Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development

The Xenopus animal cap transcriptome: building a mucociliary epithelium

With the availability of deep RNA sequencing, model organisms such as Xenopus offer an outstanding opportunity to investigate the genetic basis of vertebrate organ formation from its embryonic beginnings. Here we investigate dynamics of the RNA landscape during formation of the Xenopus tropicalis larval epidermis. Differentiation of non-neural ectoderm starts at gastrulation and takes about one day to produce a functional mucociliary epithelium, highly related to the one in human airways. To obtain RNA expression data, uncontaminated by nonepidermal tissues of the embryo, we use prospective ectodermal explants called Animal Caps (ACs), which differentiate autonomously into a ciliated epidermis. Their global transcriptome is investigated at three key timepoints, with a cumulative sequencing depth of similar to 10(8) reads per developmental stage. This database is provided as online Web Tool to the scientific community. In this paper, we report on global changes in gene expression, an unanticipated diversity of mRNA splicing isoforms, expression patterns of repetitive DNA Elements, and the complexity of circular RNAs during this process. Computationally we derive transcription factor hubs from this data set, which may help in the future to define novel genetic drivers of epidermal differentiation in vertebrates

Suv4-20h Histone Methyltransferases Promote Neuroectodermal Differentiation by Silencing the Pluripotency-Associated Oct-25 Gene

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state

Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus.

BACKGROUND: Alternative therapies for Staphylococcus aureus bacteremia and endocarditis are needed. METHODS: We randomly assigned 124 patients with S. aureus bacteremia with or without endocarditis to receive 6 mg of daptomycin intravenously per kilogram of body weight daily and 122 to receive initial low-dose gentamicin plus either an antistaphylococcal penicillin or vancomycin. The primary efficacy end point was treatment success 42 days after the end of therapy. RESULTS: Forty-two days after the end of therapy in the modified intention-to-treat analysis, a successful outcome was documented for 53 of 120 patients who received daptomycin as compared with 48 of 115 patients who received standard therapy (44.2 percent vs. 41.7 percent; absolute difference, 2.4 percent; 95 percent confidence interval, -10.2 to 15.1 percent). Our results met prespecified criteria for the noninferiority of daptomycin. The success rates were similar in subgroups of patients with complicated bacteremia, right-sided endocarditis, and methicillin-resistant S. aureus. Daptomycin therapy was associated with a higher rate of microbiologic failure than was standard therapy (19 vs. 11 patients, P=0.17). In 6 of the 19 patients with microbiologic failure in the daptomycin group, isolates with reduced susceptibility to daptomycin emerged; similarly, a reduced susceptibility to vancomycin was noted in isolates from patients treated with vancomycin. As compared with daptomycin therapy, standard therapy was associated with a nonsignificantly higher rate of adverse events that led to treatment failure due to the discontinuation of therapy (17 vs. 8, P=0.06). Clinically significant renal dysfunction occurred in 11.0 percent of patients who received daptomycin and in 26.3 percent of patients who received standard therapy (P=0.004). CONCLUSIONS: Daptomycin (6 mg per kilogram daily) is not inferior to standard therapy for S. aureus bacteremia and right-sided endocarditis. (ClinicalTrials.gov number, NCT00093067 [ClinicalTrials.gov].)

Maternal Wnt/β-Catenin Signaling Coactivates Transcription through NF-κB Binding Sites during Xenopus Axis Formation

Maternal Wnt/β-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/β-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R) signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between β-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/β-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3) is one of the genes with dual β-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus β-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous β-Catenin levels in an NF-κB motif dependent manner. These results suggest that β-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells

MoSfl1 Is Important for Virulence and Heat Tolerance in Magnaporthe oryzae

The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae

The nuclear collective motion

Current developments in nuclear structure are discussed from a theoretical perspective. First, the progress in theoretical modeling of nuclei is reviewed. This is followed by the discussion of nuclear time scales, nuclear collective modes, and nuclear deformations. Some perspectives on nuclear structure research far from stability are given. Finally, interdisciplinary aspects of the nuclear many-body problem are outlined

Dorsalisation by ectopic β-Catenin depends on endogenous NF-κB activity.

<p>Embryos were injected ventrally with high doses (100 pg, panels <b>A,B</b>) or low doses (25 pg <b>C,D</b>) of β-Catenin RNA, alone (<b>A,C</b>), or with 500 pg ΔNIκBα RNA (<b>B,D</b>). All injections included GFP RNA for lineage tracing (C′,D′). <b>A</b>) Embryos injected with high β-Catenin RNA were highly dorsalized (average DAI 6.8 for each axis), almost always lacking posterior structures (n = 160). <b>B</b>) Co-injection of 500 pg of ΔNIκBα reduced the secondary axis to an average DAI = 3.98, with a small number (8%) being single axis embryos (n = 210). <b>C</b>) The majority of embryos injected with low β-Catenin RNA produced pairs of complete axes, both of which scored as “5” on the DAI scale (n = 155). <b>D</b>) Coinjecting 500 pg of ΔNIκBα produced mostly single axis embryos (n = 162). (<b>C′,D′</b>) Lineage tracing showed that the β-Catenin RNA-injected cell progeny accumulated in the anterior region of one of the two axis (white arrow) with some posterior trailing in axial tissue (white arrowheads) (<b>C′</b>), while it was localized in posterior ventral tissues when ΔNIκBα was co-injected (<b>D′</b>). Panels <b>E</b> and <b>F</b> summarize the phenotypic penetrance for respectively high (100 pg) and low (25 pg) β-Catenin RNA (3 independent experiments in each case). Numbers on the X-axis indicate DAI-values of each embryonic Anlage (e.g. 5/5 defining a twinned embryo with two complete heads).</p

The Xnr3 promoter is regulated through a consensus NF-κB DNA binding site.

<p>Panel (<b>A</b>) - Pictorial representation of the Xnr3 promoter and its κB and Tcf binding sites, as well as representations of mutated promoters, which were used in the luciferase reporter gene assays. Bases at mutated nucleotide positions are shown in lower-case letters. (<b>B</b>) Lateral schematic view of a 32-cell stage embryo locating the B1, B4 and D1 blastomeres in black, whose progeny will later contribute to the BCNE, the ventral gastrula center (VGC) and the Nieuwkoop center (NC), respectively. (<b>C</b>) Results of injection of a group of wildtype and mutant Xnr3 promoter driven luciferase constructs, each injected into the B1, B4 and D1 blastomeres of 32-cell stage embryos; y-axis describes x-fold production of luciferase, normalized to an internal renilla control, relative to the luciferase activity of uninjected embryos (lane 1). (<b>D</b>) and (<b>E</b>) Luciferase activities of wildtype and mutant Xnr3 reporter constructs, injected into B1 or B4 blastomeres as indicated, under conditions, in which endogenous NF-κB activity is either antagonized through coninjected XrelAenR (<b>D</b>) or reduced through dominant-negative XMyD88 (<b>E</b>). Luciferase activity was measured at midgastrula (NF11).</p

Synergism of XRel and β-Catenin proteins in ectopic axis formation.

<p>Suboptimal levels of β-Catenin synergize with XRel proteins or Drosophila Easter/Spätzle to induce axial structures on the ventral side. Embryos injected ventrally with low levels (4 pg) of β-Catenin (<b>A</b>) or 500 pg of XRelA (<b>B</b>), XRel2, XRel3, XrelB RNAs were indistinguishable from uninjected sibling embryos. Co-injection of 500 pg XRelA with 3 pg of β-Catenin could induce rudimentary axial structures (<b>C</b>), as could co-injection of 100 pg each of easter and spätzle with 3 pg of β-Catenin (<b>D</b>). Graphical representation (<b>E</b>) of all injected combinations, indicating a selective synergism between ectopic β-Catenin and exogenous XrelA/p50 heterodimers, as well as with endogenous NF-κB proteins released by ectopic Ea/Spz.</p