10 research outputs found

    MOESM1 of Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia

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    Additional file 1: Figure S1. (Top) H3K4me3 peak intensity density distribution proximal to the TSS in relation to oxygen deprivation and reoxygenation. (Bottom) H3K27me3-distribution proximal to the TSS in relation to oxygen deprivation and reoxygenation. Legend: t=0: normoxia; t=8: 8 hours of hypoxia; t=24: 24 hours of hypoxia; t=+8: 8 hours of subsequent reoxygenation. These figures and underlying data have also been published in an accompanying paper [10]. Figure S2. Relation between the ratio of H3K4me3 and H3K27me3 enrichment at the transcription start site for each gene with its associated expression level at 0 hours of hypoxia (i.e. t=0, normoxia). Higher enrichment is associated with higher expression, as observed previously [46]

    The Immediate Early Gene Product EGR1 and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis

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    <div><p>Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized <i>in vivo</i> and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli <i>in vitro</i>. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal <i>Ezh2</i> and <i>Bmi1</i> expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.</p> </div

    EGR1-LOF elicits DNA damage in hyper-proliferating chondrocytes.

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    <p>(A) Immunoblot (IB) detection of enhanced phospho-CHK2 (pCHK2) in <i>shEgr1</i> cultures early in chondrogenesis; FC <i>shcon</i>/<i>shEgr1</i>: 1,1 (t = 0), 1.0 (t = 8), 0.5 (t = 24), 4.5 (t = 72 days <i>pid</i>). (B) Detection of early DNA damage by IC analysis of γH2A.X (upper panels) in ATDC5 <i>shEgr1</i> cultures at 2 days <i>pid</i>; DAPI counterstaining by DAPI (lower panels). (C) IB detection of proteins related to DNA damage responses and cell-cycle arrest in ATDC5 cells stably expressing <i>shEgr1</i>; bActin loading control (* as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058083#pone-0058083-g006" target="_blank">Figure 6B</a>). Samples corresponding to control and experiment (<i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiment shown). (D) Comparative mRNA expression analysis of genes involved in cellular senescence, DNA damage response and stress signalling in <i>shcon</i> ATDC5 cells and cells stably expressing <i>shEgr1</i> throughout chondrogenesis (arbitrary expression units).</p

    Loss of EGR1 affects chondrogenic histone modification and epigenetic modifier expression.

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    <p>(A, B) IB analysis of histone modifications ATDC5 <i>shcon</i> and <i>shEgr1</i> cultures as a function of chondrogenic differentiation time (as indicated): reduced histone acetylation (A) and abnormal histone trimethylation (B); bActin: loading control (* as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058083#pone-0058083-g005" target="_blank">Figure 5B</a>). (C) IC Detection of enhanced H3K9me3 staining (upper panels) and H3K27me3 staining in ATDC5 large flat <i>shEgr1</i> cells at 3 days <i>pid</i>; DAPI counterstaining by DAPI (lower panels). (D) IC detection of abnormal epigenetic regulator protein expression (BMI1, EZH2, KAP1) as a function of differentiation time; Tubulin loading control. Samples corresponding to control and experiment (Figures A, B, D; <i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiments shown). (E) EGR1 enrichment on BMI1 and EZH2 promoters at 0, 2 and 8 hours <i>pid</i>. *: P values (EGR1/chromatin enrichment at t = 2 vs t = 0): 0.045 and 0.05.</p

    EGR1 depletion reduces chondrogenic differentiation.

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    <p>(A) EGR1-protein expression (protein) in ATDC5 cells stably expressing control short hairpin sequences (<i>shcon</i>) (upper panel); absent EGR1 in cells expressing <i>shEgr1</i> vectors (lower panel) at 0, 1, 2 and 4 hours pid. GAPDH is used as loading control. Samples corresponding to control and experiment (<i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiment shown. Selection pressure on <i>shRNA</i> expression was maintained for the duration of the experiments. (B–D) Reduced <i>Egr1</i> (B), <i>Sox9</i> (C) and <i>Col2a1</i> (D) expression (mRNA) in ATDC5 <i>shEgr1</i> compared to <i>shcon cultures</i>; standard error is based on three independent, parallel experiments; expression was normalized to <i>cyclophilin A</i>. (E) Reduced chondrogenic marker protein expression in ATDC5 cells stably expressing <i>shEgr1</i>. Samples corresponding to control and experiment (<i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiment shown).</p

    Functional links between MK3 and PRC in proliferative life span.

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    <p>(A) Expression levels of the PRC proteins EZH2, CBX8 and PHC1 in senescing TIG3/<i>MK3</i><sup><i>WT</i></sup><i>OE</i> cells at ±3–4 weeks post-transduction; loading control b-Actin (bAct). (B) Expression levels of the PRC proteins EZH2, CBX8 and PHC1 in senescing TIG3/<i>shMK3</i> cells at ±2–3 weeks post-transduction; loading control b-Actin (bAct). (C) Chromatin immunoprecipitation (ChIP) analysis of MK3, H3K27me3, CBX8, PHC1 enrichment in <i>MK3</i><sup><i>WT</i></sup><i>OE</i> (<i>MK3</i><sup><i>WT</i></sup><i>OE</i>) expressing and control (<i>con</i>) TIG3 cells; PRC1-target loci (p<i>16 promoter</i>, <i>p16exon1</i>, <i>HOXA10</i>, <i>HOXA11)</i> and non-target loci (p15exon1, p14exon1) are indicated below the figure. Enrichments are presented as percentages of total input. Negative control HA antiserum. Experiments were performed three times; results of one representative experiment are shown (statistical significance: * p<0.05, ** p<0.01; t-test).</p

    Functional links between MK3 and PRC in proliferative life span.

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    <p>TIG3 cells were sequentially transduced with either <i>Bmi1</i>.<i>ires</i>.<i>GFP</i> (<i>Bmi1OE</i>) or <i>GFP</i> (con) virus, and <i>MK3</i>/<i>puromycin</i> (<i>MK3OE</i>) or control <i>puromycin</i> virus (con) at 48 hrs intervals. Retroviral vectors expressing murine <i>Bmi1/</i>GFP reporter were transduced first (or empty vector control), followed by a MK3/<i>puromycin</i> resistance marker (or empty vector control). Transduction of <i>Bmi1OE</i> and control transduced cells was simultaneously carried out with the same <i>MK3</i><sup><i>WT</i></sup><i>OE</i> viral preparation (or control virus) to minimize inter-experimental variation. Cells were grown on selection medium and proliferation capacity was tested ± 2–3 weeks post-transduction. (A) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i><sup><i>WT</i></sup><i>overexpression</i> vector (<i>MK3</i><sup><i>WT</i></sup><i>OE</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; triangles). (B) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i> knock-down vector (<i>shMK3</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; squares). Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Comparative morphology of TIG3 cells expression <i>Bmi1</i> and/or <i>MK3</i> versus control cells as recorded by GFP fluorescent imaging ± 3 weeks after transduction (D) Immunoblot analysis of EZH2, CBX4, RNF2 and H3K27me3 in <i>MK3</i><sup><i>WT</i></sup><i>OE</i>, <i>Bmi1OE</i>, <i>Bmi1OE/MK3</i><sup><i>WT</i></sup><i>OE</i> and <i>control</i> TIG3 cell lysates. (E) Expression analysis of BMI1, MK3, and TP53 at the indicated time points in (corresponding to experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#pone.0118840.g004" target="_blank">Fig 4C</a>). Cells were grown on selection medium and analysed at 1 or 4 weeks after serial transduction; expression vectors and antibodies are indicated in the figure. Early and late samples were loaded on the same gel for BMI1 analysis; corresponding sections are shown separately.</p

    <i>MK3</i><sup><i>WT</i></sup> overexpression induces a proliferative arrest in normal human fibroblasts.

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    <p>(A) Retroviral expression systems were applied to enhance (<i>MK3</i><sup><i>WT</i></sup><i>OE</i>) or remove (<i>shMK3</i>) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3<sup>endo</sup>) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral <i>MK3</i> expression vector (<i>MK3</i><sup><i>WT</i></sup><i>OE</i>; filled circles), an empty vector (con; open circles) or a murine <i>Bmi1</i> expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/<i>MK3</i><sup><i>WT</i></sup><i>OE</i> cells at approximately 1 week post-transduction. <i>RasV12</i>-transduced cells were used as a positive control. (D) <i>MK3</i><sup><i>WT</i></sup><i>OE</i> reduces <i>de novo</i> DNA synthesis in TIG3/<i>MK3</i><sup><i>WT</i></sup><i>OE</i> cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; <i>MK3</i><sup><i>WT</i></sup><i>OE</i>: 442 BrdU-positive cells/9 fields.</p

    Proliferative regulation by MK3 in cancer cell lines.

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    <p>(A) Proliferation curves (left) of U-2OS cells expressing a retroviral <i>MK3</i> vector (<i>MK3</i><sup><i>WT</i></sup><i>OE</i>; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (<i>MK3</i><sup><i>WT</i></sup><i>OE</i>) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/<i>MK3</i><sup><i>WT</i></sup><i>OE</i> cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21<sup>CIP1/WAF1</sup> (P21) in U-2OS/<i>MK3</i><sup><i>WT</i></sup><i>OE</i> cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/<i>MK3OE</i> versus control cells (4–6 days post-transduction; representative experiment). <i>MK3</i><sup><i>WT</i></sup>overexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/<i>MK3</i><sup><i>WT</i></sup><i>OE</i> cultures; control (top panels) or <i>MK3</i><sup><i>WT</i></sup><i>OE</i> (bottom panels).</p
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