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Presentation given by Björn Grüning at Galaxy Africa 2018 (Cape Town, South Africa)<br

Training data for ChIP-seq data analysis (Galaxy Training Material): Identification of the binding sites of the Estrogen receptor

<p>The data provided here are part of a Galaxy Training Network tutorial that analyzes ChIP-seq data from a study published by Ross-Inness et al., 2012 (DOI:10.1038/nature10730) to identify the binding sites of the Estrogen receptor, a transcription factor known to be associated with different types of breast cancer.</p

Training data for 'From peaks to gene' tutorial (Galaxy Training Material)

<p>The data provided here are part of a Galaxy Training Network tutorial that analyzes peaks from a study published by Li et al., 2012 (DOI:10.1016/j.stem.2012.04.023) to identify target genes</p

Comparative transcript profiling of EBNA2 target gene expression in CBF1 proficient and deficient DG75 cells.

<p>DG75 cells expressing ER/EBNA2 were cultivated in estrogen supplemented medium for 24 h or were left untreated. Total cellular RNA was isolated and submitted to gene expression analysis using the Human Gene 2.0 ST array. All probe sets represent single transcripts (trxs). For each condition, 3 biological replicates were examined. Each vertical column represents the results obtained after hybridizing a single microarray. Horizontal rows represent data obtained for a particular probe set across all cell lines and conditions adjusted to a scale ranging from -2.0 to + 2.0. The relative high, medium and low expression values are represented by red, white and blue color, respectively. Vertical columns are ranked according to fold changes from highest induction levels on top to highest repression levels at the bottom. (A) Expression levels of 136 transcripts which change expression levels at least 4-fold (p ≤ 0.001) in response to EBNA2 in CBF1 proficient DG75 (DG75^ER/EBNA2 CBF1 wt) cells are displayed. The transcript cluster ID and the assigned genes/transcripts, including non-coding RNAs, are annotated. (B) 21 transcripts regulated at least 4-fold (p ≤ 0.001) in CBF1 deficient DG75 (DG75^ER/EBNA2 CBF1 ko). (C) Boxplots depicting the fold change distribution of EBNA2 induced and repressed transcripts for the subset of target genes changed at least 2-fold (p ≤ 0.05) in CBF1 wt and ko cells, respectively. EBNA2 induced (D) and repressed (E) transcripts are shown to illustrate the dynamic range of each system. Boxplot whiskers extend to 1.5x interquartile range. Dotted lines mark the 2-fold change chosen as cut-off. (F) Expression levels of EBNA2 (prior to and after estrogen treatment) and CBF1 proteins were monitored by Western blot analysis. Equal amounts of total protein lysates were applied and GAPDH served as an internal loading control. One representative experiment (n = 3) is shown.</p

EBNA2 can access more than 15% of its chromatin binding sites in CBF1 deficient DG75 B cells.

<p>(A) Intersection of EBNA2 binding sites identified in CBF1 proficient or deficient cells 24 h post doxycycline induction. 1,546 peaks that were identified in CBF1 proficient but not in CBF1 deficient cells were defined as "CBF1 dependent" EBNA2 peaks. 243 EBNA2 peaks identified in CBF1 deficient and proficient DG75 cells were defined as "CBF1 independent". (B-E) Comparison of EBNA2 ChIP-seq signal distributions at CBF1 independent or dependent peaks. (B) Anchor and (C) scatter plots (mean + 95% CI) depicting ChIP-seq signal distributions at EBNA2 peak subsets. Regions flanking the peak center for 2 kb in each direction were analyzed (Data underlying panel B). Absolute means and SEMs are indicated below. (D) Anchor and (E) scatter plots (mean + 95% CI) as shown in B and C but depicting EBNA2 ChIP-seq signal intensities for the two different subsets of EBNA2 peaks as defined in A. Statistical significance for differences of all means were assessed applying unpaired two-tailed t-test for log values with Welch’s correction (**** p < 0.0001); absolute means and SEMs are indicated below. (F) List of EBNA2 mean ChIP-seq signal intensities at CBF1 independent and dependent peaks.</p

CBF1 independent and dependent EBNA2 binding sites are significantly enriched for activated chromatin marks in DG75 cells prior to EBNA2 binding.

<p>Based on published data sets on histone modification in DG75, the two EBNA2 peak subsets (CBF1 independent dark blue; CBF1 dependent light blue) were separately analyzed for histone activation marks typically found at enhancer regions. These data were compared to signal intensities of all peaks for the respective chromatin modification (red). (A) Anchor plots depict H3K4me1, H3K4me3, and H3K27ac at the respective peak centers and 20 kb flanking regions. (B) Data underlying panel (A) were used to generate boxplots showing the signal distributions encompassing the entire 40 kb genomic region. The significance of differences of means was assessed by unpaired two-tailed t-tests with Welch’s correction (**** p < 0.0001, *** p < 0.001). The differences of means for CBF1 independent compared to CBF1 dependent EBNA2 peaks for H3K4me1 (-0.3004 ± 0.7957; p = 0.706), H3K4me3 (0.4323 ± 1.411; p = 0.7595), and H3K27ac (-0.5184 ± 0.3501: p = 0.1396) were not statistically significant. Box plot whiskers extend to 1.5x interquartile range. (C) Table summarizing means and SEMs of histone modifications analyzed in (A) and (B).</p

EBNA2 and EBF1 form protein complexes in CBF1 proficient and deficient DG75 B cells.

<p>(A) DG75^doxHA-E2 CBF1 wt and CBF1 ko B cells or (B) DG75^doxHA-E2WW CBF1 wt B cells were transfected with EBF1 expression plasmids or empty vector controls. EBNA2 expression was induced by doxycycline (Dox) treatment directly after transfection or cells were left untreated. Total cellular extracts were harvested after 24 h and subjected to immunoprecipitation (IP) using an EBF specific antibody and then assayed by Western blot (WB) using EBF and EBNA2 specific antibodies. Total cell lysates (L) represent 1% of the cells used for IP (n = 2, one representative experiment is shown). (C) DG75 were transfected with EBF1 expression plasmids (+) or empty vector controls (-). Total cellular extracts were harvested 24 h post transfection, incubated with GST or GST-END domain fusion proteins coupled beads to pull down associated proteins. Western blot detection was performed with EBF and GST specific antibodies. GAPDH was used as an internal loading control (n = 2, one representative experiment is displayed).</p

EBNA2 requires EBF1 to bind to its CBF1 independent binding sites.

<p>DG75^doxHA-E2 CBF1 wt or CBF1 ko B cells were transfected with a mixture of scrambled non-targeting siRNAs (siCNTRL) or EBF1 specific siRNAs (siEBF1). 8 h post transfection, EBNA2 transcription was induced. 24 h post transfection, cells were harvested and analyzed by immunoblots (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006664#ppat.1006664.s009" target="_blank">S9 Fig</a>) and ChIP-qPCR. In the upper panel, EBNA2 (E2) binding signal and peak tracks as obtained in DG75^doxHA-E2 (DG75) as well as EBNA2, CBF1 and EBF1 peak tracks in LCLs are shown for three (A) CBF1 independent or (B) CBF1 dependent EBNA2 binding sites. ChIP-qPCR results for EBF (middle panel) and EBNA2 (lower panel) binding to chromatin before and after EBF1 knock down are shown. Data are mean values, whiskers display standard deviations, p-values, based on a two tailed, paired t-test, are indicated.</p

Gene ontology enrichment analysis for CBF1 independent EBNA2 repressed target genes.

<p>Gene ontology enrichment analysis for CBF1 independent EBNA2 repressed target genes.</p

Gene ontology enrichment analysis of CBF1 dependent EBNA2 induced target genes.

<p>Gene ontology enrichment analysis of CBF1 dependent EBNA2 induced target genes.</p