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The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL

Weimin Ci, Jose M. Polo, Leandro Cerchietti, Rita Shaknovich, Ling Wang, Shao Ning Yang, Kenny Ye, Pedro Farinha, Douglas E. Horsman, Randy D. Gascoyne, Olivier Elemento and Ari Melnick

Data supplements

  • 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) -
      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) -
      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) -
      Organized as in Table S4.

    • Table S7. BCL6 target genes that are repressed in germinal center B cells vs. na�ve B cells, and their expression status in DLBCL cells (XLS, 24.5 KB) -
      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) -
      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) -
      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+∕−




    • Figure S2. Peak identification algorithm and selection of the working threshold (JPG, 99.2 KB) -
      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) -
      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) -
      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) -
      The figures shows a graphical heat map representation of the transcript abundance of BCL6 target genes identified by ChIP-on-chip in na�ve 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) -
      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) -
      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) -
      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.

Article Figures & Data

  • Figure 1

    Distinct and overlapping BCL6 target genes in DLBCL and GC B cells. (A) The Venn diagram illustrates the numbers of target genes specific to GC B cells, DLBCL cells, and the number of the common target genes between these cell types. (B) The heatmap represents the combination of GO term enrichment identified by PAGE in GC-specific, DLBCL-specific, and common target genes. Red indicates the intensity of enrichment of GO terms (rows) among these groups of target genes (columns). The statistical significance of the heatmap color code is provided.

  • Figure 2

    Differential BCL6 binding to specific promoters in DLBCL and GC B cells. (A) An example of BCL6-binding peaks for genes that were identified as differentially bound in GC versus DLBCL cells. The y-axis indicates enrichment versus input. The x-axis indicates the location of probes within the respective loci relative to the transcriptional start site. The dark gray and light gray tracings correspond to the different replicates. (B) A subset of genes predicted to be differentially bound by ChIP-on-chip were validated by independent quantitative ChIP experiments. The x-axis measures the fold enrichment ratio of promoters in GC cells versus OCI-Ly1 (black arrow) or OCI-Ly1 versus GC cells (red arrow). The data are from 3 independent experiments. Error bars represent the SEM for triplicates.

  • Figure 3

    BCL6 target genes are preferentially repressed in BCR and GCB-type DLBCLs. The figure shows a graphical representation depicting the abundance of BCL6 targets among genes that are either repressed or up-regulated in (A) BCR versus OxPhos and (B) GCB versus ABC DLBCLs. Each panel contains a plot in which the x-axis represents all of the genes contained in our promoter arrays distributed according to whether they are repressed preferentially in BCR (A left) or OxPhos (A right), or in GCB (B left) or ABC (B right). The y-axis represents the percentage of these genes that were found to be BCL6 targets by ChIP-on-chip. The dotted line represents the percentage of genes that would be expected to be BCL6 targets if the BCL6 targets were uniformly distributed across the range of relative expression values. The statistical significance of the enrichment for BCL6 target genes is indicated by the heatmap that is below each of the plots, as shown in the heatmap color code at the bottom of the figure.

  • Figure 4

    BCL6 represses many oncogenes in centroblasts. (A) A heatmap is shown representing the transcript abundance (data from Basso26) of oncogenes and related genes that are direct transcriptional targets of BCL6 (NBC indicates naive B cells; CB, centroblasts; and CC, centrocytes). (B) The relative ratio of BCL2 to BCL6 mRNA abundance versus GAPDH was measured in 3 independent sets of human tonsilar NBC and CBs by quantitative PCR (error bars represent the SEM for triplicates), and (C) Western blots. (D) Human tonsil sections were submitted to immunohistochemistry with BCL2 and nuclear counterstain (right panel and inset), BCL6 antibody and counterstain (middle), and with PAX5 and BCL2 costaining. The data show inverse staining patterns of BCL6 and BCL2 in GCs and show that the PAX5-positive GC B cells lack BCL2, indicating the BCL2-positive GC cells are not of the B-cell lineage. Slides were viewed with a light microscope (AxioSkop2; Carl Zeiss Microimaging, Thornwood, NY) using Plan-Neofluar lens at a 10×/0.50 air objective, 40×/0.90 oil objective, 100×/1.30 oil objective. Images were taken using a color camera (AxioCam; Carl Zeiss Microimaging) and were processed using Axiovision software (Carl Zeiss Microimaging).

  • Figure 5

    BCL6 and BCL2 inverse correlation is disrupted in patients with BCL2 translocations. (A) A series of DLBCLs (columns) is plotted in a heatmap and distributed according to their abundance of BCL6 transcript (data from Lenz et al33). BCL2 mRNA is generally negatively correlated with BCL6. Cases with BCL2 translocations are indicated by the red boxes in the top row. (B) Expression of BCL2 and BCL6 was examined in a series of 157 DLBCL patients by immunohistochemistry. The top bar represents patients without BCL2 translocation; bottom bar, BCL2 translocated cases. In each case, the black bar represents the percentage of patients who express both BCL6 and BCL2, whereas the gray bar represents the percentage of tumors that express either BCL2 or BCL6 exclusively. (C) Immunohistochemistry shows that DLBCL cases with t(14;18) translocations (left panel) typically exhibit malignant cells with dual BCL2 (blue) and BCL6 (brown) protein expression (right panel). FISH results were visualized using a Zeiss LSM510 Multiphoton confocal microscope (Carl Zeiss Microimaging) equipped with 40×/0.75/0.72 and 60×/0.80/0.3 objectives and one 25-mW argon laser exciting at 458, 488, and 514 nm and one 1-mW helium-neon laser exciting at 543 nm; proprietary image acquisition software was used for image analysis. (D) The DLBCL cell lines Farage (which expresses BCL6 and has wild-type BCL2 locus) and Toledo (which is BCL6-negative) were exposed for the indicated times to the specific BCL6 peptidomimetic inhibitor RI-BPI. The y-axis represents the fold increase of mRNA change induced by RI-BPI versus a control peptide. Cells were treated as indicated, and the data were from 3 independent experiments. Error bars represent the SEM for triplicates.

  • Figure 6

    BCL6 target genes display specific genomic features and DNA elements. (A) The diagram depicts the overlap between the overlapping BCL6 genes between normal and malignant B cells (blue circle) and CpG island-containing loci among the entire pool of 24 137 promoters contained on the array. (B) Analysis of the BCL6 target gene cohort by FIRE identified 8 DNA elements significantly overrepresented among BCL6 target genes identified in GC cells (CB), DLBCL cells, the common target genes, or in the cohort of non-BCL6 targets (columns). Yellow represents highly enriched; and blue, relative depletion. The statistical significance of the heatmap color code is provided. (C) The graph represents the distribution of genes ordered according to the strength of BCL6 binding in ChIP-on-chip on the x-axis and the frequency with which genes contain the BCL6 consensus site identified by FIRE (inset), using a sliding window of 100 genes. (D) Genes are distributed along the x-axis according to BCL6 binding, and the y-axis depicts the position of the FIRE-identified BCL6 motif occurrences relative to the transcriptional start site (set at 0). Negative numbers are upstream and positive numbers downstream of the start site. Similar to panel C, the y-axis corresponds to the average position of BCL6 motif occurrences with respect to the transcription start site using a sliding window of 100 genes. Only genes containing at least one occurrence of the BCL6 motif were considered. (E) Similar to panel C, the graph represents BCL6 binding on the x-axis, but the y-axis shows the frequency at which genes contain both BCL6 and STAT3-binding elements.

  • Figure 7

    Consensus motif identification by FIRE distribute to different GO terms. PAGE was performed to identify GO terms (rows) associated with BCL6 target genes according to the presence of the several DNA elements identified by FIRE (columns). Brighter red color is indicative of enrichment of the respective motifs for the indicated GO terms. The differently colored shading of the GO terms is provided to highlight those that are preferentially associated with different subsets of binding motifs. The statistical significance of the heatmap color code is provided.

  • Table 1

    Gene ontology (GO) terms represented among BCL6 target genes

    GC-specific genesCommon genesDLBCL-specific genesGO term
    3 (0)*2 (1)1 (1)GO:0043038 amino acid activation
    3 (0)*2 (1)1 (1)GO:0006418 tRNA aminoacylation for protein translation
    3 (0)*2 (1)4 (1)GO:0006399 tRNA metabolic process
    6 (1)*5 (3)9 (4)GO:0034660 ncRNA metabolic process
    3 (0)*2 (1)1 (1)GO:0043039 tRNA aminoacylation
    0 (0)3 (0)*0 (0)GO:0045739 positive regulation of DNA repair
    0 (0)4 (1)*1 (1)GO:0006282 regulation of DNA repair
    4 (2)16 (7)*12 (8)GO:0006511 ubiquitin-dependent protein catabolic process
    4 (2)17 (8)*12 (9)GO:0030163 protein catabolic process
    4 (2)16 (7)*12 (8)GO:0044257 cellular protein catabolic process
    8 (4)22 (13)*24 (15)GO:0043285 biopolymer catabolic process
    4 (2)16 (7)*12 (8)GO:0043632 modification-dependent macromolecule catabolic process
    4 (2)16 (7)*12 (8)GO:0051603 proteolysis involved in cellular protein catabolic process
    3 (2)13 (5)*8 (6)GO:0051438 regulation of ubiquitin-protein ligase activity
    3 (2)13 (5)*8 (6)GO:0051340 regulation of ligase activity
    3 (1)11 (4)*8 (5)GO:0051436 negative regulation of ubiquitin-protein ligase activity during mitotic cell cycle
    0 (0)4 (1)*3 (1)GO:0009303 rRNA transcription
    0 (0)1 (1)7 (2)*GO:0000910 cytokinesis
    0 (0)1 (1)7 (2)*GO:0051301 cell division
    6 (3)12 (10)25 (12)*GO:0022403 cell cycle phase
    3 (1)8 (4)13 (5)*GO:0007050 cell cycle arrest
    3 (1)7 (3)10 (4)*GO:0007067 mitosis
    3 (1)8 (4)10 (4)*GO:0000087 M phase of mitotic cell cycle
    5 (2)10 (7)18 (8)*GO:0000279 M phase
    6 (4)20 (12)26 (14)*GO:0000278 mitotic cell cycle
    8 (4)20 (12)30 (14)*GO:0034984 cellular response to DNA damage stimulus
    9 (5)24 (15)33 (17)*GO:0051716 cellular response to stimulus
    8 (4)22 (14)32 (16)*GO:0033554 cellular response to stress
    10 (4)20 (14)28 (16)*GO:0006366 transcription from RNA polymerase II promoter
    6 (2)10 (7)18 (8)*GO:0006414 translational elongation
    0 (0)0 (0)3 (0)*GO:0018211 peptidyl-tryptophan modification
    0 (0)0 (0)3 (0)*GO:0018317 protein amino acid C-linked glycosylation via tryptophan
    0 (0)0 (0)3 (0)*GO:0018406 protein amino acid C-linked glycosylation via 2′-alpha-mannosyl-L-tryptophan
    0 (0)0 (0)3 (0)*GO:0035269 protein amino acid O-linked mannosylation
    0 (0)0 (0)3 (0)*GO:0035268 protein amino acid mannosylation
    2 (0)1 (1)4 (1)*GO:0043631 RNA polyadenylation
    5 (3)16 (11)26 (13)*GO:0034622 cellular macromolecular complex assembly
    9 (4)23 (14)30 (16)*GO:0044265 cellular macromolecule catabolic process
    5 (4)18 (12)26 (14)*GO:0034621 cellular macromolecular complex subunit organization
    11 (7)27 (21)39 (24)*GO:0022607 cellular component assembly
    11 (7)30 (21)39 (25)*GO:0043933 macromolecular complex subunit organization
    11 (6)27 (20)39 (23)*GO:0065003 macromolecular complex assembly
    2 (0)0 (2)7 (2)*GO:0045333 cellular respiration
    0 (1)5 (2)7 (2)*GO:0006730 one-carbon compound metabolic process
    9 (7)25 (21)45 (24)*GO:0006996 organelle organization
    • The first 3 columns list the number of targets per GO term present among GC-specific, in common between GC and DLBCL and DLBCL-specific genes. The values in parentheses denote the expected number of genes per GO term for each category (ie, GC, common, DLBCL).

    • * The numbers show significant enrichment versus expected (as demonstrated in the heatmap color code in Figure 1A).

    • The numbers are nonsignificant.

  • Table 2

    Enrichment of specific motifs identified among BCL6 target genes

    MotifMotif nameNontargets (21170 loci), depletion P valuesGC B cells (1590 loci), enrichment P valuesDLBCLs (2602 loci), enrichment P valuesCommon (1227 loci), enrichment P values
    [AT]TC[CT][AT][AC]GABCL6< 1e-04< 1e-21< 1e-25< 1e-26
    A[CT]TT[CG]CGSTAT3< 1e-21< 1e-64< 1e-09< 1e-53
    [CT]TTTC[GT]AHSF< 0.01< 1e-06< 1e-14< 1e-11
    [AT]A[CT][GT]ACGJUN< 1e-10< 1e-41< 1e-46< 1e-32
    [AG]CGA[AT]A[GT]E2F< 1e-11< 1e-44< 1e-59< 1e-39
    [AT]AG[AT][AT]CGIRF2/4< 1e-09< 1e-35< 1e-52< 1e-35
    CG[AT][CG]AA[AT]Unknown A< 1e-08< 1e-26< 1e-43< 1e-21
    CTTT[AC]C[AG][CG]Unknown B< 1e-05< 1e-15< 1e-29< 1e-15
    • The 3 rightmost columns show the statistical significance of the enrichment for each DNA element compared with their frequency among all the genes on the arrays among all BCL6 target genes in GC B cells, DLBCLs, or among the common genes between GC and DLBCL cells. The depletion column indicates the statistical significance with which these motifs are underrepresented among the non-BCL6 target genes on the arrays.

Supplementary Materials

  • Figure S1

    Supplementary PDF file available online.

  • Figure S2

    Supplementary PDF file available online.

  • Figure S3

    Supplementary PDF file available online.

  • Figure S4

    Supplementary PDF file available online.

  • Figure S5

    Supplementary PDF file available online.

  • Figure S6

    Supplementary PDF file available online.

  • Figure S9

    Supplementary PDF file available online.

  • Figure S10

    Supplementary PDF file available online.

  • Table S1

    Supplementary PDF file available online.

  • Table S2

    Supplementary PDF file available online.

  • Table S3

    Supplementary PDF file available online.

  • Table S4

    Supplementary PDF file available online.

  • Table S5

    Supplementary PDF file available online.

  • Table S6

    Supplementary PDF file available online.

  • Table S7

    Supplementary PDF file available online.

  • Table S8

    Supplementary PDF file available online.