Article Figures & Data
Figures
- FIG. 1.
The BCL6Cmpd mutant is defective in its corepressor interactions and transcription repression activity. (A) Schematic representation of mutations carried by BCL6Cmpd and affected corepressors. (B) BCL6Cmpd has significantly decreased binding to HDAC2 and SMRT. 293T cells were transiently transfected to express SMRT together with Flag-BCL6 or Flag-BCL6Cmpd. Alternatively, Flag-HDAC2 was expressed with either HA-BCL6 or HA-BCL6Cmpd. Whole-cell lysates were immunoprecipitated with Flag beads and analyzed by Western blotting (WB) using the indicated antibodies. Vertical lines are inserted to indicate a repositioned gel lane. (C) BCL6Cmpd was incapable of restoring aberrantly expressed CCL3 in BCL6−/− BMM. BCL6+/+ and BCL6−/− BMM were infected with either a control murine stem cell virus retrovirus or viral constructs expressing BCL6 or BCL6Cmpd. Semiquantitative RT-PCR was performed using twofold serially diluted cDNA prepared from GFP+ cells sorted by fluorescence-activated cell sorting. β-Actin was used as a control for normalization. Ctrl, control.
- FIG. 2.
BCL6 autoregulation has a distinct corepressor requirement. (A) BCL6Cmpd potently repressed transcription from the BCL6 reporter but had limited activity on a number of other target gene reporters. Reporter assays were performed 293T cells with the indicated reporter constructs and increasing amounts of either wt BCL6 (wt) or BCL6Cmpd (Cmpd) expression plasmids. Activity of all reporters in the absence of BCL6 was set to 100. (B) Reporter assays were performed similar to those described in panel A but in the BCL6-negative Mutu III cells. (C) BPI treatment of SUDHL6 cells derepressed the expression of CCL3 but not BCL6. qRT-PCR was performed using primers specific for the three indicated genes. β2M, a gene not known to be regulated by BCL6, was examined as a specificity control. For each gene analyzed, the increase is relative to untreated cells. pTAT is a control peptide with only the carrier sequence. All graphs in this figure represent means and standard deviations of triplicate tests.
- FIG. 3.
CtBP1 corepressor interacts with both wt BCL6 and BCL6Cmpd. 293T cells were transiently transfected to express Flag-CtBP1 together with either HA-BCL6 or HA-BCL6Cmpd (A). Alternatively, myc-CtBP1 was expressed with either Flag-BCL6 or Flag-BCL6Cmpd (B). Whole-cell lysates were immunoprecipitated with Flag beads and analyzed by Western blotting using the indicated antibodies. WB, Western blotting.
- FIG. 4.
Interaction with CtBP requires both the POZ and the RDII domains of BCL6. (A) Schematic representation of BCL6 domain structure and the BCL6 deletion mutants used in the mapping studies. Relative strength of the interaction is depicted above the line structures: +++, strong, nearly wt level of binding; ++, moderate binding; +, weak binding; −, no binding. (B) Interaction between BCL6 and CtBP was studied in GST pull-down assays where immobilized GST or GST-BCL6 proteins were incubated with 35S-labeled CtBP1. (C) Co-IP assays were performed in 293T cells that were transiently transfected to express Flag-CtBP1 together with the indicated BCL6 deletion mutants. Full-length BCL6 was also expressed alone to serve as negative control for Co-IP (lanes 1 and 6). Whole-cell lysates were immunoprecipitated with anti-Flag beads and analyzed by Western blotting (WB) using either polyclonal anti-BCL6 (sc-858 plus sc-368) or CtBP antibodies. IgH, cross-reactive signals from the Ig heavy chain of the IP antibody. They are more pronounced in the right panel. Within each panel, vertical lines have been inserted to indicate a repositioned gel lane. FL, full-length BCL6.
- FIG. 5.
CtBP forms a complex with BCL6 in B cells. (A) Western blot analysis of BCL6 and CtBP expression in cell lines representing pre-B, GC-derived mature B, and plasma cells (PC). GAPDH was included as a loading control. Human centroblasts and centrocytes were purified by magnetic cell separation using the MidiMACS system (Miltenyi Biotechnology) following a published protocol (21). (B) CtBP1 is present in a complex with BCL6 in the BCL6-positive Ly7 cells. Co-IP assays in Ly7 and Mutu III cells were carried out using an antibody for BCL6 or two control antibodies (BCL3 and STAT1). The presence of BCL6 and CtBP1 was examined in the precipitates by Western blotting (WB). In the BCL6-negative Mutu III cells, neither BCL6 nor CtBP was immunoprecipitated by the panel of antibodies we used although a weak band with similar migration to BCL6 was nonspecifically pulled down by both the BCL6 and the BCL3 antibodies (n.s.). Vertical lines are inserted to indicate a repositioned gel lane. Ab, antibody.
- FIG. 6.
Recruitment of CtBP to the BCL6 exon 1 region and other BCL6 target loci. (A) CtBP is preferentially targeted to the wt BCL6 promoter in Ly1 cells. (Left) Shown is a schematic of the BCL6 exon 1 region in Ly1 cells which carry an “activating mutation” in the mutant allele (mut) that generates an EcoNI site. Other mutations (asterisks) are also present but do not affect BCL6 transcription (33). The mutant and wt alleles can be distinguished by EcoN1 digestion, which enables allelic analysis of amplified DNA fragments from ChIP. (Middle) Agarose gel images from a ChIP experiment show the DNA fragments before and after EcoN1 digestion. A ChIP assay was also carried out in the Mutu III cells to provide antibody specificity control. (Right) Following quantitation of the band intensity by the ImageQuant program, allelic enrichment was calculated as follows: (wt IP/wt input)/(mut IP/mut input). The bar graph shows the means and standard deviations of two independent ChIP experiments. (B) Occupancy of BCL6 and CtBP at selected BCL6 target loci was examined by ChIP-on-chip analysis using a custom-designed high-density tiling array. A graphic view of BCL6 and CtBP binding was generated in the University of California Santa Cruz web browser with the structure of the genes shown at the top of the histograms. Arrows indicate transcriptional orientation. In the BCL6 locus, the orange triangle indicates the exon 1 BCL6 binding sites studied in panel A. Opaque masking windows represent the cutoff defined as 2.5 times the standard deviation above the average relative enrichment on the entire array.
- FIG. 7.
CtBP is a required corepressor for BCL6 negative autoregulation. Both alleles of BCL6 in Ly7 are in germ line configuration, while in Val and Ly8 there is one translocated (xlocated) and one wt allele. (A) Effect of siRNA-mediated CtBP knockdown in 4 DLBCL cell lines. Western blot (WB) analysis was performed on cell lysates 40 h after transfection with CtBP siRNA oligonucleotides (CtBPi 1 and CtBPi 2) or a control oligonucleotide (−). (B) Gene expression changes caused by CtBP siRNA were analyzed by qRT-PCR. The ΔΔCT method was used to calculate the increase relative to control-treated cells defined as 1.0. Plotted in the graph are the means and standard deviations of two independent experiments. At the bottom of the graph, gene names appearing in bold indicate occupancy by both BCL6 and CtBP.
