sections from treated mice compared to untreated and infected mice. Scale bar = 200 μm (H&E) and 50 μm (immunofluorescence). (<b>E</b>) Kinetics of neutrophil recruitment and bacterial clearance in the urine of IL-1RA or MMPI treated <i>Asc</i>^-/- mice, compared to untreated mice. (<i>n</i> = 7 mice per group, means ± SEMs, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, two-tailed unpaired <i>t</i>-test).</p