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Blood, Vol. 113, Issue 22, 5536-5548, May 28, 2009
The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL
Blood Ci et al.
113: 5536
Supplementary materials for: Ci et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 26.4 KB)
- Table S1. Primers used for QChIP (PDF, 574 KB)
- Table S2. Primers used for QPCR (PDF, 64.7 KB)
- Table S3. Master table (attached separately as an excel file) listing all BCL6 target genes identified in germinal center B cells, OCI-Ly1, and OCI-Ly7 cells (XLS, 562 KB)
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The columns with headers shaded in light blue indicate the NimbleGen locus name, the open reading frame (ORF) name for each gene. The columns with headers shaded in light green indicate whether the gene was captured in germinal center B cells, OCI-Ly1, or OCI-Ly7 cells. The columns shaded in orange indicate whether the genes contain consensus DNA elements for BCL6, HSF, IRF2/4, or STAT3. The column shaded in yellow contains the gene ontology (GO) terms for genes where available.
- Table S4. Genes differentially bound by BCL6 in DLBCL cells (XLS, 273 KB)
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The columns with headers shaded in light blue indicate the NimbleGen probeset, and the open reading frame (ORF) name for each gene. Certain probesets correspond to bidirectional promoters, in which case more than one ORF is listed per probeset (as described in the methods section). The columns with headers shaded in light green indicate whether the gene was captured in germinal center B cells, OCI-Ly1, or OCI-Ly7 cells. The columns shaded in gray indicate whether the genes contain consensus DNA elements for BCL6, HSF, IRF2/4, or STAT3. The column shaded in yellow contains the gene ontology (GO) terms for each gene. This table was extracted from Table 3 to make it easier to read and is provided as a separate excel file.
- Table S5. Genes preferentially bound in GC B cells (XLS, 83 KB)
- Table S6. Genes bound in both DLBCL and GC B cells (XLS, 241 KB)
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Organized as in Table S4.
- Table S7. BCL6 target genes that are repressed in germinal center B cells vs. naďve B cells, and their expression status in DLBCL cells (XLS, 24.5 KB)
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The columns indicate the ORF name and whether each of these genes is repressed or activated in primary DLBCLs. The transcript abundance was determined from previously published gene expression arrays as indicated and referenced in the text. A graphical representation of these data is shown in Fig. S4.
- Table S8. BCL6 co-distributes to specific pathways in association with particular DNA binding elements (XLS, 25.5 KB)
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Each column represents the statistical significance for the frequency with which each respective DNA element is represented within each of the GO terms listed on the right. This is the tabular representation of the heat map shown in Fig. 7. NS: not significant.
- Figure S1. Phenotypic characterization of primary B-cell populations (JPG, 227 KB)
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Purity of primary B-cell populations isolated from human tonsils using the MidiMACS system was determined by FACS analysis. Germinal center cells include both centroblast and centrocytes, which are CD38+ and CD77+∕−. TMCs (tonsilar mononuclear cells) are shown as controls.
- Figure S2. Peak identification algorithm and selection of the working threshold (JPG, 99.2 KB)
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On the Y axis is depicted the number of hits called, the X axis represents the top percentile as (1 – quantile). The probability of finding false positives increases exponentially when the threshold is increased. The Y axis represents the Ratio between “randomly expected calls” over “Called hits” (RRC). The X axis: log scale of the top percentile as (1 – quantile).
- Figure S3. Examples of BCL6 promoter binding peaks (JPG, 161 KB)
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Graphical representation of BCL6 binding peaks in replicate ChIP-on-chip studies at selected promoters (replicate 1 dark gray and replicate 2 in light gray). (A) In the case of the STAT3 promoter, the BCL6 peak reached threshold (indicated by the black line) in all replicates in germinal center (GC) B cells, OCI-Ly1, and OCI-Ly7 DLBCLs. (B) In the case of the FOXO4 promoter in GC cells or the MYC promoter in OCI-Ly1 cells one of the replicates did not reach threshold but was captured by the peak-rescue algorithm as positive since there was a similar distribution of probe variation in the sub-threshold peak. (C) The HPRT promoter is shown as an example of a non-BCL6 target promoter in which the signal did not pass the cutoff in either replicates. The Y axis depicts the ratio of BCL6 signal/Input. The X axis depicts probe location on each promoter relative to the transcriptional start site.
- Figure S4. QCHIP validation of BCL6 target genes predicted by ChIP-on-chip (JPG, 217 KB)
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Thirty-six candidate BCL6 target genes identified by ChIP-on-chip were selected for validation by QChIP assay control. In OCI-Ly1 cells (shown here), while 32/26 displayed greater than 3-fold enrichment. The results were similar in centroblasts and OCI-Ly7 cells (not shown). The X axis represents the fold enrichment for each promoter region vs. actin control antibody after normalization to input DNA. GAPDH is included as a negative control (fold enrichment=1, green). The gray field indicates enrichment greater than control. The target gene amplicons represent the enriched regions based on the localization of the BCL6 peak on the promoter array.
- Figure S5. Expression of BCL6 target genes in normal and malignant B cells (JPG, 1.27 MB)
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The figures shows a graphical heat map representation of the transcript abundance of BCL6 target genes identified by ChIP-on-chip in naďve B cells (NBC), centroblasts (CB), centrocytes, and DLBCL patients. The data are derived from previously published expression arrays studies as cited in the text. Red indicates high abundance and green indicates low abundance of each transcript. The name of each gene is indicated in the rows to the right of the heat map. This file is also supplied as a JPEG to allow higher resolution visualization.
- Figure S6. Confirmation that BCL6 can bind to the BCL2 promoter (JPG, 104 KB)
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QChIP was performed in independent GC B-cell, OCI-Ly1, and OCI-Ly7 samples using BCL6 (black bars) or negative control (actin) antibodies (gray bars) to pull down the section of the BCL2 promoter identified through ChIP-on-chip. The Y axis represents the % input of the promoter fragments captured by the two different antibodies.
- Figure S7. RI-BPI induces expression of MYC, JUNB, and IRF4 (JPG, 169 KB)
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OCI-Ly1 cells were exposed to 15 micromolar RI-BPI or control peptide for the indicated time points, and the mRNA extracted for QPCR to determine the transcription abundance of JUNB, MYC, and IRF4. The Y axis represents the fold induction of mRNA vs. control peptide at each timepoint. Direct binding of BCL6 to MYC and JUNB is shown in Fig. S3.
- Figure S8. BCL6 binding peaks coincide with BCL6 DNA elements predicted by FIRE at the BCL6 locus (JPG, 239 KB)
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Top: A graphical representation of the BCL6 locus is shown indicating at the top (black lines) the location of BCL6 binding sites predicted by the FIRE algorithm as described in the text. BCL6 ChIP-on-chip was performed in OCI-Ly1 cells and hybridized to a custom microarray that tiles across the BCL6 locus (as reported in Mendez et. al., 2008 Mol Cell Biol). The fold enrichment of BCL6 binding is represented by the red tracing. Most of the binding peaks correspond to the predicted BCL6 consensus elements. The location of BCL6 exon 1 and intron 1 is indicated by the cartoon representation at the lower part of the picture. Bottom: QChIP was performed with BCL6 or IgG control antibody in OCI-Ly1 cells generating amplicons the location of which are indicated by the green and blue asterisks in the top panel. The blue bars represent amplicons that are more proximal to the predicted BCL6 binding sites and binding peaks, while the green bars represent sites that are slightly more removed from the main binding peaks. These data confirm the preferential binding of BCL6 generally in association with the presence of the FIRE predicted DNA consensus motif.
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