10 research outputs found

    Reprogramming of H3K27me3 Is Critical for Acquisition of Pluripotency from Cultured <em>Arabidopsis</em> Tissues

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    <div><p>In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3) is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2) is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants <em>curly leaf swinger</em> (<em>clf swn</em>) and <em>embryonic flower2</em> (<em>emf2</em>) were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP–chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of <em>clf swn</em>. In contrast to the leaf explants, the root explants of both <em>clf swn</em> and <em>emf2</em> formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition, suggesting a general mechanism for cell fate transition in plants.</p> </div

    H3K27me3 hypomethylations occur at several root-regulatory genes.

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    <p>(A) qRT-PCR analyses show increased expression levels of root-regulatory genes <i>WOX5</i> and <i>SHR</i>. (B) ChIP analysis of <i>WOX5</i> and <i>SHR</i>. Bars show s.e. (C–G) GUS staining of the <i>WOX5<sub>pro</sub>:GUS</i> transgenic line in the root (C) and leaf explants cultured for 2 to 8 days on CIM (D–G). (H–M) GUS staining of the <i>SHR<sub>pro</sub>:GUS</i> transgenic line in the root (H), and in leaf explants at time 0 to 8 DAC (I–M). (N) ChIP-chip results in calli showed the reduced H3K27me3 levels at <i>WOX5, SHR, LOB33</i> and <i>AGL21</i>. Bars = 100 µm in (C, H) and 2 mm in (D–G, I–M).</p

    Leaf explants of the PcG double mutant <i>clf-50 swn-1</i> lose the ability to form a callus.

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    <p>Sterilized seeds were grown on plain MS media. The third and fourth rosette leaves of 20-day-old seedlings were cut at the junction between the blade and the petiole and only the blade parts were cultured on CIM for another 20 days. (A–F) Wild-type Col-0 (A), <i>atx1-2</i> (B), <i>sdg2-3</i> (C), <i>sdg8-2</i> (D), wild-type Ws (E), and <i>clf-50 swn-1</i> (F). Note that only leaf explants, which were from the <i>clf-50 swn-1</i> rosette leaves, failed to form callus. For each genotype, more than 30 rosette leaves were used in one experiment, and they all exhibited the consistent phenotype. Shown are three randomly picked leaves out of the 30 tested leaves for each genotype. Bars = 2 mm in (A–E) and 400 µm in (F).</p

    ChIP–chip analysis to identify genes with changes in the H3K27me3 levels between leaf and callus.

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    <p>(A) Leaf explants (left) and calli (right) were used for the ChIP-chip experiment. For preparation of experimental materials, leaf explants that were just cut from leaves of 20-day-old wild-type Col-0 seedlings and calli that were on the margins of leaf explants cultured on CIM for 20 days were harvested for nuclei extraction. (B) A comparison of H3K27me3 covered loci taken from three independent genome-wide analyses (seedlings from Zhang et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002911#pgen.1002911-Zhang1" target="_blank">[13]</a>, and leaves and calli in this work). (C) Classifications by annotated functions of the identified H3K27me3-covered genes with either decreased (left) or increased (right) levels. (D) Expression profiles of the genes identified by the ChIP-chip experiment with the decreased (left) or increased (right) H3K27me3 levels. The original expression data were downloaded from the online program Genevestigator <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002911#pgen.1002911-Hruz1" target="_blank">[30]</a>.</p

    The leaf-to-callus transition process could be divided into pre-2 DAC and post-2 DAC stages based on the status of cell proliferation.

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    <p>(A–H) SEM analysis of morphology of leaf explants from 2 to 10 DAC, respectively. Green arrowheads indicate the midvein. Red arrowheads indicate leaf epidermal pavement cells. Yellow arrowheads show the dividing callus cells. Images in (B), (D), and (F) are close-ups of the boxed regions in (A), (C), and (E), respectively. (I–L) GUS staining of leaf explants from the <i>CYCB1;1:GUS</i> transgenic line from 2 to 8 DAC. Note that the 2 DAC explants show no GUS staining. Bars = 100 µm in (B, D, F), and 1 mm in (A, C, E, G, H) and 2 mm in (I–L).</p

    H3K27me3 reprogramming affects cell fate transition in the post-2 DAC stage.

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    <p>(A) The online program Genevestigator <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002911#pgen.1002911-Hruz1" target="_blank">[30]</a> was used to analyze expression patterns of the putative transcription factor genes that show changes in H3K27me3 levels during callus formation. Asterisks show significant statistical differences by t-tests (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001). Among the putative transcription factors with decreased H3K27me3 levels, 21 out of 67 (31.3%) genes have significantly higher expression patterns in the root tissues (left), and among those with increased H3K27me3 levels, 16 out of 19 (84.2%) genes have significantly higher expression patterns in the leaf tissues (right). (B) qRT-PCR analysis of <i>SAW1</i> and <i>SAW2</i>. (C) ChIP analysis of <i>SAW1</i> and <i>SAW2</i>. Bars show s.e. (D) ChIP-chip results in calli showed increased H3K27me3 levels at <i>SAW1, SAW2, ATH1</i>, and <i>TCP10</i>.</p

    PcG is required for silencing leaf-regulatory genes during the leaf-to-callus transition.

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    <p>(A–F) Expression level changes of <i>GH3.2</i> (A), <i>IAA2</i> (B), <i>SAW1</i> (C), <i>SAW2</i> (D), <i>WOX5</i> (E), and <i>SHR</i> (F) in leaf explants of <i>clf-50 swn-1</i> from time 0 to 8 DAC, revealed by qRT-PCR analyses. Bars show s.e.</p

    Synergistic Effect of MoS<sub>2</sub> Nanosheets and VS<sub>2</sub> for the Hydrogen Evolution Reaction with Enhanced Humidity-Sensing Performance

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    As a typical transition-metal dichalcogenides, MoS<sub>2</sub> has been a hotspot of research in many fields. In this work, the MoS<sub>2</sub> nanosheets were compounded on 1T-VS<sub>2</sub> nanoflowers (VS<sub>2</sub>@MoS<sub>2</sub>) successfully by a two-step hydrothermal method for the first time, and their hydrogen evolution properties were studied mainly. The higher charge-transfer efficiency benefiting from the metallicity of VS<sub>2</sub> and the greater activity due to more exposed active edge sites of MoS<sub>2</sub> improve the hydrogen evolution reaction performance of the nanocomposite electrocatalyst. Adsorption and transport of an intermediate hydrogen atom by VS<sub>2</sub> also enhances the hydrogen evolution efficiency. The catalyst shows a low onset potential of 97 mV, a Tafel slope as low as 54.9 mV dec<sup>–1</sup>, and good stability. Combining the electric conductivity of VS<sub>2</sub> with the physicochemical stability of MoS<sub>2</sub>, VS<sub>2</sub>@MoS<sub>2</sub> also exhibits excellent humidity properties

    DataSheet_2_OPN promotes pro-inflammatory cytokine expression via ERK/JNK pathway and M1 macrophage polarization in Rosacea.docx

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    Rosacea is a chronic inflammatory dermatosis that involves dysregulation of innate and adaptive immune systems. Osteopontin (OPN) is a phosphorylated glycoprotein produced by a broad range of immune cells such as macrophages, keratinocytes, and T cells. However, the role of OPN in rosacea remains to be elucidated. In this study, it was found that OPN expression was significantly upregulated in rosacea patients and LL37-induced rosacea-like skin inflammation. Transcriptome sequencing results indicated that OPN regulated pro-inflammatory cytokines and promoted macrophage polarization towards M1 phenotype in rosacea-like skin inflammation. In vitro, it was demonstrated that intracellular OPN (iOPN) promoted LL37-induced IL1B production through ERK1/2 and JNK pathways in keratinocytes. Moreover, secreted OPN (sOPN) played an important role in keratinocyte-macrophage crosstalk. In conclusion, sOPN and iOPN were identified as key regulators of the innate immune system and played different roles in the pathogenesis of rosacea.</p

    DataSheet_1_OPN promotes pro-inflammatory cytokine expression via ERK/JNK pathway and M1 macrophage polarization in Rosacea.xls

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    Rosacea is a chronic inflammatory dermatosis that involves dysregulation of innate and adaptive immune systems. Osteopontin (OPN) is a phosphorylated glycoprotein produced by a broad range of immune cells such as macrophages, keratinocytes, and T cells. However, the role of OPN in rosacea remains to be elucidated. In this study, it was found that OPN expression was significantly upregulated in rosacea patients and LL37-induced rosacea-like skin inflammation. Transcriptome sequencing results indicated that OPN regulated pro-inflammatory cytokines and promoted macrophage polarization towards M1 phenotype in rosacea-like skin inflammation. In vitro, it was demonstrated that intracellular OPN (iOPN) promoted LL37-induced IL1B production through ERK1/2 and JNK pathways in keratinocytes. Moreover, secreted OPN (sOPN) played an important role in keratinocyte-macrophage crosstalk. In conclusion, sOPN and iOPN were identified as key regulators of the innate immune system and played different roles in the pathogenesis of rosacea.</p
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