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  • 93
    Diagenode bioruptor
    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a <t>Bioruptor</t> and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
    Bioruptor, supplied by Diagenode, used in various techniques. Bioz Stars score: 93/100, based on 17061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode lysis buffer
    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a <t>Bioruptor</t> and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
    Lysis Buffer, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 2457 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode ezh2
    Immunostaining pattern of FOXO3 expression in breast cancers with different BRCA mutation status. ( a ) BRCA mutation makeup of tissue microarray constructed from 308 cases of Korean breast cancer samples. ( b ) Representative staining images of FOXO3 and <t>EZH2</t> immunohistochemical staining of BRCA1-mutated, BRCA2-mutated or wild-type breast cancer samples. Images (original magnification, × 20); insets (original magnification, × 100).
    Ezh2, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagenode h3k4me3
    reChIP-seq and normR analysis reveals bivalent promoters in primary human CD4 + central memory T cells. ( a ) Anticipated outcomes after analysis. (X)/(Y), where X denotes (re)ChIP-seq track (A ChIP, B ChIP, BA reChIP or AB reChIP) normalized by the control Y (I=Input, A or B). Yellow, blue and blue/yellow boxes indicate enrichment, no enrichment or borderline enrichment, respectively. The colours next to the (re)ChIP enrichment patterns denote: full co-occupancy (black); A antigen partial co-occupancy (beige); B antigen partial co-occupancy (blue); low co-occupancy (yellow); pseudo co-occupancy (orange); only A antigen (purple); only B antigen (red); or no occupancy (grey). ( b ) TSS clustered by their enrichment pattern for <t>H3K4me3,</t> H3K27me3, reChIP H3K4me3 and reChIP H3K27me3 over respective controls resulting in seven classes (see a ): black, full bivalent; beige, H3K4me3 partial bivalent; blue, H3K27me3 partial bivalent; orange, pseudo bivalent; purple, H3K4me3-only; red, H3K27me3-only and grey; unmodified. Within a class, the TSS are ordered from low (bottom) to high DNA methylation. The gene names on the right denote the gene state of critical regulators of T Helper subtype differentiation 45 . Enrichment was calculated by normR (see the ‘Methods' section) ( c ) Enrichment/Depletion analysis of the classes in the top 1,000 most variably expressed genes. Stars indicate statistical significance (***) P value
    H3k4me3, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagenode h3k27me3
    MeCP2 binds to <t>H3K27me3</t> enriched loci independent of DNA methylation. a MeCP2 enrichment at differential methylated regions (DMRs) between HCT116 and DKO1 cells. N = 2 (per genotype, HCT116 vs DKO1) from biologically independent cells. n = 91,106, p
    H3k27me3, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 500 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode genomic dna
    Genome-wide <t>5hmC</t> patterns in mouse whole brain and liver <t>DNA</t> following enrichment by either antibody, chemical capture or protein affinity-based methods. ( a ) An overview of the three commercially available techniques for 5hmC enrichment. In our study, following enrichment we carried out whole genome amplification and dye labelling for micro-array hybridization. ( b ) qPCR validation of the relative enrichment efficiencies over candidate loci previously identified as being either enriched or depleted in 5hmC in the mouse liver ( 26 ). Following normalization to the negative region at the Gapdh promoter, all three techniques report similar findings; however, the JBP-1-based affinity technique gives very low enrichment values compared to the hmeDIP and hMeSeal methods. Red dotted line denotes no enrichment over Gapdh . ( c ) Pearson correlation analysis and clustering among the microarray datasets. Biological replicates cluster closely while tissues clustered independently confirming the tissue-specific nature of 5hmC patterns. JBP-1 affinity purified 5hmC datasets correlate poorly with the hmeDIP and hMeSeal sets ( d ) Autocorrelation analysis of 5hmC patterns determined by hmeDIP, hMeSeal and JBP-1-binding in a single mouse brain sample. Autocorrelation was determined to a distance of 40 probes (∼10 kb). A ‘random’ sample for comparison was generated by randomization of the hMeSeal data. Filled circles represent relative probe position. ( e ) Example of microarray datasets showing tissue specificity and biological replicate reproducibility between each technique over the liver specific gene Cyp2b10 . Data are plotted on log2 scales from −3 to +3. Biological replicates are numbered 1 and 2, respectively. Gene structure is shown below by blue bars. Boxed regions are expanded upon on the right to display regions independently validated by gRES-qPCR. Plots represent the percentage of each modification at a single CpG in the sequence CCGG following normalization (purple; 5hmC, red; 5mC, green; C). Error bars display the standard error of the biological replicates. ( f ) Percentage plots of the distributions of 5hmC enriched regions following hmeDIP, hMeSeal and JBP-1 5hmC purification. Peak probes of 5hmC enrichment were defined (see ‘Materials and Methods’ section) and then mapped to one of five unique genomic loci (promoter cores, proximal and distal regions as well as intra- and inter-genic regions; box on right). Red dotted lines highlight changes in the distributions between techniques. Boxed region is expanded upon to reveal technique dependant differences over promoter-core, -proximal and -distal peaks. ( g ) The number and distribution of 5hmC peak probes generated for the three techniques are low over CpG islands (CGI) and largely non-promoter associated. Pie charts representative of the dataset size reveal low numbers of CGI related 5hmC peak probes following hmeDIP or hMeSeal in the brain (i) and the liver (ii). The total number of peaks mapping to CGIs are shown in square brackets while round brackets denote the total per cent of probes on the arrays which overlapped with CGI enriched peaks. Pink = peak probes mapping to promoter CGI regions, brown = peak probes mapping to orphan CGI (non-promoter) regions.
    Genomic Dna, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 1680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode h3k27ac
    Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and <t>H3K27ac‐positive)</t> enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.
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    91
    Diagenode nrf2
    <t>NRF2</t> silencing positively regulates autophagy. The connection of NRF2-dependent oxidative stress and autophagy induction was checked by immunofluorescence microscopy. LC3 was stained by green fluorescence dye (Alexa Fluor 488); therefore, the green dots in the cells represent functional autophagosomes. A ) Time dependency of TBHP-induced (100 μM) autophagy regulation by NRF2. Rapamycin (Rap, 100 nM, 2 h) and bafilomycin (Baf, 10 μM, 2 h) were used as positive controls. B ) Quantification and statistical analysis of immunofluorescence microscopy data. Error bars represent ± sem . ** P
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    92
    Diagenode immunoprecipitation
    <t>NRF2</t> silencing positively regulates autophagy. The connection of NRF2-dependent oxidative stress and autophagy induction was checked by immunofluorescence microscopy. LC3 was stained by green fluorescence dye (Alexa Fluor 488); therefore, the green dots in the cells represent functional autophagosomes. A ) Time dependency of TBHP-induced (100 μM) autophagy regulation by NRF2. Rapamycin (Rap, 100 nM, 2 h) and bafilomycin (Baf, 10 μM, 2 h) were used as positive controls. B ) Quantification and statistical analysis of immunofluorescence microscopy data. Error bars represent ± sem . ** P
    Immunoprecipitation, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Diagenode ipure kit
    <t>NRF2</t> silencing positively regulates autophagy. The connection of NRF2-dependent oxidative stress and autophagy induction was checked by immunofluorescence microscopy. LC3 was stained by green fluorescence dye (Alexa Fluor 488); therefore, the green dots in the cells represent functional autophagosomes. A ) Time dependency of TBHP-induced (100 μM) autophagy regulation by NRF2. Rapamycin (Rap, 100 nM, 2 h) and bafilomycin (Baf, 10 μM, 2 h) were used as positive controls. B ) Quantification and statistical analysis of immunofluorescence microscopy data. Error bars represent ± sem . ** P
    Ipure Kit, supplied by Diagenode, used in various techniques. Bioz Stars score: 90/100, based on 689 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode chip kit
    <t>NRF2</t> silencing positively regulates autophagy. The connection of NRF2-dependent oxidative stress and autophagy induction was checked by immunofluorescence microscopy. LC3 was stained by green fluorescence dye (Alexa Fluor 488); therefore, the green dots in the cells represent functional autophagosomes. A ) Time dependency of TBHP-induced (100 μM) autophagy regulation by NRF2. Rapamycin (Rap, 100 nM, 2 h) and bafilomycin (Baf, 10 μM, 2 h) were used as positive controls. B ) Quantification and statistical analysis of immunofluorescence microscopy data. Error bars represent ± sem . ** P
    Chip Kit, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode h3k4me1
    Decreased H3K27ac at immune and cancer associated enhancers with age. (A) Heatmap depicting the 12 clusters identified using k-means clustering of regions with significant (LLR > 3 and absolute fold-change > 1.5) changes of <t>H3K4me1,</t> H3K4me3, H3K27me3, or H3K27ac with age (n=37,058 peaks). The fold-change(aged/young) signal is plotted for each histone modification for each peak. Annotation to active and poised enhancers as well as bivalent promoters identified in young HSCe is also shown. (B) Heatmap of H3K4me1 and H3K27ac signal at the enhancer enriched clusters J-L. The log 2 (Pooled IP/Pooled Input) signal is plotted for each age group, centered on the differential peak +/− 5kb. Select genes within each cluster are denoted on the right of the heatmap. (C) Bubble plot representation of select KEGG pathways that are enriched in clusters J-L. The size of each bubble corresponds to the number of genes within the cluster that are within the given gene set. Highly significant (FDR
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    Diagenode protease inhibitors
    Decreased H3K27ac at immune and cancer associated enhancers with age. (A) Heatmap depicting the 12 clusters identified using k-means clustering of regions with significant (LLR > 3 and absolute fold-change > 1.5) changes of <t>H3K4me1,</t> H3K4me3, H3K27me3, or H3K27ac with age (n=37,058 peaks). The fold-change(aged/young) signal is plotted for each histone modification for each peak. Annotation to active and poised enhancers as well as bivalent promoters identified in young HSCe is also shown. (B) Heatmap of H3K4me1 and H3K27ac signal at the enhancer enriched clusters J-L. The log 2 (Pooled IP/Pooled Input) signal is plotted for each age group, centered on the differential peak +/− 5kb. Select genes within each cluster are denoted on the right of the heatmap. (C) Bubble plot representation of select KEGG pathways that are enriched in clusters J-L. The size of each bubble corresponds to the number of genes within the cluster that are within the given gene set. Highly significant (FDR
    Protease Inhibitors, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 548 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagenode h3k9me3
    KAP1 recruitment at individual ERE loci UCSC genome browser snapshots of KAP1 and <t>H3K9me3</t> ChIP-seq profiles in resting and activated CD4 + T cells at individual ERE loci. Results are representative of two independent ChIP-seq experiments. ERE annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. ERE integrants on which KAP1 binding is centered are highlighted in red.
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    89
    Diagenode microplex library preparation kit v2
    KAP1 recruitment at individual ERE loci UCSC genome browser snapshots of KAP1 and <t>H3K9me3</t> ChIP-seq profiles in resting and activated CD4 + T cells at individual ERE loci. Results are representative of two independent ChIP-seq experiments. ERE annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. ERE integrants on which KAP1 binding is centered are highlighted in red.
    Microplex Library Preparation Kit V2, supplied by Diagenode, used in various techniques. Bioz Stars score: 89/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagenode bioruptor sonication system
    KAP1 recruitment at individual ERE loci UCSC genome browser snapshots of KAP1 and <t>H3K9me3</t> ChIP-seq profiles in resting and activated CD4 + T cells at individual ERE loci. Results are representative of two independent ChIP-seq experiments. ERE annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. ERE integrants on which KAP1 binding is centered are highlighted in red.
    Bioruptor Sonication System, supplied by Diagenode, used in various techniques. Bioz Stars score: 90/100, based on 681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagenode microplex library preparation kit
    Length distribution of interrupted palindromes at 5′ and 3′-ends in Illumina HiSeq 2000 reads of Atlantic cod ( Gadus morhua ). Reads were generated from 11 historic samples using TruSeq library creation protocols (red lines), four historic samples using <t>Microplex</t> protocols (black lines) and one modern sample using TruSeq protocols (grey line). Terminal palindromic sequences longer than three basepair are rare in the Microplex and modern samples.
    Microplex Library Preparation Kit, supplied by Diagenode, used in various techniques. Bioz Stars score: 89/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagenode magmedip kit
    ABO promoter methylation and ABO transcript expression analysis. The ABO promoter methylation status (n = 4 clinical grade MSCs, 2 samples each) and the expression of ABO blood group gene transcripts in clinical grade MSCs (n = 2) of known blood group and secretor genotype (K16- A 1 /A 1 -Se/se , K25- B/O 1 -Se/Se ) was tested with <t>MagMeDIP</t> Kit and quantitative real time PCR (qPCR), respectively. ( A ) DNA methylation (%, metDIP/input), of methylated TSH2B , unmethylated GAPDH , and ABO proximal promoter region. ( B ) ABO transcript analysis (qPCR) on resting MSCs compared to cells subjected to different types of induction treatments. MSCs were either stimulated for 5 days with 5 ng/ml of interferon-gamma (INFg), or activated for 5 days by inflammatory mediators released through a cell-impermeable membrane in trans-well mixed lymphocyte reactions (MLRs). MSCs were also subjected to 14-day in vitro differentiation with adipogenic (ADI) and osteogenic (OST) induction medium, or respective control medium. Adenocarcinoma cell line HPAF-II was used as positive control for ABO transcript expression and distilled H 2 O served as negative control. Control genes ICAM1 and aP2 served as positive controls for cytokine activation and adipogenic induction, respectively. Relative gene expression is shown compared to control gene beta-actin. Adipogenic and osteogenic differentiation was also confirmed with Oil red O staining for lipid rich vacuoles and von Kossa staining for mineralized matrix, respectively. Mean ± SD, ** * P
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    Image Search Results


    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Article Snippet: Sonication was performed using a Bioruptor (Diagenode).

    Techniques: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    Library preparation using the ligation method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of RNA oligomers. A total of six tags ( Table 3 : TAG02, TAG04, TAG05, TAG06, TAG07, TAG12) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor, collection of biotinylated 5′ ends using beads, and sample pooling for multiplexing. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the ligation method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of RNA oligomers. A total of six tags ( Table 3 : TAG02, TAG04, TAG05, TAG06, TAG07, TAG12) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor, collection of biotinylated 5′ ends using beads, and sample pooling for multiplexing. H) Illumina sequencing library preparation.

    Article Snippet: Sonication was performed using a Bioruptor (Diagenode).

    Techniques: Ligation, De-Phosphorylation Assay, Amplification, Polymerase Chain Reaction, Multiplexing, Sequencing

    Library preparation using the Non-CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the Non-CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Article Snippet: Sonication was performed using a Bioruptor (Diagenode).

    Techniques: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    Library preparation using the SMART method. A) The protocol used either poly A+ (0.025–0.5 µg) or total (0.05–1.0 µg) RNA. B) First-strand cDNA synthesis, together with template switching and continuous replication to the end of the oligonucleotide. C) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. D) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. E) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the SMART method. A) The protocol used either poly A+ (0.025–0.5 µg) or total (0.05–1.0 µg) RNA. B) First-strand cDNA synthesis, together with template switching and continuous replication to the end of the oligonucleotide. C) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. D) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. E) Illumina sequencing library preparation.

    Article Snippet: Sonication was performed using a Bioruptor (Diagenode).

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing

    Quantifying hybrid particles presenting both ER and PM markers . The membrane integrity of MelJuSo cells was compromised by EMBL 8.020mm “cell cracker” homogenizer. For all experiments, the percentage of hybrid vesicles labeling for TAP1-GFP and MHC class II-L243 relative to single or non-labeled vesicles was determined by a Beckman-Coulter MoFlo fluorescent cell sorter and represented in the graphs. All experiments are performed in multiplo. Shown is mean + SD. (A) Discernable cell populations with either ER (Tap1-GFP) or PM (stained MHC-II) markers were broken in separate tubes and than mixed ('Stained: Separately, Fractionated: Separately') or broken in the same tube simultaneously ('Stained: Separately, Fractionated: Together'). As a control cells labeled with both markers were used ('Stained: Together, Fractionated: Together'). (B) Fractionation strokes were performed 2, 10 or 30 times (ball size 8.010mm) or (D) with ball sizes of 8.010, 8.008 or 8.004mm (30 strokes). As a soft detergent 0.1% Triton X-100 in PBS (Triton) was used for 5 or 10 minutes on ice. (C) Sonication pulses of approximately 0.5 second by a Branson sonifier 250 (Duty cycle 50%, output control 5) or Diagenode Bioruptor (High level 0.5 seconds on/off interval), for either 4 (short), 8 (intermediate) or 12 (long) pulses. The released cell fragments were spun down and both supernatant and PBS resuspended pellets were analyzed. (E) The effect of temperature variations on formation of hybrid vesicles test was performed by EMBL cell cracker with 8.010mm ball size. Fractionation was 30 times at 4, 10, 18, 25 and 37 o C.

    Journal: International Journal of Biological Sciences

    Article Title: Mechanical Forces Used for Cell Fractionation Can Create Hybrid Membrane Vesicles

    doi:

    Figure Lengend Snippet: Quantifying hybrid particles presenting both ER and PM markers . The membrane integrity of MelJuSo cells was compromised by EMBL 8.020mm “cell cracker” homogenizer. For all experiments, the percentage of hybrid vesicles labeling for TAP1-GFP and MHC class II-L243 relative to single or non-labeled vesicles was determined by a Beckman-Coulter MoFlo fluorescent cell sorter and represented in the graphs. All experiments are performed in multiplo. Shown is mean + SD. (A) Discernable cell populations with either ER (Tap1-GFP) or PM (stained MHC-II) markers were broken in separate tubes and than mixed ('Stained: Separately, Fractionated: Separately') or broken in the same tube simultaneously ('Stained: Separately, Fractionated: Together'). As a control cells labeled with both markers were used ('Stained: Together, Fractionated: Together'). (B) Fractionation strokes were performed 2, 10 or 30 times (ball size 8.010mm) or (D) with ball sizes of 8.010, 8.008 or 8.004mm (30 strokes). As a soft detergent 0.1% Triton X-100 in PBS (Triton) was used for 5 or 10 minutes on ice. (C) Sonication pulses of approximately 0.5 second by a Branson sonifier 250 (Duty cycle 50%, output control 5) or Diagenode Bioruptor (High level 0.5 seconds on/off interval), for either 4 (short), 8 (intermediate) or 12 (long) pulses. The released cell fragments were spun down and both supernatant and PBS resuspended pellets were analyzed. (E) The effect of temperature variations on formation of hybrid vesicles test was performed by EMBL cell cracker with 8.010mm ball size. Fractionation was 30 times at 4, 10, 18, 25 and 37 o C.

    Article Snippet: Alternatively, cell disintegration was achieved by sonication pulses of approximately 0.5 second by a Branson sonifier 250 (Duty cycle 50%, output control 5) or Diagenode Bioruptor (High level 0.5 seconds on/off interval), for either 4 (short), 8 (intermediate) or 12 (long) pulses.

    Techniques: Labeling, Staining, Fractionation, Sonication

    Sonication efficiency and antibody specificity. a Chromatin samples were fragmented at 20 sec ON/30 sec OFF using Bioruptor® Pico. Samples were decrosslinked, DNA was purified by phenol:chloroform:isoamyl alcohol extraction and loaded on 1.5% agarose gel. Lane 1: non-sonicated; lane 2: sonicated 7 times; lane 3: sonicated 15 times; lane 4: sonicated 20 times; lane M: 5000 bp DNA marker. Scales indicate the DNA fragments to be between 100–500 bp. b Western blot figure showing lane M: protein molecular weight marker; lane 1: crude protein extract (40 μg); lane 2: IP in sample 1; lane 3: IP in sample 2; lane 4: mock control in sample 1; lane 5: mock control in sample 2. Arrow indicates the protein marker band 50 KD. c Western blot showing the lane M protein weight marker; lane 1: crude protein extract (40 μg) from endosperms of 35 DAP castor bean seeds; lane 2: IP sample; lane 3: IP sample (20 μl from the IP sample used for lane 2); lane 4: mock control sample. Arrow indicates the protein marker band 50 KD.

    Journal: PLoS ONE

    Article Title: Development of an efficient chromatin immunoprecipitation method to investigate protein-DNA interaction in oleaginous castor bean seeds

    doi: 10.1371/journal.pone.0197126

    Figure Lengend Snippet: Sonication efficiency and antibody specificity. a Chromatin samples were fragmented at 20 sec ON/30 sec OFF using Bioruptor® Pico. Samples were decrosslinked, DNA was purified by phenol:chloroform:isoamyl alcohol extraction and loaded on 1.5% agarose gel. Lane 1: non-sonicated; lane 2: sonicated 7 times; lane 3: sonicated 15 times; lane 4: sonicated 20 times; lane M: 5000 bp DNA marker. Scales indicate the DNA fragments to be between 100–500 bp. b Western blot figure showing lane M: protein molecular weight marker; lane 1: crude protein extract (40 μg); lane 2: IP in sample 1; lane 3: IP in sample 2; lane 4: mock control in sample 1; lane 5: mock control in sample 2. Arrow indicates the protein marker band 50 KD. c Western blot showing the lane M protein weight marker; lane 1: crude protein extract (40 μg) from endosperms of 35 DAP castor bean seeds; lane 2: IP sample; lane 3: IP sample (20 μl from the IP sample used for lane 2); lane 4: mock control sample. Arrow indicates the protein marker band 50 KD.

    Article Snippet: In our experiment, we tried to optimize the shearing conditions using Bioruptor® Pico (DIAGENODE, BELGIUM) with a combination of 20 sec ON/30 sec OFF for several times, and obtained maximum fragments in the range of 100–500 bp in 20 times on agarose gel ( ).

    Techniques: Sonication, Size-exclusion Chromatography, Purification, Agarose Gel Electrophoresis, Marker, Western Blot, Molecular Weight

    Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: TF-ChIP Method for Tissue-Specific Gene Targets

    doi: 10.3389/fncel.2019.00095

    Figure Lengend Snippet: Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

    Article Snippet: Bioruptor Plus (Diagenode, Belgium): Power: High Runs: 3× 10 cycles Interval: 30″ ON/30″ OFF Note: Vortex gently and spin down between each run.

    Techniques: Expressing, Immunofluorescence, Transgenic Assay, Activation Assay, Staining, Sonication, Nucleic Acid Electrophoresis

    Immunostaining pattern of FOXO3 expression in breast cancers with different BRCA mutation status. ( a ) BRCA mutation makeup of tissue microarray constructed from 308 cases of Korean breast cancer samples. ( b ) Representative staining images of FOXO3 and EZH2 immunohistochemical staining of BRCA1-mutated, BRCA2-mutated or wild-type breast cancer samples. Images (original magnification, × 20); insets (original magnification, × 100).

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: Immunostaining pattern of FOXO3 expression in breast cancers with different BRCA mutation status. ( a ) BRCA mutation makeup of tissue microarray constructed from 308 cases of Korean breast cancer samples. ( b ) Representative staining images of FOXO3 and EZH2 immunohistochemical staining of BRCA1-mutated, BRCA2-mutated or wild-type breast cancer samples. Images (original magnification, × 20); insets (original magnification, × 100).

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Immunostaining, Expressing, Mutagenesis, Microarray, Construct, Staining, Immunohistochemistry

    Inhibition of EZH2 induced FOXO3 expression in basal-type cell lines. The basal type cell lines ( a ) HCC70 and ( b ) MDA-MB-468, as well as the luminal ( c ) MCF-7 cells were treated with 0, 1 and 5 μ M of the EZH2 inhibitor GSK126 for 72 h with culture medium changed every day. Total protein was extracted from these cells and analysed by western blotting with the indicated antibodies. In parallel, total RNA was also extracted and expression of BRCA1, EZH2 and FOXO3 mRNA was analysed by qRT–PCR. The experiments were repeated three times independently and qRT–PCR results were normalized against L19 mRNA levels and the results expressed as mean±s.d. * P ⩽0.05 and ** P ⩽0.001; NS, not significant Students' t -test.

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: Inhibition of EZH2 induced FOXO3 expression in basal-type cell lines. The basal type cell lines ( a ) HCC70 and ( b ) MDA-MB-468, as well as the luminal ( c ) MCF-7 cells were treated with 0, 1 and 5 μ M of the EZH2 inhibitor GSK126 for 72 h with culture medium changed every day. Total protein was extracted from these cells and analysed by western blotting with the indicated antibodies. In parallel, total RNA was also extracted and expression of BRCA1, EZH2 and FOXO3 mRNA was analysed by qRT–PCR. The experiments were repeated three times independently and qRT–PCR results were normalized against L19 mRNA levels and the results expressed as mean±s.d. * P ⩽0.05 and ** P ⩽0.001; NS, not significant Students' t -test.

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Inhibition, Expressing, Multiple Displacement Amplification, Western Blot, Quantitative RT-PCR

    Depletion of EZH2 induced FOXO3 expression in HCC70 but not in MCF-7 cells. Western blotting and qRT–PCR analysis was performed on ( a ) HCC70 and ( b ) MCF-7 cells mock transfected or transfected with EZH2-specific siRNA pool or non-silencing control (NSC) siRNA pool for 48 h. Depletion of EZH2 by siRNA in HCC70 significantly induced FOXO3 expression but did not affect FOXO3 expression in MCF-7 cells. These experiments have been repeated three times and the representative western blottings were shown. qRT–PCR data were expressed as mean±s.d; * P

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: Depletion of EZH2 induced FOXO3 expression in HCC70 but not in MCF-7 cells. Western blotting and qRT–PCR analysis was performed on ( a ) HCC70 and ( b ) MCF-7 cells mock transfected or transfected with EZH2-specific siRNA pool or non-silencing control (NSC) siRNA pool for 48 h. Depletion of EZH2 by siRNA in HCC70 significantly induced FOXO3 expression but did not affect FOXO3 expression in MCF-7 cells. These experiments have been repeated three times and the representative western blottings were shown. qRT–PCR data were expressed as mean±s.d; * P

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection

    ( a ) FOXO3 gene promoter is hypermethylated in BRCA1 mutation tumours. Frequency of FOXO3 promoter methylation in clinical samples with mutations in BRCA1, BRCA2 and BRCAx tumours was analysed using the kConFab database. In 33 familial breast tumour samples, significant higher percentage of FOXO3 promoter methylation was found in BRCA1-mutated tumours compared with BRCA2- or BRCAx-mutated tumour. Boxplots represent median (centre line), interquartile range (box) and 95th percentiles (whisker), and samples out with this range are represented as points. FOXO3 methylation scores were significantly higher in BRCA1-mutated samples when compared with BRCA2 or BRCAx ( P =0.019 and P =0.053, respectively, Students' t -test) or BRCA2/x ( P =0.026, Students' t -test). ( b ) Prognostic significance of FOXO3 and EZH2 mRNA in breast cancer. Examination of FOXO3 and EZH2 transcript expression in a previously published cohort (3455 breast cancer patients) 28 revealed that both low FOXO3 and high EZH2 mRNA expression levels are very significantly associated with poor survival ( P =0.033 and P =3.8 × 10 −1 , respectively, for overall survival, Kaplan–Meier analysis). The significance of both FOXO3 and EZH2in survival analyses provides further evidence for the involvement of both genes in breast cancer progression and drug response.

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: ( a ) FOXO3 gene promoter is hypermethylated in BRCA1 mutation tumours. Frequency of FOXO3 promoter methylation in clinical samples with mutations in BRCA1, BRCA2 and BRCAx tumours was analysed using the kConFab database. In 33 familial breast tumour samples, significant higher percentage of FOXO3 promoter methylation was found in BRCA1-mutated tumours compared with BRCA2- or BRCAx-mutated tumour. Boxplots represent median (centre line), interquartile range (box) and 95th percentiles (whisker), and samples out with this range are represented as points. FOXO3 methylation scores were significantly higher in BRCA1-mutated samples when compared with BRCA2 or BRCAx ( P =0.019 and P =0.053, respectively, Students' t -test) or BRCA2/x ( P =0.026, Students' t -test). ( b ) Prognostic significance of FOXO3 and EZH2 mRNA in breast cancer. Examination of FOXO3 and EZH2 transcript expression in a previously published cohort (3455 breast cancer patients) 28 revealed that both low FOXO3 and high EZH2 mRNA expression levels are very significantly associated with poor survival ( P =0.033 and P =3.8 × 10 −1 , respectively, for overall survival, Kaplan–Meier analysis). The significance of both FOXO3 and EZH2in survival analyses provides further evidence for the involvement of both genes in breast cancer progression and drug response.

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Mutagenesis, Methylation, Whisker Assay, Expressing

    FOXO3 and EZH2 expression levels in breast cancers with different BRCA mutation. ( a ) Comparison of FOXO3 expression levels with different BRCA mutation status by Mann–Whitney U -rank test using all samples. ( b ) Comparison of FOXO3 expression levels with different BRCA mutation status using samples that express low levels of nuclear EZH2 by Mann–Whitney U -rank test. ( c ) Comparison of FOXO3 expression levels with different BRCA mutation status compared using samples that express high levels of nuclear EZH2 by Mann–Whitney U -rank test.

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: FOXO3 and EZH2 expression levels in breast cancers with different BRCA mutation. ( a ) Comparison of FOXO3 expression levels with different BRCA mutation status by Mann–Whitney U -rank test using all samples. ( b ) Comparison of FOXO3 expression levels with different BRCA mutation status using samples that express low levels of nuclear EZH2 by Mann–Whitney U -rank test. ( c ) Comparison of FOXO3 expression levels with different BRCA mutation status compared using samples that express high levels of nuclear EZH2 by Mann–Whitney U -rank test.

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Expressing, Mutagenesis, MANN-WHITNEY

    BRCA1 depletion causes the accumulation of H3K27me3, DNMT1/3a/3b and DNA methylation on the FOXO3 promoter in MCF-7 cells. BRCA1 was transiently knocked down using specific siRNA pool in MCF-7 for 48 h. ( a ) MCF-7 cells transfected with BRCA1 and non-silencing control (NSC) siRNA pools independently were analysed for the accumulation of H3K27me3 on the endogenous FOXO3 promoter by ChIP–qRT–PCR analysis. The results showed that despite the variable changes in EZH2 recruitment, there was always an increase in the accumulation of H3K27me3 marks on BRCA1 depletion. ( b ) MCF-7 cells transfected with BRCA1 and NSC siRNA pools independently were analysed for the recruitment of DNMT1/3a/3b to the endogenous FOXO3 promoter by ChIP–qRT–PCR analysis. The results revealed that BRCA1 knockdown culminated in an increase in DNMT1/3a/3b recruitment. ( c ) MCF-7 cells transfected with BRCA1 and NSC siRNA pools independently were analysed for FOXO3 promoter methylation by methylated DNA immunoprecipitation (MeDIP) qRT–PCR analysis. Despite the primers 2 and 4 consistently failed to generate reliable results, the results from primer sets 1 and 3 showed that BRCA1, but not EZH2, knockdown significantly enhanced FOXO3 promoter methylation. The results were normalized to the amount of Input and compared with the IgG-negative controls. These experiments were repeated three times independently and the qRT–PCR results presented as mean±s.d. * P

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: BRCA1 depletion causes the accumulation of H3K27me3, DNMT1/3a/3b and DNA methylation on the FOXO3 promoter in MCF-7 cells. BRCA1 was transiently knocked down using specific siRNA pool in MCF-7 for 48 h. ( a ) MCF-7 cells transfected with BRCA1 and non-silencing control (NSC) siRNA pools independently were analysed for the accumulation of H3K27me3 on the endogenous FOXO3 promoter by ChIP–qRT–PCR analysis. The results showed that despite the variable changes in EZH2 recruitment, there was always an increase in the accumulation of H3K27me3 marks on BRCA1 depletion. ( b ) MCF-7 cells transfected with BRCA1 and NSC siRNA pools independently were analysed for the recruitment of DNMT1/3a/3b to the endogenous FOXO3 promoter by ChIP–qRT–PCR analysis. The results revealed that BRCA1 knockdown culminated in an increase in DNMT1/3a/3b recruitment. ( c ) MCF-7 cells transfected with BRCA1 and NSC siRNA pools independently were analysed for FOXO3 promoter methylation by methylated DNA immunoprecipitation (MeDIP) qRT–PCR analysis. Despite the primers 2 and 4 consistently failed to generate reliable results, the results from primer sets 1 and 3 showed that BRCA1, but not EZH2, knockdown significantly enhanced FOXO3 promoter methylation. The results were normalized to the amount of Input and compared with the IgG-negative controls. These experiments were repeated three times independently and the qRT–PCR results presented as mean±s.d. * P

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: DNA Methylation Assay, Transfection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation

    H3K27me3 is only enriched on the FOXO3 promoter in the BRCA1-deficient HCC70 and MDA-MB-468 cells but not in the BRCA1-competent MCF-7 cells, qRT–PCR analysis of immunoprecipitated chromatin for the recruitment of BRCA1, EZH2 and H3K27me3 to the endogenous FOXO3 promoter in HCC70, MDA-MB-468 and MCF-7 cells. ( a ) In HCC70, the ChIP–qPCR results showed that BRCA1, EZH2 and H3K27me3 were all recruited to the FOXO3 promoter albeit BRCA1 at low levels. ( b ) BRCA1 (C61G mutant), EZH2 and H3K27me3 were recruited to the FOXO3 promoter in MDA-MB-468 cells as revealed ChIP–qPCR analysis. ( c ) In MCF-7, BRCA1 and EZH2 were associated with the FOXO3 promoter but H3K27me3 was not. The results were normalized to the amount of Input and compared with the IgG-negative controls. IgG (R), rabbit IgG-negative control; IgG (M), mouse IgG-negative control. These experiments were repeated three times independently and the qRT–PCR results presented as mean±s.d. * P

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: H3K27me3 is only enriched on the FOXO3 promoter in the BRCA1-deficient HCC70 and MDA-MB-468 cells but not in the BRCA1-competent MCF-7 cells, qRT–PCR analysis of immunoprecipitated chromatin for the recruitment of BRCA1, EZH2 and H3K27me3 to the endogenous FOXO3 promoter in HCC70, MDA-MB-468 and MCF-7 cells. ( a ) In HCC70, the ChIP–qPCR results showed that BRCA1, EZH2 and H3K27me3 were all recruited to the FOXO3 promoter albeit BRCA1 at low levels. ( b ) BRCA1 (C61G mutant), EZH2 and H3K27me3 were recruited to the FOXO3 promoter in MDA-MB-468 cells as revealed ChIP–qPCR analysis. ( c ) In MCF-7, BRCA1 and EZH2 were associated with the FOXO3 promoter but H3K27me3 was not. The results were normalized to the amount of Input and compared with the IgG-negative controls. IgG (R), rabbit IgG-negative control; IgG (M), mouse IgG-negative control. These experiments were repeated three times independently and the qRT–PCR results presented as mean±s.d. * P

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Multiple Displacement Amplification, Quantitative RT-PCR, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mutagenesis, Negative Control

    5′-Aza-dC treatment induces FOXO3 expression in basal-type cell lines. The basal type cell lines ( a ) HCC70 and ( b ) MDA-MB-468, as well as the luminal ( c ) MCF-7 cells were treated with 0, 1 and 5 μ M of 5′-aza-dC for 72 h with culture medium changed every day. Total protein was extracted from these cells and analysed by western blotting with the indicated antibodies. In parallel, total RNA was also extracted and expression of BRCA1, EZH2 and FOXO3 mRNA was analysed by qRT–PCR. The experiments were repeated three times independently and qRT–PCR results were normalized against L19 mRNA levels and the results expressed as mean±s.d. * P ⩽0.05 and ** P ⩽0.001; NS, not significant by Students' t -test.

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: 5′-Aza-dC treatment induces FOXO3 expression in basal-type cell lines. The basal type cell lines ( a ) HCC70 and ( b ) MDA-MB-468, as well as the luminal ( c ) MCF-7 cells were treated with 0, 1 and 5 μ M of 5′-aza-dC for 72 h with culture medium changed every day. Total protein was extracted from these cells and analysed by western blotting with the indicated antibodies. In parallel, total RNA was also extracted and expression of BRCA1, EZH2 and FOXO3 mRNA was analysed by qRT–PCR. The experiments were repeated three times independently and qRT–PCR results were normalized against L19 mRNA levels and the results expressed as mean±s.d. * P ⩽0.05 and ** P ⩽0.001; NS, not significant by Students' t -test.

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot, Quantitative RT-PCR

    BRCA1 status correlates with FOXO3 expression in breast cancer cell lines. ( a ) Western blotting and ( b ) qRT–PCR analysis was performed on a panel of five different breast cancer cell lines including the luminal-type cell line MCF-7, which expresses wild-type BRCA1, basal-type cell lines HCC70, MDA-MB-231, MDA-MB-468 and MDA-MB-436, expressing either low or mutated BRCA1. ( a ) The expression of BRCA1, FOXO3, EZH2, ERα, GATA3 and Tubulin was examined by western blotting. ( b ) The experiments were repeated three times independently and qRT–PCR results were normalized against L19 mRNA levels and the results presented as bars representing mean±s.d.

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: BRCA1 status correlates with FOXO3 expression in breast cancer cell lines. ( a ) Western blotting and ( b ) qRT–PCR analysis was performed on a panel of five different breast cancer cell lines including the luminal-type cell line MCF-7, which expresses wild-type BRCA1, basal-type cell lines HCC70, MDA-MB-231, MDA-MB-468 and MDA-MB-436, expressing either low or mutated BRCA1. ( a ) The expression of BRCA1, FOXO3, EZH2, ERα, GATA3 and Tubulin was examined by western blotting. ( b ) The experiments were repeated three times independently and qRT–PCR results were normalized against L19 mRNA levels and the results presented as bars representing mean±s.d.

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Multiple Displacement Amplification

    Schematic representation of the alignment of the binding profiles for EZH2, BRCA1 and H3K27me3, and the locations of the ChIP primers with the human FOXO3 promoter. A schematic illustration of the human FOXO3 promoter region, showing the two transcription start sites (Chr 6: 108,559,835- and Chr 6: 108,560,866-) (Top). ENCODE (the Encyclopedia of DNA Elements) project ChIP sequencing data of EZH2, BRCA1 and H3K27me3 binding in the liver carcinoma HepG2 (ATCC Number HB-8065) cells were used for predicting global genome-binding profiles for EZH2, BRCA1 and H3K27me3. The predicted binding profiles of EZH2, BRCA1 and H3K27me3 on the human FOXO3 promoter are shown (below the FOXO3 promoter). The positions of the black boxes represent the amplicons of the designed ChIP primer pairs (primers 1–4; further below). The predicted binding profiles of EZH2, BRCA1 and H3K27me3, and the locations of amplicons from the designed ChIP primer pairs (primers 1–4) are aligned to the FOXO3 promoter.

    Journal: Oncogenesis

    Article Title: BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    doi: 10.1038/oncsis.2016.23

    Figure Lengend Snippet: Schematic representation of the alignment of the binding profiles for EZH2, BRCA1 and H3K27me3, and the locations of the ChIP primers with the human FOXO3 promoter. A schematic illustration of the human FOXO3 promoter region, showing the two transcription start sites (Chr 6: 108,559,835- and Chr 6: 108,560,866-) (Top). ENCODE (the Encyclopedia of DNA Elements) project ChIP sequencing data of EZH2, BRCA1 and H3K27me3 binding in the liver carcinoma HepG2 (ATCC Number HB-8065) cells were used for predicting global genome-binding profiles for EZH2, BRCA1 and H3K27me3. The predicted binding profiles of EZH2, BRCA1 and H3K27me3 on the human FOXO3 promoter are shown (below the FOXO3 promoter). The positions of the black boxes represent the amplicons of the designed ChIP primer pairs (primers 1–4; further below). The predicted binding profiles of EZH2, BRCA1 and H3K27me3, and the locations of amplicons from the designed ChIP primer pairs (primers 1–4) are aligned to the FOXO3 promoter.

    Article Snippet: Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK) , EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    reChIP-seq and normR analysis reveals bivalent promoters in primary human CD4 + central memory T cells. ( a ) Anticipated outcomes after analysis. (X)/(Y), where X denotes (re)ChIP-seq track (A ChIP, B ChIP, BA reChIP or AB reChIP) normalized by the control Y (I=Input, A or B). Yellow, blue and blue/yellow boxes indicate enrichment, no enrichment or borderline enrichment, respectively. The colours next to the (re)ChIP enrichment patterns denote: full co-occupancy (black); A antigen partial co-occupancy (beige); B antigen partial co-occupancy (blue); low co-occupancy (yellow); pseudo co-occupancy (orange); only A antigen (purple); only B antigen (red); or no occupancy (grey). ( b ) TSS clustered by their enrichment pattern for H3K4me3, H3K27me3, reChIP H3K4me3 and reChIP H3K27me3 over respective controls resulting in seven classes (see a ): black, full bivalent; beige, H3K4me3 partial bivalent; blue, H3K27me3 partial bivalent; orange, pseudo bivalent; purple, H3K4me3-only; red, H3K27me3-only and grey; unmodified. Within a class, the TSS are ordered from low (bottom) to high DNA methylation. The gene names on the right denote the gene state of critical regulators of T Helper subtype differentiation 45 . Enrichment was calculated by normR (see the ‘Methods' section) ( c ) Enrichment/Depletion analysis of the classes in the top 1,000 most variably expressed genes. Stars indicate statistical significance (***) P value

    Journal: Nature Communications

    Article Title: reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4+ memory T cells

    doi: 10.1038/ncomms12514

    Figure Lengend Snippet: reChIP-seq and normR analysis reveals bivalent promoters in primary human CD4 + central memory T cells. ( a ) Anticipated outcomes after analysis. (X)/(Y), where X denotes (re)ChIP-seq track (A ChIP, B ChIP, BA reChIP or AB reChIP) normalized by the control Y (I=Input, A or B). Yellow, blue and blue/yellow boxes indicate enrichment, no enrichment or borderline enrichment, respectively. The colours next to the (re)ChIP enrichment patterns denote: full co-occupancy (black); A antigen partial co-occupancy (beige); B antigen partial co-occupancy (blue); low co-occupancy (yellow); pseudo co-occupancy (orange); only A antigen (purple); only B antigen (red); or no occupancy (grey). ( b ) TSS clustered by their enrichment pattern for H3K4me3, H3K27me3, reChIP H3K4me3 and reChIP H3K27me3 over respective controls resulting in seven classes (see a ): black, full bivalent; beige, H3K4me3 partial bivalent; blue, H3K27me3 partial bivalent; orange, pseudo bivalent; purple, H3K4me3-only; red, H3K27me3-only and grey; unmodified. Within a class, the TSS are ordered from low (bottom) to high DNA methylation. The gene names on the right denote the gene state of critical regulators of T Helper subtype differentiation 45 . Enrichment was calculated by normR (see the ‘Methods' section) ( c ) Enrichment/Depletion analysis of the classes in the top 1,000 most variably expressed genes. Stars indicate statistical significance (***) P value

    Article Snippet: We used the enrichment calls for H3K4me3, H3K27me3, H3K4me3 reChIP and H3K27me3 reChIP over input to cluster the TSSs or CpG islands in eight classes: (i) no enrichment in any (re)ChIP, (ii) only enrichment in H3K27me3, (iii) only enrichment in H3K4me3, (iv) enrichment in H3K4me3 and H3K27me3 but not in the two reChIPs, (v) only enrichment in both the reChIPs but no enrichment in the primary ChIPs, (vi) enrichment in H3K27me3 and both the reChIPs, (vii) enrichment in H3K4me3 and both the reChIPs and (viii) enrichment in all the four (re)ChIPs.

    Techniques: Chromatin Immunoprecipitation, DNA Methylation Assay

    Bivalency is the default response to unmethylated CpG-rich DNA sequences. CpG islands clustered by their enrichment pattern for H3K4me3, H3K27me3, reChIP H3K4me3 and reChIP H3K27me3 over respective controls. Fragment coverage for the tracks indicated above each column (1 to 4) for each CpG island (rows, sorted by CpG island length) ±2,000 base pairs centred around the centre of the CpG island. The fifth column represents DNA methylation (0 denotes no DNA methylation and 1 denotes full DNA methylation). The last two columns represent the CpG content and the CpGA/TpG content in percentage.

    Journal: Nature Communications

    Article Title: reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4+ memory T cells

    doi: 10.1038/ncomms12514

    Figure Lengend Snippet: Bivalency is the default response to unmethylated CpG-rich DNA sequences. CpG islands clustered by their enrichment pattern for H3K4me3, H3K27me3, reChIP H3K4me3 and reChIP H3K27me3 over respective controls. Fragment coverage for the tracks indicated above each column (1 to 4) for each CpG island (rows, sorted by CpG island length) ±2,000 base pairs centred around the centre of the CpG island. The fifth column represents DNA methylation (0 denotes no DNA methylation and 1 denotes full DNA methylation). The last two columns represent the CpG content and the CpGA/TpG content in percentage.

    Article Snippet: We used the enrichment calls for H3K4me3, H3K27me3, H3K4me3 reChIP and H3K27me3 reChIP over input to cluster the TSSs or CpG islands in eight classes: (i) no enrichment in any (re)ChIP, (ii) only enrichment in H3K27me3, (iii) only enrichment in H3K4me3, (iv) enrichment in H3K4me3 and H3K27me3 but not in the two reChIPs, (v) only enrichment in both the reChIPs but no enrichment in the primary ChIPs, (vi) enrichment in H3K27me3 and both the reChIPs, (vii) enrichment in H3K4me3 and both the reChIPs and (viii) enrichment in all the four (re)ChIPs.

    Techniques: DNA Methylation Assay

    Cross-talk between genome and epigenome. ( a – c ) The filled circles denote the relative fraction of TSSs in the indicated state. Purple indicates H3K4me3-only, red H3K27me3-only, black bivalent and grey the unmodified state. Continuous lines indicate possible transitions, while broken lines indicate a low probability of a transition between the states. ( d , e ) Possible models promoting either a bivalent ( d ) or H3K4me3-only state ( e ) as depicted in a . H3K4me3 X and H3K27me3 Y indicate that they reside on two different H3-tails, while H3K4me3 XY indicates that H3K4me3 is on both H3-tails of a nucleosome.

    Journal: Nature Communications

    Article Title: reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4+ memory T cells

    doi: 10.1038/ncomms12514

    Figure Lengend Snippet: Cross-talk between genome and epigenome. ( a – c ) The filled circles denote the relative fraction of TSSs in the indicated state. Purple indicates H3K4me3-only, red H3K27me3-only, black bivalent and grey the unmodified state. Continuous lines indicate possible transitions, while broken lines indicate a low probability of a transition between the states. ( d , e ) Possible models promoting either a bivalent ( d ) or H3K4me3-only state ( e ) as depicted in a . H3K4me3 X and H3K27me3 Y indicate that they reside on two different H3-tails, while H3K4me3 XY indicates that H3K4me3 is on both H3-tails of a nucleosome.

    Article Snippet: We used the enrichment calls for H3K4me3, H3K27me3, H3K4me3 reChIP and H3K27me3 reChIP over input to cluster the TSSs or CpG islands in eight classes: (i) no enrichment in any (re)ChIP, (ii) only enrichment in H3K27me3, (iii) only enrichment in H3K4me3, (iv) enrichment in H3K4me3 and H3K27me3 but not in the two reChIPs, (v) only enrichment in both the reChIPs but no enrichment in the primary ChIPs, (vi) enrichment in H3K27me3 and both the reChIPs, (vii) enrichment in H3K4me3 and both the reChIPs and (viii) enrichment in all the four (re)ChIPs.

    Techniques:

    MeCP2 binds to H3K27me3 enriched loci independent of DNA methylation. a MeCP2 enrichment at differential methylated regions (DMRs) between HCT116 and DKO1 cells. N = 2 (per genotype, HCT116 vs DKO1) from biologically independent cells. n = 91,106, p

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: MeCP2 binds to H3K27me3 enriched loci independent of DNA methylation. a MeCP2 enrichment at differential methylated regions (DMRs) between HCT116 and DKO1 cells. N = 2 (per genotype, HCT116 vs DKO1) from biologically independent cells. n = 91,106, p

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: DNA Methylation Assay, Methylation

    Distribution of MeCP2, H3K27me3 and H3K9ac. a – c Distribution of MeCP2, H3K27me3 and H3K9ac within differentially expressed genes between WT and Mecp2 KO. N = 3 (per genotype, WT vs Mecp2 KO) from biologically independent animals. MeCP2 ( a ), H3K27me3 ( b ), and H3K9ac ( c ) distribution in wild-type chromatin plotted 1 kb up- and 10 kb downstream of the TSS of genes that are up- (purple, n = 890) or down-regulated (green, n = 699) in Mecp2 KO. N = 2 (WT) from biologically independent animals. For MeCP2 ( a ), p = 6.62 × 10 −30 (Up, TSS vs proximal regions); p = 4.13 × 10 −7 (Up, Gene body vs proximal regions); p = 0.05 (Down, TSS vs proximal regions); p = 0.02 (Down, Gene body vs proximal regions). For H3K27me3 ( b ), p = 1.79 × 10 −43 (Up, TSS vs proximal regions); p = 2.02 × 10 −6 (Up, Gene body vs proximal regions); p = 2.53 × 10 −5 (Down, TSS vs proximal regions); p = 0.26 (Down, Gene body vs proximal regions). For H3K9ac ( c ), p = 2.90 × 10 −32 (Up, TSS vs proximal regions); p = 0.17 (Up, Gene body vs proximal regions); p = 1.88 × 10 −6 (Down, TSS vs proximal regions); p = 3.21 × 10 −4 (Down, Gene body vs proximal regions). (two-tailed Mann–Whitney test, *** p

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: Distribution of MeCP2, H3K27me3 and H3K9ac. a – c Distribution of MeCP2, H3K27me3 and H3K9ac within differentially expressed genes between WT and Mecp2 KO. N = 3 (per genotype, WT vs Mecp2 KO) from biologically independent animals. MeCP2 ( a ), H3K27me3 ( b ), and H3K9ac ( c ) distribution in wild-type chromatin plotted 1 kb up- and 10 kb downstream of the TSS of genes that are up- (purple, n = 890) or down-regulated (green, n = 699) in Mecp2 KO. N = 2 (WT) from biologically independent animals. For MeCP2 ( a ), p = 6.62 × 10 −30 (Up, TSS vs proximal regions); p = 4.13 × 10 −7 (Up, Gene body vs proximal regions); p = 0.05 (Down, TSS vs proximal regions); p = 0.02 (Down, Gene body vs proximal regions). For H3K27me3 ( b ), p = 1.79 × 10 −43 (Up, TSS vs proximal regions); p = 2.02 × 10 −6 (Up, Gene body vs proximal regions); p = 2.53 × 10 −5 (Down, TSS vs proximal regions); p = 0.26 (Down, Gene body vs proximal regions). For H3K9ac ( c ), p = 2.90 × 10 −32 (Up, TSS vs proximal regions); p = 0.17 (Up, Gene body vs proximal regions); p = 1.88 × 10 −6 (Down, TSS vs proximal regions); p = 3.21 × 10 −4 (Down, Gene body vs proximal regions). (two-tailed Mann–Whitney test, *** p

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: Two Tailed Test, MANN-WHITNEY

    Coexistence of MeCP2, H3K27me3, and H3K9ac in transcription regulatory regions. a Genome-wide distribution of MeCP2 binding loci, determined by PING. Mouse OE is the tissue source for all subsequent analyses in this figure. b MeCP2 binding frequency determined by Z-score in each annotated gene structural regions. c UCSC genome-browser view (mm9) of MeCP2, H3K27me3, H3K9ac ChIP-seq, and DNA methylation levels at a region on chr17: 45,675,956–45,700,322. The transparent blue bars indicate loci where MeCP2 and H3K27me3 coexist. The transparent orange bars point to the co-enrichment of MeCP2, H3K27me3, and H3K9ac. d Percentage of overlap between MeCP2 binding and H3K27me3 (blue) or H3K27me3 with H3K9ac (orange) within each annotated regions. e Methylation levels of CpG, CAG and CAH in 1 kb up- and 4 kb downstream of the TSS. f Z-score transformed average enrichment of MeCP2, H3K27me3, and H3K9ac, calculated 1 kb up- and 4 kb downstream of the TSS. g Methylation levels of CpG, CAG and CAH in 3 kb up- and 3 kb downstream of the enhancer loci (defined in Supplementary Fig. 9G ). h Z-score transformed average enrichment of MeCP2, H3K27me3 and H3K9ac, calculated −3 kb to +3 kb around the midpoint of each enhancer (defined in Supplementary Fig. 9G ). The shaded areas are up to ±S.D. from the average profile ( e - h ).

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: Coexistence of MeCP2, H3K27me3, and H3K9ac in transcription regulatory regions. a Genome-wide distribution of MeCP2 binding loci, determined by PING. Mouse OE is the tissue source for all subsequent analyses in this figure. b MeCP2 binding frequency determined by Z-score in each annotated gene structural regions. c UCSC genome-browser view (mm9) of MeCP2, H3K27me3, H3K9ac ChIP-seq, and DNA methylation levels at a region on chr17: 45,675,956–45,700,322. The transparent blue bars indicate loci where MeCP2 and H3K27me3 coexist. The transparent orange bars point to the co-enrichment of MeCP2, H3K27me3, and H3K9ac. d Percentage of overlap between MeCP2 binding and H3K27me3 (blue) or H3K27me3 with H3K9ac (orange) within each annotated regions. e Methylation levels of CpG, CAG and CAH in 1 kb up- and 4 kb downstream of the TSS. f Z-score transformed average enrichment of MeCP2, H3K27me3, and H3K9ac, calculated 1 kb up- and 4 kb downstream of the TSS. g Methylation levels of CpG, CAG and CAH in 3 kb up- and 3 kb downstream of the enhancer loci (defined in Supplementary Fig. 9G ). h Z-score transformed average enrichment of MeCP2, H3K27me3 and H3K9ac, calculated −3 kb to +3 kb around the midpoint of each enhancer (defined in Supplementary Fig. 9G ). The shaded areas are up to ±S.D. from the average profile ( e - h ).

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: Genome Wide, Binding Assay, Chromatin Immunoprecipitation, DNA Methylation Assay, Methylation, Transformation Assay

    MeCP2 differentially regulates gene expression depending on H3K27me3 and H3K9ac pattern. a Gene clusters classified by co-modification of MeCP2, H3K27me3, and H3K9ac within 1 kb up- and 5 kb downstream of the TSS. Genes sorted by descending order of mean signal intensity (numbers of genes in each cluster indicated on the left). Average profile of MeCP2 (bottom left), H3K27me3 (bottom center), and H3K9ac (bottom right). The shaded areas are up to ±S.D. from the average profile. Mouse OE is the tissue source for all subsequent analyses in this figure. b The expression levels of genes from cluster1 (purple, n = 4,159), cluster2 (gray, n = 628), and cluster3 (green, n = 1747). N = 3 (WT) from biologically independent animals. p = 7.70 × 10 −73 (cluster1 vs cluster2); p = 2.25 × 10 −218 (cluster1 vs group3); p = 2.93 × 10 −5 (cluster2 vs cluster3). (two-tailed Mann–Whitney testing with Bonferroni corrections, *** p

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: MeCP2 differentially regulates gene expression depending on H3K27me3 and H3K9ac pattern. a Gene clusters classified by co-modification of MeCP2, H3K27me3, and H3K9ac within 1 kb up- and 5 kb downstream of the TSS. Genes sorted by descending order of mean signal intensity (numbers of genes in each cluster indicated on the left). Average profile of MeCP2 (bottom left), H3K27me3 (bottom center), and H3K9ac (bottom right). The shaded areas are up to ±S.D. from the average profile. Mouse OE is the tissue source for all subsequent analyses in this figure. b The expression levels of genes from cluster1 (purple, n = 4,159), cluster2 (gray, n = 628), and cluster3 (green, n = 1747). N = 3 (WT) from biologically independent animals. p = 7.70 × 10 −73 (cluster1 vs cluster2); p = 2.25 × 10 −218 (cluster1 vs group3); p = 2.93 × 10 −5 (cluster2 vs cluster3). (two-tailed Mann–Whitney testing with Bonferroni corrections, *** p

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: Expressing, Modification, Two Tailed Test, MANN-WHITNEY

    Epigenetic characteristics of MeCP2 binding or non-binding mononucleosomes. a Examples of MeCP2 ChIP-seq and MNase-seq tracks for MeCP2-absent mononucleosome occupied loci (left), MeCP2-enriched mononucleosome occupied loci (center), and MeCP2 binding in unstable nucleosome loci (right). b Venn diagram showing the number of peaks in MeCP2-absent mononucleosome loci (group1), MeCP2-enriched mononucleosome loci (group2) and nucleosome-free MeCP2-binding loci (group3). c Comparison of methylation levels of CpG, CAG, and CAH in each of 10,000 loci randomly selected from the three groups defined in b . N = 2 (WT) from biologically independent mice. For CpG, p = 1.11 × 10 −115 (group1 vs group2); p = 2.4 × 10 −85 (group1 vs group3); p = 1.16 × 10 −4 (group2 vs group3). For CAG, p = 1.06 × 10 −75 (group1 vs group2); p = 1.55 × 10 −63 (group1 vs group3); p = 0.17 (group2 vs group3). For CAH, p = 1.69 × 10 −22 (group1 vs group2); p = 9.85 × 10 −27 (group1 vs group3); p = 1.08 (group2 vs group3). d Heatmap of Input, MeCP2, H3K27me3, H3K9ac ChIP-seq peaks and MNase-seq with flanking regions, of Chr19 by the types of regions defined in b . Aggregate plot of average Input, MeCP2, H3K27me3, H3K9ac and MNase-seq reads below. e , f Comparison of H3K27me3 ( e ) or H3K9ac ( f ) levels among the three groups of loci at chr19 (group1, n = 101,549; group2, n = 67,051; group3, n = 33,644). N = 2 (WT) from biologically independent mice. For H3K27me3, p

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: Epigenetic characteristics of MeCP2 binding or non-binding mononucleosomes. a Examples of MeCP2 ChIP-seq and MNase-seq tracks for MeCP2-absent mononucleosome occupied loci (left), MeCP2-enriched mononucleosome occupied loci (center), and MeCP2 binding in unstable nucleosome loci (right). b Venn diagram showing the number of peaks in MeCP2-absent mononucleosome loci (group1), MeCP2-enriched mononucleosome loci (group2) and nucleosome-free MeCP2-binding loci (group3). c Comparison of methylation levels of CpG, CAG, and CAH in each of 10,000 loci randomly selected from the three groups defined in b . N = 2 (WT) from biologically independent mice. For CpG, p = 1.11 × 10 −115 (group1 vs group2); p = 2.4 × 10 −85 (group1 vs group3); p = 1.16 × 10 −4 (group2 vs group3). For CAG, p = 1.06 × 10 −75 (group1 vs group2); p = 1.55 × 10 −63 (group1 vs group3); p = 0.17 (group2 vs group3). For CAH, p = 1.69 × 10 −22 (group1 vs group2); p = 9.85 × 10 −27 (group1 vs group3); p = 1.08 (group2 vs group3). d Heatmap of Input, MeCP2, H3K27me3, H3K9ac ChIP-seq peaks and MNase-seq with flanking regions, of Chr19 by the types of regions defined in b . Aggregate plot of average Input, MeCP2, H3K27me3, H3K9ac and MNase-seq reads below. e , f Comparison of H3K27me3 ( e ) or H3K9ac ( f ) levels among the three groups of loci at chr19 (group1, n = 101,549; group2, n = 67,051; group3, n = 33,644). N = 2 (WT) from biologically independent mice. For H3K27me3, p

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Methylation, Mouse Assay

    MeCP2 binding to chromatin is regulated by the levels of H3K27me3. a SH-SY5Y cells treated with either DMSO or GSK343 were immunoprecipitated with either histone H3 or MeCP2 antibody. Samples of input were analyzed with Western blot for MeCP2, histone H3 and H3K27me3. IgG and immunoprecipitates were analyzed with Western blot for MeCP2 and histone H3. Star indicates a non-specific band. Source data are provided as a Source Data file. b Scatter plots show the correlation between H3K27me3 ChIP-seq signal and MeCP2 enrichment from SH-SY5Y cells (binning size = 1000 bp). N = 2 (WT) from biologically independent experiments. c Differences of exogenous reference normalized H3K27me3-enrichment between GSK343 treatment and control DMSO are plotted. N = 2 (per treatment) from biologically independent experiments. The x axis corresponds to the average of H3K27me3 signals in both GSK343 treated and DMSO control; the y axis corresponds to the difference of H3K27me3 signal between GSK343 treated and DMSO control SH-SY5Y cells. Based on the H3K27me3 difference, loci are categorized as unchanged (gray), moderate (green) or severe (purple). d MeCP2 enrichment of DMSO or GSK343-treated samples, separated by groups defined in c . N = 2 (per treatment, DMSO vs GSK343) from biologically independent experiments. n = 333,212, p

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: MeCP2 binding to chromatin is regulated by the levels of H3K27me3. a SH-SY5Y cells treated with either DMSO or GSK343 were immunoprecipitated with either histone H3 or MeCP2 antibody. Samples of input were analyzed with Western blot for MeCP2, histone H3 and H3K27me3. IgG and immunoprecipitates were analyzed with Western blot for MeCP2 and histone H3. Star indicates a non-specific band. Source data are provided as a Source Data file. b Scatter plots show the correlation between H3K27me3 ChIP-seq signal and MeCP2 enrichment from SH-SY5Y cells (binning size = 1000 bp). N = 2 (WT) from biologically independent experiments. c Differences of exogenous reference normalized H3K27me3-enrichment between GSK343 treatment and control DMSO are plotted. N = 2 (per treatment) from biologically independent experiments. The x axis corresponds to the average of H3K27me3 signals in both GSK343 treated and DMSO control; the y axis corresponds to the difference of H3K27me3 signal between GSK343 treated and DMSO control SH-SY5Y cells. Based on the H3K27me3 difference, loci are categorized as unchanged (gray), moderate (green) or severe (purple). d MeCP2 enrichment of DMSO or GSK343-treated samples, separated by groups defined in c . N = 2 (per treatment, DMSO vs GSK343) from biologically independent experiments. n = 333,212, p

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: Binding Assay, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

    MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c Co-immunoprecipitation of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c Co-immunoprecipitation of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).

    Article Snippet: Each sheared genomic DNA preparation was incubated with 10 μg of H3K27me3 (Diagenode, pAb-069-050) or 20 μg of MeCP2 antibody (Diagenode, pAb-052-050).

    Techniques: Binding Assay, In Vitro, Pull Down Assay, Staining, Construct, Immunoprecipitation, Transfection, Purification, Recombinant, SDS Page

    Genome-wide 5hmC patterns in mouse whole brain and liver DNA following enrichment by either antibody, chemical capture or protein affinity-based methods. ( a ) An overview of the three commercially available techniques for 5hmC enrichment. In our study, following enrichment we carried out whole genome amplification and dye labelling for micro-array hybridization. ( b ) qPCR validation of the relative enrichment efficiencies over candidate loci previously identified as being either enriched or depleted in 5hmC in the mouse liver ( 26 ). Following normalization to the negative region at the Gapdh promoter, all three techniques report similar findings; however, the JBP-1-based affinity technique gives very low enrichment values compared to the hmeDIP and hMeSeal methods. Red dotted line denotes no enrichment over Gapdh . ( c ) Pearson correlation analysis and clustering among the microarray datasets. Biological replicates cluster closely while tissues clustered independently confirming the tissue-specific nature of 5hmC patterns. JBP-1 affinity purified 5hmC datasets correlate poorly with the hmeDIP and hMeSeal sets ( d ) Autocorrelation analysis of 5hmC patterns determined by hmeDIP, hMeSeal and JBP-1-binding in a single mouse brain sample. Autocorrelation was determined to a distance of 40 probes (∼10 kb). A ‘random’ sample for comparison was generated by randomization of the hMeSeal data. Filled circles represent relative probe position. ( e ) Example of microarray datasets showing tissue specificity and biological replicate reproducibility between each technique over the liver specific gene Cyp2b10 . Data are plotted on log2 scales from −3 to +3. Biological replicates are numbered 1 and 2, respectively. Gene structure is shown below by blue bars. Boxed regions are expanded upon on the right to display regions independently validated by gRES-qPCR. Plots represent the percentage of each modification at a single CpG in the sequence CCGG following normalization (purple; 5hmC, red; 5mC, green; C). Error bars display the standard error of the biological replicates. ( f ) Percentage plots of the distributions of 5hmC enriched regions following hmeDIP, hMeSeal and JBP-1 5hmC purification. Peak probes of 5hmC enrichment were defined (see ‘Materials and Methods’ section) and then mapped to one of five unique genomic loci (promoter cores, proximal and distal regions as well as intra- and inter-genic regions; box on right). Red dotted lines highlight changes in the distributions between techniques. Boxed region is expanded upon to reveal technique dependant differences over promoter-core, -proximal and -distal peaks. ( g ) The number and distribution of 5hmC peak probes generated for the three techniques are low over CpG islands (CGI) and largely non-promoter associated. Pie charts representative of the dataset size reveal low numbers of CGI related 5hmC peak probes following hmeDIP or hMeSeal in the brain (i) and the liver (ii). The total number of peaks mapping to CGIs are shown in square brackets while round brackets denote the total per cent of probes on the arrays which overlapped with CGI enriched peaks. Pink = peak probes mapping to promoter CGI regions, brown = peak probes mapping to orphan CGI (non-promoter) regions.

    Journal: Nucleic Acids Research

    Article Title: Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

    doi: 10.1093/nar/gkt1080

    Figure Lengend Snippet: Genome-wide 5hmC patterns in mouse whole brain and liver DNA following enrichment by either antibody, chemical capture or protein affinity-based methods. ( a ) An overview of the three commercially available techniques for 5hmC enrichment. In our study, following enrichment we carried out whole genome amplification and dye labelling for micro-array hybridization. ( b ) qPCR validation of the relative enrichment efficiencies over candidate loci previously identified as being either enriched or depleted in 5hmC in the mouse liver ( 26 ). Following normalization to the negative region at the Gapdh promoter, all three techniques report similar findings; however, the JBP-1-based affinity technique gives very low enrichment values compared to the hmeDIP and hMeSeal methods. Red dotted line denotes no enrichment over Gapdh . ( c ) Pearson correlation analysis and clustering among the microarray datasets. Biological replicates cluster closely while tissues clustered independently confirming the tissue-specific nature of 5hmC patterns. JBP-1 affinity purified 5hmC datasets correlate poorly with the hmeDIP and hMeSeal sets ( d ) Autocorrelation analysis of 5hmC patterns determined by hmeDIP, hMeSeal and JBP-1-binding in a single mouse brain sample. Autocorrelation was determined to a distance of 40 probes (∼10 kb). A ‘random’ sample for comparison was generated by randomization of the hMeSeal data. Filled circles represent relative probe position. ( e ) Example of microarray datasets showing tissue specificity and biological replicate reproducibility between each technique over the liver specific gene Cyp2b10 . Data are plotted on log2 scales from −3 to +3. Biological replicates are numbered 1 and 2, respectively. Gene structure is shown below by blue bars. Boxed regions are expanded upon on the right to display regions independently validated by gRES-qPCR. Plots represent the percentage of each modification at a single CpG in the sequence CCGG following normalization (purple; 5hmC, red; 5mC, green; C). Error bars display the standard error of the biological replicates. ( f ) Percentage plots of the distributions of 5hmC enriched regions following hmeDIP, hMeSeal and JBP-1 5hmC purification. Peak probes of 5hmC enrichment were defined (see ‘Materials and Methods’ section) and then mapped to one of five unique genomic loci (promoter cores, proximal and distal regions as well as intra- and inter-genic regions; box on right). Red dotted lines highlight changes in the distributions between techniques. Boxed region is expanded upon to reveal technique dependant differences over promoter-core, -proximal and -distal peaks. ( g ) The number and distribution of 5hmC peak probes generated for the three techniques are low over CpG islands (CGI) and largely non-promoter associated. Pie charts representative of the dataset size reveal low numbers of CGI related 5hmC peak probes following hmeDIP or hMeSeal in the brain (i) and the liver (ii). The total number of peaks mapping to CGIs are shown in square brackets while round brackets denote the total per cent of probes on the arrays which overlapped with CGI enriched peaks. Pink = peak probes mapping to promoter CGI regions, brown = peak probes mapping to orphan CGI (non-promoter) regions.

    Article Snippet: Purification of 5hmC and 5mC enriched DNA fragments Prior to purification, genomic DNA was extracted from frozen (−80°C), ground-up livers and fragmented to an average of 500 bp for hmeDIP protocols and 300 bp for JBP-1 and hMeSeal protocols (Bioruptor, Diagenode).

    Techniques: Genome Wide, Whole Genome Amplification, Microarray, Hybridization, Real-time Polymerase Chain Reaction, Affinity Purification, Binding Assay, Generated, Modification, Sequencing, Purification

    Regions of 5hmC enrichment are associated with select histone modifications. ( a ) Overlap between peaks of histone modifications/DNA binding proteins with peaks of 5hmC derived through the three purification techniques in the mouse brain. Plots show percentage of total histone modification peaks which overlap with a peak of 5hmC by at least 1 bp. Total number of histone modification peaks are shown below in square brackets. ( b ) Average 5hmC profiles over 10 kb windows at ‘poised’ and active enhancers compared with active promoters in the brain for each enrichment technique. ( c ) Genome browser visualization of 5hmC patterns (hmeDIP: purple, hMeSeal: teal, JBP-1 affinity: orange) overlap with select histone modifications (H3K4me1:light blue, H3K4me3: dark blue, H3K27me3: red, H3K27ac: green, H3K36me3: pink) in the mouse brain. Array data plotted on log2 scale while ChIP-seq the number of reads. Refseq genes are displayed below.

    Journal: Nucleic Acids Research

    Article Title: Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

    doi: 10.1093/nar/gkt1080

    Figure Lengend Snippet: Regions of 5hmC enrichment are associated with select histone modifications. ( a ) Overlap between peaks of histone modifications/DNA binding proteins with peaks of 5hmC derived through the three purification techniques in the mouse brain. Plots show percentage of total histone modification peaks which overlap with a peak of 5hmC by at least 1 bp. Total number of histone modification peaks are shown below in square brackets. ( b ) Average 5hmC profiles over 10 kb windows at ‘poised’ and active enhancers compared with active promoters in the brain for each enrichment technique. ( c ) Genome browser visualization of 5hmC patterns (hmeDIP: purple, hMeSeal: teal, JBP-1 affinity: orange) overlap with select histone modifications (H3K4me1:light blue, H3K4me3: dark blue, H3K27me3: red, H3K27ac: green, H3K36me3: pink) in the mouse brain. Array data plotted on log2 scale while ChIP-seq the number of reads. Refseq genes are displayed below.

    Article Snippet: Purification of 5hmC and 5mC enriched DNA fragments Prior to purification, genomic DNA was extracted from frozen (−80°C), ground-up livers and fragmented to an average of 500 bp for hmeDIP protocols and 300 bp for JBP-1 and hMeSeal protocols (Bioruptor, Diagenode).

    Techniques: DNA Binding Assay, Derivative Assay, Purification, Modification, Chromatin Immunoprecipitation

    Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    Spatial chromatin interactions in the IL8 locus are rewired by deleting p65‐binding cis ‐elements within enhancers i4C profiles in the 1 Mbp around the IL8 locus on chromosome 4 ( ideogram ) from control (empty vector) and enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines ± IL‐1α stimulation for 60 min, generated using the IL8 promoter ( pink highlight ) or enhancer ( blue highlight ) as a viewpoint. For the IL8 promoter, the average of two biological replicates is shown, while for the IL8 enhancer, data from one replicate are shown. Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig are indicated. All profiles are shown aligned to gene models ( blue ) and to CTCF, H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE HeLa‐S3 ChIP‐seq profiles. The breadth of TADs in the locus is indicated above. Meta‐plots showing coverage of H3K27ac ChIP‐seq signal at i4C fragments ± 1 kbp contacted by the IL8 promoter or enhancer in control cells (empty vector) in the presence ( magenta ) or absence ( gray ) of IL‐1α stimulation for 60 min, and in enhancer‐mutant cells (Δp65 eIL8 , blue ; Δp65 eCXCL2 , green ) after IL‐1α stimulation. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Spatial chromatin interactions in the IL8 locus are rewired by deleting p65‐binding cis ‐elements within enhancers i4C profiles in the 1 Mbp around the IL8 locus on chromosome 4 ( ideogram ) from control (empty vector) and enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines ± IL‐1α stimulation for 60 min, generated using the IL8 promoter ( pink highlight ) or enhancer ( blue highlight ) as a viewpoint. For the IL8 promoter, the average of two biological replicates is shown, while for the IL8 enhancer, data from one replicate are shown. Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig are indicated. All profiles are shown aligned to gene models ( blue ) and to CTCF, H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE HeLa‐S3 ChIP‐seq profiles. The breadth of TADs in the locus is indicated above. Meta‐plots showing coverage of H3K27ac ChIP‐seq signal at i4C fragments ± 1 kbp contacted by the IL8 promoter or enhancer in control cells (empty vector) in the presence ( magenta ) or absence ( gray ) of IL‐1α stimulation for 60 min, and in enhancer‐mutant cells (Δp65 eIL8 , blue ; Δp65 eCXCL2 , green ) after IL‐1α stimulation. Source data are available online for this figure.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Plasmid Preparation, Mutagenesis, Generated, Chromatin Immunoprecipitation

    Deletion of the p65‐binding site from the IL8 promoter only affects IL8 expression Genome browser view of the IL8 chemokine locus on human chromosome 4 showing H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 gene model ( blue ). The location of the deleted NF‐κB binding site in the IL8 promoter is indicated ( orange ), and Sanger sequencing of PCR amplicons generated from genomic DNA of the Δp65 pIL8 cell line shows removal of 57 bp. Immunoblot analysis of extracts from parental HeLa cells (wt), vector controls, or Δp65 pIL8 cells was compared to cells stably expressing a sgRNA targeting the RELA (Δ RELA ) gene which encodes for the p65 NF‐κB subunit in the presence (+) or absence (−) of IL‐1α stimulation for 30 min or 60 min. Antibodies against p65 confirm the strong suppression of p65 in Δ RELA cells. Antibodies against P(S536)‐p65, P‐IκBα, and IκBα reveal normal IL‐1α‐mediated signaling in Δp65 pIL8 cells compared to control cells, while Δ RELA cells show reduced levels of the IL‐1α and the p65 target gene IκBα. β‐Actin levels provide a loading control. Cas9 antibodies reveal the levels of FLAG‐Cas9 present in the stable cell cultures. mRNA levels of seven IL‐1α‐responsive genes in parental (wt), control (empty vector), or promoter‐mutant (Δp65 pIL8 ) HeLa lines were assessed by RT–qPCR (mean levels ± SEM, n = 4) at 60 min after IL‐1α stimulation. Asterisks show significanc e of changes in basal and IL‐1α‐stimulated conditions compared to the corresponding vector control samples ( P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Deletion of the p65‐binding site from the IL8 promoter only affects IL8 expression Genome browser view of the IL8 chemokine locus on human chromosome 4 showing H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 gene model ( blue ). The location of the deleted NF‐κB binding site in the IL8 promoter is indicated ( orange ), and Sanger sequencing of PCR amplicons generated from genomic DNA of the Δp65 pIL8 cell line shows removal of 57 bp. Immunoblot analysis of extracts from parental HeLa cells (wt), vector controls, or Δp65 pIL8 cells was compared to cells stably expressing a sgRNA targeting the RELA (Δ RELA ) gene which encodes for the p65 NF‐κB subunit in the presence (+) or absence (−) of IL‐1α stimulation for 30 min or 60 min. Antibodies against p65 confirm the strong suppression of p65 in Δ RELA cells. Antibodies against P(S536)‐p65, P‐IκBα, and IκBα reveal normal IL‐1α‐mediated signaling in Δp65 pIL8 cells compared to control cells, while Δ RELA cells show reduced levels of the IL‐1α and the p65 target gene IκBα. β‐Actin levels provide a loading control. Cas9 antibodies reveal the levels of FLAG‐Cas9 present in the stable cell cultures. mRNA levels of seven IL‐1α‐responsive genes in parental (wt), control (empty vector), or promoter‐mutant (Δp65 pIL8 ) HeLa lines were assessed by RT–qPCR (mean levels ± SEM, n = 4) at 60 min after IL‐1α stimulation. Asterisks show significanc e of changes in basal and IL‐1α‐stimulated conditions compared to the corresponding vector control samples ( P

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Generated, Plasmid Preparation, Stable Transfection, Mutagenesis, Quantitative RT-PCR

    The IL‐1α–TAK1 pathway regulates spatial chromatin interactions by the CXCL2 locus Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , 2014 ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , 2015 ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: The IL‐1α–TAK1 pathway regulates spatial chromatin interactions by the CXCL2 locus Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , 2014 ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , 2015 ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Generated, Software, Chromatin Immunoprecipitation

    Differential regulation of enhancers and promoters of IL‐1α target loci NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Differential regulation of enhancers and promoters of IL‐1α target loci NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Deletion of NF‐κB binding elements from the IL8 and CXCL2 proximal enhancers in HeLa suppresses inducible mRNA expression and secretion of IL‐1α target genes Genome browser views of the CXCL2 and IL8 chemokine loci on human chromosome 4 show H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 and CXCL2 gene models ( blue ). The locations of the deleted NF‐κB binding sites in their flanking enhancer regions are indicated ( orange ). Both loci were mutated using pairs of sgRNAs in stably transfected HeLa cell lines, and Sanger sequencing results of PCR‐amplified genomic regions using DNA of both enhancer‐mutant cell lines (Δp65 eIL8 and Δp65 eCXCL2 ) confirmed removal of 56 and 59 bp, respectively. Blue shades mark the targeted NF‐κB binding sites. mRNA levels of seven IL‐1α‐responsive genes in control (empty vector) or enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines was assessed by RT–qPCR (mean levels ± SEM, normalized to GUSB ; n = 4 (vector, Δp65 eIL8 ), n = 3 (Δp65 eCXCL2 )) at the indicated times after IL‐1α stimulation. *: significantly different to control; P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Deletion of NF‐κB binding elements from the IL8 and CXCL2 proximal enhancers in HeLa suppresses inducible mRNA expression and secretion of IL‐1α target genes Genome browser views of the CXCL2 and IL8 chemokine loci on human chromosome 4 show H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 and CXCL2 gene models ( blue ). The locations of the deleted NF‐κB binding sites in their flanking enhancer regions are indicated ( orange ). Both loci were mutated using pairs of sgRNAs in stably transfected HeLa cell lines, and Sanger sequencing results of PCR‐amplified genomic regions using DNA of both enhancer‐mutant cell lines (Δp65 eIL8 and Δp65 eCXCL2 ) confirmed removal of 56 and 59 bp, respectively. Blue shades mark the targeted NF‐κB binding sites. mRNA levels of seven IL‐1α‐responsive genes in control (empty vector) or enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines was assessed by RT–qPCR (mean levels ± SEM, normalized to GUSB ; n = 4 (vector, Δp65 eIL8 ), n = 3 (Δp65 eCXCL2 )) at the indicated times after IL‐1α stimulation. *: significantly different to control; P

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation, Stable Transfection, Transfection, Sequencing, Polymerase Chain Reaction, Amplification, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR

    NRF2 silencing positively regulates autophagy. The connection of NRF2-dependent oxidative stress and autophagy induction was checked by immunofluorescence microscopy. LC3 was stained by green fluorescence dye (Alexa Fluor 488); therefore, the green dots in the cells represent functional autophagosomes. A ) Time dependency of TBHP-induced (100 μM) autophagy regulation by NRF2. Rapamycin (Rap, 100 nM, 2 h) and bafilomycin (Baf, 10 μM, 2 h) were used as positive controls. B ) Quantification and statistical analysis of immunofluorescence microscopy data. Error bars represent ± sem . ** P

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: NRF2 silencing positively regulates autophagy. The connection of NRF2-dependent oxidative stress and autophagy induction was checked by immunofluorescence microscopy. LC3 was stained by green fluorescence dye (Alexa Fluor 488); therefore, the green dots in the cells represent functional autophagosomes. A ) Time dependency of TBHP-induced (100 μM) autophagy regulation by NRF2. Rapamycin (Rap, 100 nM, 2 h) and bafilomycin (Baf, 10 μM, 2 h) were used as positive controls. B ) Quantification and statistical analysis of immunofluorescence microscopy data. Error bars represent ± sem . ** P

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Immunofluorescence, Microscopy, Staining, Fluorescence, Functional Assay

    Proposed computational model of NRF2-dependent AMPK down-regulation during oxidative stress. A ) The core network of AMPK and NRF2 regulation with respect to oxidative stress. The dotted line denotes how the components can influence each other. B ) Numerical simulations of relative protein levels and activities of the HEK293T cell line with a mathematical model during oxidative stress. Both AMPK and NRF2 have a transient activation profile resulting in the down-regulation of autophagy after 4 h of oxidative stress. C ) Numerical simulations of relative protein levels and activities of the HEK293T + siNRF2 cell line with a mathematical model during oxidative stress. The active AMPK is able to maintain the autophagic process high in the absence of NRF2.

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: Proposed computational model of NRF2-dependent AMPK down-regulation during oxidative stress. A ) The core network of AMPK and NRF2 regulation with respect to oxidative stress. The dotted line denotes how the components can influence each other. B ) Numerical simulations of relative protein levels and activities of the HEK293T cell line with a mathematical model during oxidative stress. Both AMPK and NRF2 have a transient activation profile resulting in the down-regulation of autophagy after 4 h of oxidative stress. C ) Numerical simulations of relative protein levels and activities of the HEK293T + siNRF2 cell line with a mathematical model during oxidative stress. The active AMPK is able to maintain the autophagic process high in the absence of NRF2.

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Activation Assay

    NRF2 down-regulates both mRNA and protein levels of AMPK in the HEK293T cell line after long exposure to oxidative stress. A ) Time dependency of both NRF2 and AMPK mRNA levels in oxidative stress with/without silencing of NRF2 by siRNA. HEK293T cells were treated with 100 µM TBHP. The relative level of mRNA was measured by quantitative real-time PCR. Nrf2 gene expression was depleted by Nrf2 siRNA. TBHP-treated samples were compared with control (Ctrl) samples. B ) Time dependency of total AMPK protein levels (AMPK-T), NRF2, NQO1, and HO-1 during oxidative stress. Cells were treated with 100 µM TBHP, while the AMPK level was detected by immunoblotting. Nrf2 gene expression was depleted by Nrf2 siRNA. GAPDH was used as a loading control. C ) Quantification and statistical analysis of Western blot assays. Densitometry data represent the intensity of AMPK-T, NRF2, NQO1, and HO-1 normalized for GAPDH. Samples were compared with their partner with siNRF2 background. Error bars represent ± sem . * P

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: NRF2 down-regulates both mRNA and protein levels of AMPK in the HEK293T cell line after long exposure to oxidative stress. A ) Time dependency of both NRF2 and AMPK mRNA levels in oxidative stress with/without silencing of NRF2 by siRNA. HEK293T cells were treated with 100 µM TBHP. The relative level of mRNA was measured by quantitative real-time PCR. Nrf2 gene expression was depleted by Nrf2 siRNA. TBHP-treated samples were compared with control (Ctrl) samples. B ) Time dependency of total AMPK protein levels (AMPK-T), NRF2, NQO1, and HO-1 during oxidative stress. Cells were treated with 100 µM TBHP, while the AMPK level was detected by immunoblotting. Nrf2 gene expression was depleted by Nrf2 siRNA. GAPDH was used as a loading control. C ) Quantification and statistical analysis of Western blot assays. Densitometry data represent the intensity of AMPK-T, NRF2, NQO1, and HO-1 normalized for GAPDH. Samples were compared with their partner with siNRF2 background. Error bars represent ± sem . * P

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

    NRF2 binds to AMPK query and consensus oligonucleotide sequences. Nuclear extracts (NE) of Caco-2 treated with TBHP were incubated with query (Q) (lane 4) or consensus (C) (lane 5) oligonucleotide probes and compared with nuclear extract only control (lane 1). Image of the EMSA gel stained with SYBR Green EMSA stain identified nucleic acids (green), and subsequent SYPRO Ruby EMSA stain identified protein (red) components. NRF2 protein–probe complexes (yellow) formed only in lanes containing nuclear extracts + probe. B ) Caco-2 nuclear extracts were incubated with 2 μg of anti-NRF2 antibody (Ab.) before the addition of oligonucleotide probe (lanes 3 and 5) and analyzed for complex retardation.

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: NRF2 binds to AMPK query and consensus oligonucleotide sequences. Nuclear extracts (NE) of Caco-2 treated with TBHP were incubated with query (Q) (lane 4) or consensus (C) (lane 5) oligonucleotide probes and compared with nuclear extract only control (lane 1). Image of the EMSA gel stained with SYBR Green EMSA stain identified nucleic acids (green), and subsequent SYPRO Ruby EMSA stain identified protein (red) components. NRF2 protein–probe complexes (yellow) formed only in lanes containing nuclear extracts + probe. B ) Caco-2 nuclear extracts were incubated with 2 μg of anti-NRF2 antibody (Ab.) before the addition of oligonucleotide probe (lanes 3 and 5) and analyzed for complex retardation.

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Incubation, Staining, SYBR Green Assay

    NRF2 down-regulates autophagy proteins after long exposure to oxidative stress in the HEK293T cell line. A ) Time dependency of autophagy down-regulation by NRF2 in oxidative stress. Western blot results of HEK293T cells in 100 µM TBHP-containing media. The Western blot shows the time course levels of autophagy-related proteins. Cells were cultured with TBHP for 0.5, 1, 2, 3, and 4 h, while NRF2 gene expression was depleted by NRF2 siRNA. GAPDH was used as a loading control. B ) Quantification and statistical analysis of the Western blot assays. Densitometry data represent the intensity of p62 normalized for GAPDH, LC3-II normalized for LC3-I, ULK555-P normalized for total level of ULK, and AMPK-P normalized for GAPDH. Samples were compared with their partner with siNRF2 background. Error bars represent ± sem . * P

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: NRF2 down-regulates autophagy proteins after long exposure to oxidative stress in the HEK293T cell line. A ) Time dependency of autophagy down-regulation by NRF2 in oxidative stress. Western blot results of HEK293T cells in 100 µM TBHP-containing media. The Western blot shows the time course levels of autophagy-related proteins. Cells were cultured with TBHP for 0.5, 1, 2, 3, and 4 h, while NRF2 gene expression was depleted by NRF2 siRNA. GAPDH was used as a loading control. B ) Quantification and statistical analysis of the Western blot assays. Densitometry data represent the intensity of p62 normalized for GAPDH, LC3-II normalized for LC3-I, ULK555-P normalized for total level of ULK, and AMPK-P normalized for GAPDH. Samples were compared with their partner with siNRF2 background. Error bars represent ± sem . * P

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Western Blot, Cell Culture, Expressing

    SKN-1/NRF2 down-regulates aak-2/AMPK expression upon oxidative stress in C. elegans. A ) The genomic region of aak-2 with predicted SKN-1 binding sites, and the structure of the translational aak-2::gfp reporter gene, is shown. Black boxes correspond to exonic sequences, connecting lines represent introns. Red triangles indicate conserved SKN-1 binding sites. B ) Expression of aak-2::gfp transgene was increased in skn-1(-) mutant background. C ) In acute oxidative conditions (2 mM TBHP, 5 h), aak-2::gfp expression was decreased in animals with wild-type background. The absence of skn-1 gene products eliminated this change of gene expression. D ) In chronic oxidative conditions (2 mM TBHP, 24 h), aak-2::gfp gene expression was not inhibited. E ) Paralysis assay of worms held on 10 mM final concentration TBHP plates. Data are expressed as the ratio of paralyzed/nonparalyzed worms. The number of animals examined: n = 15–100. Error bars represent ± sem . All experiments were independently reproduced 3 times. * P

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: SKN-1/NRF2 down-regulates aak-2/AMPK expression upon oxidative stress in C. elegans. A ) The genomic region of aak-2 with predicted SKN-1 binding sites, and the structure of the translational aak-2::gfp reporter gene, is shown. Black boxes correspond to exonic sequences, connecting lines represent introns. Red triangles indicate conserved SKN-1 binding sites. B ) Expression of aak-2::gfp transgene was increased in skn-1(-) mutant background. C ) In acute oxidative conditions (2 mM TBHP, 5 h), aak-2::gfp expression was decreased in animals with wild-type background. The absence of skn-1 gene products eliminated this change of gene expression. D ) In chronic oxidative conditions (2 mM TBHP, 24 h), aak-2::gfp gene expression was not inhibited. E ) Paralysis assay of worms held on 10 mM final concentration TBHP plates. Data are expressed as the ratio of paralyzed/nonparalyzed worms. The number of animals examined: n = 15–100. Error bars represent ± sem . All experiments were independently reproduced 3 times. * P

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Expressing, Binding Assay, Mutagenesis, Concentration Assay

    Putative conserved NRF2/SKN-1 binding sites were found in aak-2/AMPK genes. A ) Location and sequences of the conserved putative NRF2/SKN-1 transcription factor binding sites, relative to the transcription start site (+1) in different species. (N denotes arbitrary nucleotides, R indicates adenine or guanine, and S indicates cytosine or guanine.) B ) The genomic region of AMPK and aak-2 gene with predicted NRF2 and SKN-1 binding sites. Black boxes correspond to exonic sequences; connecting lines represent introns. Red triangles indicate conserved NRF2/SKN-1 binding sites. C ) The genomic region of the aak-2 gene with 2 possible SKN-1 binding sites. The blue lines indicate the genomic regions where Brdlik et al. ( 44 ) identified binding sites for SKN-1 in their chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq ) assay in any of the 4 larval stages of C. elegans . Black boxes correspond to exons; connecting lines represent introns. Red triangles indicate conserved SKN-1 binding sites. TSS indicates the transcription start site. L1, 2, 3, and 4 are the 4 larval stages of C. elegans before reaching adulthood.

    Journal: The FASEB Journal

    Article Title: Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress

    doi: 10.1096/fj.201800565RR

    Figure Lengend Snippet: Putative conserved NRF2/SKN-1 binding sites were found in aak-2/AMPK genes. A ) Location and sequences of the conserved putative NRF2/SKN-1 transcription factor binding sites, relative to the transcription start site (+1) in different species. (N denotes arbitrary nucleotides, R indicates adenine or guanine, and S indicates cytosine or guanine.) B ) The genomic region of AMPK and aak-2 gene with predicted NRF2 and SKN-1 binding sites. Black boxes correspond to exonic sequences; connecting lines represent introns. Red triangles indicate conserved NRF2/SKN-1 binding sites. C ) The genomic region of the aak-2 gene with 2 possible SKN-1 binding sites. The blue lines indicate the genomic regions where Brdlik et al. ( 44 ) identified binding sites for SKN-1 in their chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq ) assay in any of the 4 larval stages of C. elegans . Black boxes correspond to exons; connecting lines represent introns. Red triangles indicate conserved SKN-1 binding sites. TSS indicates the transcription start site. L1, 2, 3, and 4 are the 4 larval stages of C. elegans before reaching adulthood.

    Article Snippet: To further analyze the effect of NRF2 on AMPK during oxidative stress, Western blot experiments were also performed when NRF2 was silenced by using siRNA.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Multiplex Assay, Sequencing

    Decreased H3K27ac at immune and cancer associated enhancers with age. (A) Heatmap depicting the 12 clusters identified using k-means clustering of regions with significant (LLR > 3 and absolute fold-change > 1.5) changes of H3K4me1, H3K4me3, H3K27me3, or H3K27ac with age (n=37,058 peaks). The fold-change(aged/young) signal is plotted for each histone modification for each peak. Annotation to active and poised enhancers as well as bivalent promoters identified in young HSCe is also shown. (B) Heatmap of H3K4me1 and H3K27ac signal at the enhancer enriched clusters J-L. The log 2 (Pooled IP/Pooled Input) signal is plotted for each age group, centered on the differential peak +/− 5kb. Select genes within each cluster are denoted on the right of the heatmap. (C) Bubble plot representation of select KEGG pathways that are enriched in clusters J-L. The size of each bubble corresponds to the number of genes within the cluster that are within the given gene set. Highly significant (FDR

    Journal: Cancer discovery

    Article Title: Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia

    doi: 10.1158/2159-8290.CD-18-1474

    Figure Lengend Snippet: Decreased H3K27ac at immune and cancer associated enhancers with age. (A) Heatmap depicting the 12 clusters identified using k-means clustering of regions with significant (LLR > 3 and absolute fold-change > 1.5) changes of H3K4me1, H3K4me3, H3K27me3, or H3K27ac with age (n=37,058 peaks). The fold-change(aged/young) signal is plotted for each histone modification for each peak. Annotation to active and poised enhancers as well as bivalent promoters identified in young HSCe is also shown. (B) Heatmap of H3K4me1 and H3K27ac signal at the enhancer enriched clusters J-L. The log 2 (Pooled IP/Pooled Input) signal is plotted for each age group, centered on the differential peak +/− 5kb. Select genes within each cluster are denoted on the right of the heatmap. (C) Bubble plot representation of select KEGG pathways that are enriched in clusters J-L. The size of each bubble corresponds to the number of genes within the cluster that are within the given gene set. Highly significant (FDR

    Article Snippet: Chromatin was immunoprecipitated for 12 hr at 4° C using 1μg H3K27me3 (Millipore 07–449, lot #21494165), 1μg H3K4me3 (Abcam ab8580, lot # –1), 0.5 μg H3K4me1 (Diagenode C15410194, lot #A1862D), 0.5 μg H3K27ac (Abcam ab4729, lot # –2), or 0.5 μg H3 (Abcam ab10799, lot # –1).

    Techniques: Modification

    Focal histone and DNA methylation alterations with HSCe aging. (A) Heatmap representation of regions with either loss or gain (log 10 likelihood ratio > 3, absolute fold-change > 1.5) of H3K4me1, H3K27ac, H3K4me3, or H3K27me3 signal in aged HSCe compared to young. The log 2 (IP/Input) signal is plotted for each replicate, centered on the differential peak +/− 5 kb. Each column is representative of an individual donor. (B) Functional annotation using ChIP-enrich Gene Ontology Biological Processes for genes annotated to peaks that have reduced H3K4me1, H3K27ac, H3K4me3, or H3K27me3 signal in aged HSCe compared to young. Select significant (FDR

    Journal: Cancer discovery

    Article Title: Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia

    doi: 10.1158/2159-8290.CD-18-1474

    Figure Lengend Snippet: Focal histone and DNA methylation alterations with HSCe aging. (A) Heatmap representation of regions with either loss or gain (log 10 likelihood ratio > 3, absolute fold-change > 1.5) of H3K4me1, H3K27ac, H3K4me3, or H3K27me3 signal in aged HSCe compared to young. The log 2 (IP/Input) signal is plotted for each replicate, centered on the differential peak +/− 5 kb. Each column is representative of an individual donor. (B) Functional annotation using ChIP-enrich Gene Ontology Biological Processes for genes annotated to peaks that have reduced H3K4me1, H3K27ac, H3K4me3, or H3K27me3 signal in aged HSCe compared to young. Select significant (FDR

    Article Snippet: Chromatin was immunoprecipitated for 12 hr at 4° C using 1μg H3K27me3 (Millipore 07–449, lot #21494165), 1μg H3K4me3 (Abcam ab8580, lot # –1), 0.5 μg H3K4me1 (Diagenode C15410194, lot #A1862D), 0.5 μg H3K27ac (Abcam ab4729, lot # –2), or 0.5 μg H3 (Abcam ab10799, lot # –1).

    Techniques: DNA Methylation Assay, Functional Assay, Chromatin Immunoprecipitation

    Loss of activating histone modifications at promoter regions with age. (A) Heatmap of H3K4me1, H3K27ac, H3K4me3 and H3K27me3 signals at age-associated clusters that are enriched for active promoters (top and middle) and bivalent promoters (bottom) . The log 2 (Pooled IP/Pooled Input) signal is plotted for each age group, centered on the differential peak +/− 5kb. (B) Density plots of the log 2 (Pooled IP/Pooled Input) signal for the characteristic histone marks for the peaks within the Active TSS I ( top ), Active TSS II ( middle ) and Bivalent promoter ( bottom ) categories. (C) UCSC genome browser tracks of genes with altered promoters from the Active TSS I cluster ( GFI1 and NFATC4 ) and Active TSS II cluster ( CARM1 and PER1 ). Tracks are of pooled replicates for each age group, normalized to reads per million and to the corresponding Input for ChIP-seq. Light green bars below tracks represent the differential promoter regions identified from the cluster analysis. (D) Bubble plot representation of select KEGG pathways that are enriched in the active promoter clusters (clusters A-D). The size of each bubble corresponds to the number of genes within the cluster that are within the given gene set. Highly significant (FDR

    Journal: Cancer discovery

    Article Title: Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia

    doi: 10.1158/2159-8290.CD-18-1474

    Figure Lengend Snippet: Loss of activating histone modifications at promoter regions with age. (A) Heatmap of H3K4me1, H3K27ac, H3K4me3 and H3K27me3 signals at age-associated clusters that are enriched for active promoters (top and middle) and bivalent promoters (bottom) . The log 2 (Pooled IP/Pooled Input) signal is plotted for each age group, centered on the differential peak +/− 5kb. (B) Density plots of the log 2 (Pooled IP/Pooled Input) signal for the characteristic histone marks for the peaks within the Active TSS I ( top ), Active TSS II ( middle ) and Bivalent promoter ( bottom ) categories. (C) UCSC genome browser tracks of genes with altered promoters from the Active TSS I cluster ( GFI1 and NFATC4 ) and Active TSS II cluster ( CARM1 and PER1 ). Tracks are of pooled replicates for each age group, normalized to reads per million and to the corresponding Input for ChIP-seq. Light green bars below tracks represent the differential promoter regions identified from the cluster analysis. (D) Bubble plot representation of select KEGG pathways that are enriched in the active promoter clusters (clusters A-D). The size of each bubble corresponds to the number of genes within the cluster that are within the given gene set. Highly significant (FDR

    Article Snippet: Chromatin was immunoprecipitated for 12 hr at 4° C using 1μg H3K27me3 (Millipore 07–449, lot #21494165), 1μg H3K4me3 (Abcam ab8580, lot # –1), 0.5 μg H3K4me1 (Diagenode C15410194, lot #A1862D), 0.5 μg H3K27ac (Abcam ab4729, lot # –2), or 0.5 μg H3 (Abcam ab10799, lot # –1).

    Techniques: Chromatin Immunoprecipitation

    KAP1 recruitment at individual ERE loci UCSC genome browser snapshots of KAP1 and H3K9me3 ChIP-seq profiles in resting and activated CD4 + T cells at individual ERE loci. Results are representative of two independent ChIP-seq experiments. ERE annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. ERE integrants on which KAP1 binding is centered are highlighted in red.

    Journal: bioRxiv

    Article Title: The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

    doi: 10.1101/523597

    Figure Lengend Snippet: KAP1 recruitment at individual ERE loci UCSC genome browser snapshots of KAP1 and H3K9me3 ChIP-seq profiles in resting and activated CD4 + T cells at individual ERE loci. Results are representative of two independent ChIP-seq experiments. ERE annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. ERE integrants on which KAP1 binding is centered are highlighted in red.

    Article Snippet: Immunoprecipitations were performed using rabbit polyclonal antibodies specific for KAP1 (Tronolab SY326768, S23470, or Abcam), H3K9me3 (Diagenode), H3K4me1 and H3K27ac (Abcam).

    Techniques: Chromatin Immunoprecipitation, Modification, Binding Assay

    KAP1 binding at ERE-based, activation-dependent regulatory elements (A) UCSC Genome Browser view of KAP1-bound ERE integrants characterized by a switch from active to repressive (left panel) or from repressive to actve (right panel) chromatin marks following T cell activation. Displayed tracks include KAP1, H3K9me3 and H3K27ac ChIP-seq profiles for resting and activated CD4* T cells. Repeat annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. EREs integrants on which KAP1 binding is centered are highlighted in red. Results are representative of two independent ChIP-seq experiments. (B) Distance from TSS of resting cell enhancers (left panel) or activated cell enhancers (right panel). For the random set, EREs were shuffled 1000 times within chromosomes. The lists of EREs possessing resting or activated cell enhancer-like signatures were obtained by interrogating them for the presence of KAP1 and H3K9me3-enrichment in one condition and the presence of active H3K27ac mark in the other. EREs shared between the lists were discarded. The total number of identified loci is indicated above each panel.

    Journal: bioRxiv

    Article Title: The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

    doi: 10.1101/523597

    Figure Lengend Snippet: KAP1 binding at ERE-based, activation-dependent regulatory elements (A) UCSC Genome Browser view of KAP1-bound ERE integrants characterized by a switch from active to repressive (left panel) or from repressive to actve (right panel) chromatin marks following T cell activation. Displayed tracks include KAP1, H3K9me3 and H3K27ac ChIP-seq profiles for resting and activated CD4* T cells. Repeat annotation, downloaded from the UCSC Genome Browser and modified as described in the Methods, is also displayed. EREs integrants on which KAP1 binding is centered are highlighted in red. Results are representative of two independent ChIP-seq experiments. (B) Distance from TSS of resting cell enhancers (left panel) or activated cell enhancers (right panel). For the random set, EREs were shuffled 1000 times within chromosomes. The lists of EREs possessing resting or activated cell enhancer-like signatures were obtained by interrogating them for the presence of KAP1 and H3K9me3-enrichment in one condition and the presence of active H3K27ac mark in the other. EREs shared between the lists were discarded. The total number of identified loci is indicated above each panel.

    Article Snippet: Immunoprecipitations were performed using rabbit polyclonal antibodies specific for KAP1 (Tronolab SY326768, S23470, or Abcam), H3K9me3 (Diagenode), H3K4me1 and H3K27ac (Abcam).

    Techniques: Binding Assay, Activation Assay, Chromatin Immunoprecipitation, Modification

    KAP1 genomic recruitment is widely redistributed following T cell activation (A) Number of KAP1 ChIP-seq peaks located at the 5’ end of genes, within 10kb or at further distance. Left, KAP1 peaks overlapping with EREs. Right, KAP1 peaks not overlapping with EREs. KAP1 peaks were grouped according to whether they were detected exclusively in resting (REST only) or activated (ACT only) CD4 + T cell ChIP-seq experiments or in all data sets (common). As a control, we plotted the distribution of KAP1 peaks that we obtained by averaging a 1000-time permutation of random assignation of KAP1 peak coordinates. (B) UCSC Genome Browser snapshots of KAP1 ChIP-seq profiles providing example of conserved or differential binding between the resting and activated T cell conditions. Results of replicate experiments are shown. (C) Positional relationship between KAP1 peaks on EREs and the indicated histone marks, at a 100-bp resolution and over a 10kb window centered on KAP1 ChIP-seq peaks. Left, KAP1 peaks detected exclusively in resting cells. Middle, KAP1 peaks detected exclusively in activated cells. Right, KAP1 peaks detected in both datasets. The profiles were normalized for the total number of ChIP-seq peaks in each sample. (D) H3K9me3 and H3K4me1 ChIP-qPCR analysis in control and KAP1 knockdown cells at selected KAP1-bound ERE integrants. Values are normalized to their respective total inputs and to the total H3 levels. EVX1 and ZNF554 3’ were chosen, respectively, as control regions for H3K4me1 and H3K9me3. Error bars represent SEM of biological replicates (n=3), *p

    Journal: bioRxiv

    Article Title: The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

    doi: 10.1101/523597

    Figure Lengend Snippet: KAP1 genomic recruitment is widely redistributed following T cell activation (A) Number of KAP1 ChIP-seq peaks located at the 5’ end of genes, within 10kb or at further distance. Left, KAP1 peaks overlapping with EREs. Right, KAP1 peaks not overlapping with EREs. KAP1 peaks were grouped according to whether they were detected exclusively in resting (REST only) or activated (ACT only) CD4 + T cell ChIP-seq experiments or in all data sets (common). As a control, we plotted the distribution of KAP1 peaks that we obtained by averaging a 1000-time permutation of random assignation of KAP1 peak coordinates. (B) UCSC Genome Browser snapshots of KAP1 ChIP-seq profiles providing example of conserved or differential binding between the resting and activated T cell conditions. Results of replicate experiments are shown. (C) Positional relationship between KAP1 peaks on EREs and the indicated histone marks, at a 100-bp resolution and over a 10kb window centered on KAP1 ChIP-seq peaks. Left, KAP1 peaks detected exclusively in resting cells. Middle, KAP1 peaks detected exclusively in activated cells. Right, KAP1 peaks detected in both datasets. The profiles were normalized for the total number of ChIP-seq peaks in each sample. (D) H3K9me3 and H3K4me1 ChIP-qPCR analysis in control and KAP1 knockdown cells at selected KAP1-bound ERE integrants. Values are normalized to their respective total inputs and to the total H3 levels. EVX1 and ZNF554 3’ were chosen, respectively, as control regions for H3K4me1 and H3K9me3. Error bars represent SEM of biological replicates (n=3), *p

    Article Snippet: Immunoprecipitations were performed using rabbit polyclonal antibodies specific for KAP1 (Tronolab SY326768, S23470, or Abcam), H3K9me3 (Diagenode), H3K4me1 and H3K27ac (Abcam).

    Techniques: Activation Assay, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

    KAP1–mediated control of a ERE-based regulatory element modulates the lineage-specific expression of TLR1 (A) UCSC Genome Browser view of TLR1 locus. Displayed tracks include RNA-seq for control (shLUC) and KAP1 knockdown (shKAP1) activated CD4 + T cells; RefSeq genes and retroelement annotation (modified from UCSC Genome Browser as described in the Methods); DNase cluster (ENCODE); KAP1 and H3K9me3 ChIP-seq tracks for resting and activated CD4 + T cells; public H3K9me3, H3K4me3, H3K4me1, and H3K27ac ChIP-seq tracks for Th1 activated CD4 + T cells and/or mature neutrophils; primers used in (B) located at varying distances relative to the TSS. The dashed box highlights the KAP1-bound region. The repetitive elements on which KAP1 binding is centered are shown in red. (B) ChIP-qPCR analysis of H3K9me3, H3K4me1 and H3K27ac around KAP1 binding site at the TLR1 locus in control and KAP1 knockdown cells. Values are normalized to their respective total inputs, to the total H3 protein levels and to GAPDH . (C) RT-qPCR analysis of KAP1 and TLR1 mRNA expression in activated CD4 + T lymphocytes transduced with shLUC or shKAP1-expressing LVs. (D) RT-qPCR analysis of TLR1 mRNA expression in activated CD4 + T lymphocytes and in the human promyelocytic leukemia cell line HL-60. HL-60 cells were differentiated into neutrophil-like cells in culture medium in the presence of 1.3% DMSO for five days. (E) RT-qPCR analysis of KAP1 and TLR1 mRNA expression in untreated or DMSO-treated HL60 cells, transduced with shLUC or shKAP1-expressing LVs. Error bars represent SEM of biological replicates (n=3), *p

    Journal: bioRxiv

    Article Title: The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

    doi: 10.1101/523597

    Figure Lengend Snippet: KAP1–mediated control of a ERE-based regulatory element modulates the lineage-specific expression of TLR1 (A) UCSC Genome Browser view of TLR1 locus. Displayed tracks include RNA-seq for control (shLUC) and KAP1 knockdown (shKAP1) activated CD4 + T cells; RefSeq genes and retroelement annotation (modified from UCSC Genome Browser as described in the Methods); DNase cluster (ENCODE); KAP1 and H3K9me3 ChIP-seq tracks for resting and activated CD4 + T cells; public H3K9me3, H3K4me3, H3K4me1, and H3K27ac ChIP-seq tracks for Th1 activated CD4 + T cells and/or mature neutrophils; primers used in (B) located at varying distances relative to the TSS. The dashed box highlights the KAP1-bound region. The repetitive elements on which KAP1 binding is centered are shown in red. (B) ChIP-qPCR analysis of H3K9me3, H3K4me1 and H3K27ac around KAP1 binding site at the TLR1 locus in control and KAP1 knockdown cells. Values are normalized to their respective total inputs, to the total H3 protein levels and to GAPDH . (C) RT-qPCR analysis of KAP1 and TLR1 mRNA expression in activated CD4 + T lymphocytes transduced with shLUC or shKAP1-expressing LVs. (D) RT-qPCR analysis of TLR1 mRNA expression in activated CD4 + T lymphocytes and in the human promyelocytic leukemia cell line HL-60. HL-60 cells were differentiated into neutrophil-like cells in culture medium in the presence of 1.3% DMSO for five days. (E) RT-qPCR analysis of KAP1 and TLR1 mRNA expression in untreated or DMSO-treated HL60 cells, transduced with shLUC or shKAP1-expressing LVs. Error bars represent SEM of biological replicates (n=3), *p

    Article Snippet: Immunoprecipitations were performed using rabbit polyclonal antibodies specific for KAP1 (Tronolab SY326768, S23470, or Abcam), H3K9me3 (Diagenode), H3K4me1 and H3K27ac (Abcam).

    Techniques: Expressing, RNA Sequencing Assay, Modification, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transduction

    CD4-specific KZFPs contribute to tissue-specific regulation of gene expression (A) Heatmap showing the Fisher’s Exact test adjusted P-values for the overlap between KZFP-bound EREs (X-axis, where each column represents a different KZFP ChIP-exo sample) and H3K9me3 from the NIH roadmap dataset (Y-axis). The score is -log10 of Benjamini-Hochberg-corrected P-values. (B) Volcano plot displaying KZFP genes expressed in activated CD4 + T lymphocytes and K562 cells based on RNA-seq data. Red and green data points depict genes with a significant higher expression in CD4 + or K562 cells, respectively ( > 100 normalized counts and > 10-fold changes where P adj

    Journal: bioRxiv

    Article Title: The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

    doi: 10.1101/523597

    Figure Lengend Snippet: CD4-specific KZFPs contribute to tissue-specific regulation of gene expression (A) Heatmap showing the Fisher’s Exact test adjusted P-values for the overlap between KZFP-bound EREs (X-axis, where each column represents a different KZFP ChIP-exo sample) and H3K9me3 from the NIH roadmap dataset (Y-axis). The score is -log10 of Benjamini-Hochberg-corrected P-values. (B) Volcano plot displaying KZFP genes expressed in activated CD4 + T lymphocytes and K562 cells based on RNA-seq data. Red and green data points depict genes with a significant higher expression in CD4 + or K562 cells, respectively ( > 100 normalized counts and > 10-fold changes where P adj

    Article Snippet: Immunoprecipitations were performed using rabbit polyclonal antibodies specific for KAP1 (Tronolab SY326768, S23470, or Abcam), H3K9me3 (Diagenode), H3K4me1 and H3K27ac (Abcam).

    Techniques: Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay

    KAP1-regulated expression and histone mark changes at FGL2 and YPEL3 loci (A) UCSC Genome Browser view of FGL2 locus. Displayed tracks include RNA-seq for control (shLUC) and KAP1 knockdown (shKAP1) activated CD4* T cells; RefSeq genes and retroelement annotation (modified from UCSC Genome Browser as described in the Methods); DNase cluster (ENCODE); KAP1 and H3K9me3 ChIP-seq tracks for resting activated CD4* T cells; public H3K9me3, H3K4me3, H3K4me1, and H3K27ac ChIP-seq tracks for Th1 activated CD4 + T cells and/or peripheral blood mononuclear cells (PBMC); primers used in (B) located at varying distances relative to the TSS. The dashed box highlights the KAP1-bound region. The retroelement on which KAP1 binding is centered is shown in red. (B) ChIP-qPCR analysis of H3K9me3, H3K4me1 and H3K27ac at the FGL2 locus in control and KAP1 knockdown cells. Values are normalized to their respective total inputs, to the total H3 protein levels and to EVX1 . (C) UCSC Genome Browser view of YPEL3 locus. (D) ChIP-qPCR analysis of H3K9me3, H3K4me1 and H3K27ac at the YPEL3 locus in control and KAP1 knockdown cells.

    Journal: bioRxiv

    Article Title: The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

    doi: 10.1101/523597

    Figure Lengend Snippet: KAP1-regulated expression and histone mark changes at FGL2 and YPEL3 loci (A) UCSC Genome Browser view of FGL2 locus. Displayed tracks include RNA-seq for control (shLUC) and KAP1 knockdown (shKAP1) activated CD4* T cells; RefSeq genes and retroelement annotation (modified from UCSC Genome Browser as described in the Methods); DNase cluster (ENCODE); KAP1 and H3K9me3 ChIP-seq tracks for resting activated CD4* T cells; public H3K9me3, H3K4me3, H3K4me1, and H3K27ac ChIP-seq tracks for Th1 activated CD4 + T cells and/or peripheral blood mononuclear cells (PBMC); primers used in (B) located at varying distances relative to the TSS. The dashed box highlights the KAP1-bound region. The retroelement on which KAP1 binding is centered is shown in red. (B) ChIP-qPCR analysis of H3K9me3, H3K4me1 and H3K27ac at the FGL2 locus in control and KAP1 knockdown cells. Values are normalized to their respective total inputs, to the total H3 protein levels and to EVX1 . (C) UCSC Genome Browser view of YPEL3 locus. (D) ChIP-qPCR analysis of H3K9me3, H3K4me1 and H3K27ac at the YPEL3 locus in control and KAP1 knockdown cells.

    Article Snippet: Immunoprecipitations were performed using rabbit polyclonal antibodies specific for KAP1 (Tronolab SY326768, S23470, or Abcam), H3K9me3 (Diagenode), H3K4me1 and H3K27ac (Abcam).

    Techniques: Expressing, RNA Sequencing Assay, Modification, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

    Length distribution of interrupted palindromes at 5′ and 3′-ends in Illumina HiSeq 2000 reads of Atlantic cod ( Gadus morhua ). Reads were generated from 11 historic samples using TruSeq library creation protocols (red lines), four historic samples using Microplex protocols (black lines) and one modern sample using TruSeq protocols (grey line). Terminal palindromic sequences longer than three basepair are rare in the Microplex and modern samples.

    Journal: PLoS ONE

    Article Title: Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

    doi: 10.1371/journal.pone.0089676

    Figure Lengend Snippet: Length distribution of interrupted palindromes at 5′ and 3′-ends in Illumina HiSeq 2000 reads of Atlantic cod ( Gadus morhua ). Reads were generated from 11 historic samples using TruSeq library creation protocols (red lines), four historic samples using Microplex protocols (black lines) and one modern sample using TruSeq protocols (grey line). Terminal palindromic sequences longer than three basepair are rare in the Microplex and modern samples.

    Article Snippet: The Microplex Library Preparation kit ( www.diagenode.com ) differs from the TruSeq protocol in that it is a single tube protocol, uses blunt-end ligation of stem-loop adapters, and no intermediate purification procedures are used before library amplification.

    Techniques: Generated

    Proportion of reads aligning to the Atlantic cod genome for TruSeq and Microplex libraries. The proportions of reads aligning (relative to the number of untrimmed read pairs) were calculated for libraries including interrupted palindromes (light grey) and those for which these palindromes (dark gray) were removed at the 3′-end. Only reads with a minimum mapping quality (MapQ) value of 25 were considered.

    Journal: PLoS ONE

    Article Title: Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

    doi: 10.1371/journal.pone.0089676

    Figure Lengend Snippet: Proportion of reads aligning to the Atlantic cod genome for TruSeq and Microplex libraries. The proportions of reads aligning (relative to the number of untrimmed read pairs) were calculated for libraries including interrupted palindromes (light grey) and those for which these palindromes (dark gray) were removed at the 3′-end. Only reads with a minimum mapping quality (MapQ) value of 25 were considered.

    Article Snippet: The Microplex Library Preparation kit ( www.diagenode.com ) differs from the TruSeq protocol in that it is a single tube protocol, uses blunt-end ligation of stem-loop adapters, and no intermediate purification procedures are used before library amplification.

    Techniques:

    Frequency of nucleotide substitutions along historic reads of Atlantic cod. Reads were generated using the TruSeq V2 library creation protocol ( a ) or the Microplex single tube protocol (see methods) ( b ). Misalignments to the reference at the 5′ and 3′-end of sequencing reads are the result of elevated proportions of C to T substitutions (red), G to A substitutions (blue) and other possible substitutions (grey). The figure was generated using the program mapDamage V2.0.0 using 1 million randomly chosen reads for merged Illumina and Microplex libraries [13] .

    Journal: PLoS ONE

    Article Title: Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

    doi: 10.1371/journal.pone.0089676

    Figure Lengend Snippet: Frequency of nucleotide substitutions along historic reads of Atlantic cod. Reads were generated using the TruSeq V2 library creation protocol ( a ) or the Microplex single tube protocol (see methods) ( b ). Misalignments to the reference at the 5′ and 3′-end of sequencing reads are the result of elevated proportions of C to T substitutions (red), G to A substitutions (blue) and other possible substitutions (grey). The figure was generated using the program mapDamage V2.0.0 using 1 million randomly chosen reads for merged Illumina and Microplex libraries [13] .

    Article Snippet: The Microplex Library Preparation kit ( www.diagenode.com ) differs from the TruSeq protocol in that it is a single tube protocol, uses blunt-end ligation of stem-loop adapters, and no intermediate purification procedures are used before library amplification.

    Techniques: Generated, Sequencing

    ABO promoter methylation and ABO transcript expression analysis. The ABO promoter methylation status (n = 4 clinical grade MSCs, 2 samples each) and the expression of ABO blood group gene transcripts in clinical grade MSCs (n = 2) of known blood group and secretor genotype (K16- A 1 /A 1 -Se/se , K25- B/O 1 -Se/Se ) was tested with MagMeDIP Kit and quantitative real time PCR (qPCR), respectively. ( A ) DNA methylation (%, metDIP/input), of methylated TSH2B , unmethylated GAPDH , and ABO proximal promoter region. ( B ) ABO transcript analysis (qPCR) on resting MSCs compared to cells subjected to different types of induction treatments. MSCs were either stimulated for 5 days with 5 ng/ml of interferon-gamma (INFg), or activated for 5 days by inflammatory mediators released through a cell-impermeable membrane in trans-well mixed lymphocyte reactions (MLRs). MSCs were also subjected to 14-day in vitro differentiation with adipogenic (ADI) and osteogenic (OST) induction medium, or respective control medium. Adenocarcinoma cell line HPAF-II was used as positive control for ABO transcript expression and distilled H 2 O served as negative control. Control genes ICAM1 and aP2 served as positive controls for cytokine activation and adipogenic induction, respectively. Relative gene expression is shown compared to control gene beta-actin. Adipogenic and osteogenic differentiation was also confirmed with Oil red O staining for lipid rich vacuoles and von Kossa staining for mineralized matrix, respectively. Mean ± SD, ** * P

    Journal: PLoS ONE

    Article Title: Do ABO Blood Group Antigens Hamper the Therapeutic Efficacy of Mesenchymal Stromal Cells?

    doi: 10.1371/journal.pone.0085040

    Figure Lengend Snippet: ABO promoter methylation and ABO transcript expression analysis. The ABO promoter methylation status (n = 4 clinical grade MSCs, 2 samples each) and the expression of ABO blood group gene transcripts in clinical grade MSCs (n = 2) of known blood group and secretor genotype (K16- A 1 /A 1 -Se/se , K25- B/O 1 -Se/Se ) was tested with MagMeDIP Kit and quantitative real time PCR (qPCR), respectively. ( A ) DNA methylation (%, metDIP/input), of methylated TSH2B , unmethylated GAPDH , and ABO proximal promoter region. ( B ) ABO transcript analysis (qPCR) on resting MSCs compared to cells subjected to different types of induction treatments. MSCs were either stimulated for 5 days with 5 ng/ml of interferon-gamma (INFg), or activated for 5 days by inflammatory mediators released through a cell-impermeable membrane in trans-well mixed lymphocyte reactions (MLRs). MSCs were also subjected to 14-day in vitro differentiation with adipogenic (ADI) and osteogenic (OST) induction medium, or respective control medium. Adenocarcinoma cell line HPAF-II was used as positive control for ABO transcript expression and distilled H 2 O served as negative control. Control genes ICAM1 and aP2 served as positive controls for cytokine activation and adipogenic induction, respectively. Relative gene expression is shown compared to control gene beta-actin. Adipogenic and osteogenic differentiation was also confirmed with Oil red O staining for lipid rich vacuoles and von Kossa staining for mineralized matrix, respectively. Mean ± SD, ** * P

    Article Snippet: The promoter methylation status was analyzed with MagMeDIP Kit (mc-magme-048; Diagenode, Belgium) according to manual.

    Techniques: Methylation, Expressing, Real-time Polymerase Chain Reaction, DNA Methylation Assay, In Vitro, Positive Control, Negative Control, Activation Assay, Staining