Negative autoregulation of BCL-6 is bypassed by genetic alterations in diffuse large B cell lymphomas

Abstract

Thirty to forty percent of diffuse large B cell lymphomas (DLBCL) carry BCL-6 translocations that disrupt its 5' regulatory region. This same region is also subject to somatic hypermutations, although only a small fraction of these mutations have a detectable effect on transcription. Here, we show that transcription of the BCL-6 gene is negatively self-regulated in multiple cell types. This mechanism operates by means of the interaction of two BCL-6-binding sites within exon 1 of the gene and the BCL-6 protein itself, which is a potent transcription repressor. Because the DLBCL-associated "activating mutations" specifically target these exon 1 binding sites, and because the entire exon 1 is usually removed in the BCL-6-translocated tumors, this autoregulation is bypassed in 30-40% of all DLBCL cases. Our results not only demonstrate an important mechanism governing the expression of BCL-6, but also explain how BCL-6 is deregulated in a large number of DLBCL patients, providing a better understanding of BCL-6-related lymphomagenesis.

Fig 1.

The truncated BCL-6 gene is overexpressed in BCL-6 KO mice. (A) Structure of the truncated BCL-6 protein made in the BCL-6 KO mice compared with the wild type. (B) Northern blot analysis of BCL-6 expression in multiple tissues of the KO mice. Ethidium bromide staining of the 28S rRNA is used as a loading control. Mac, macrophage.

Fig 2.

BCL-6 exon 1 contains two functional BCL-6 binding sites that are targeted by activating mutations in lymphomas. (A) Sequences surrounding the transcriptional start site of the major promoter (Pb) (2), showing the locations of the two BCL-6 binding sites (SRB and SR20), and the four activating mutations from three lymphoma cases 1682, 1652, and Ly1. The two alleles of 1682 each carry a mutation in this region. Arrows underlie the 9-bp core sequences of the motifs. The consensus B6BS is also given for comparison. (B) Gel shift analysis of the SRB and SR20 motifs. The ³²P-labeled B6BS probe was used with nuclear extract from the BCL-6-positive Ly1 cells. Specificity of the major complex, indicated by an arrow, was verified by supershift assay with the anti-BCL-6 serum, 73-6. An excess amount of cold B6BS, SRB, and SR20 oligos was used to assess their relative affinity for BCL-6. (C) Reporter assay of Pab-Luc construct in 3T3 cells. Pab-Luc and Pab-dm1-Luc were transfected with increasing amounts of a BCL-6 expression plasmid. (D and E) Gel shift analysis of the mutated SRB and SR20 sites. Labels are used as described for B.

Fig 3.

Overexpression of exogenous BCL-6 down-regulates the endogenous gene. The heavy-metal-inducible pMEP4-HA-BCL-6 and control pMEP4 plasmids were stably transfected into Ly7 (A) and BT549 (B) cells. Representative results from three independent inductions are shown. In each experiment, cells were sampled before and at various time points after induction and analyzed by Northern blot for expression of both the endogenous and exogenous BCL-6 transcripts. Results were quantitated and normalized by the GAPDH signal. Plotted in the bar graph are ratios of various BCL-6 signals vs. the endogenous BCL-6 mRNA in the control cells, which was set as 1.0. All signals were first normalized to GAPDH for loading.

Fig 4.

“Activating mutations” confer resistance to BCL-6 repression in transient reporter assays. (A) Schematic representation of the reporter constructs used. (B) Activities of the wild-type and mutated reporters in Mutu I vs. Mutu III cells. (C) Reporter assays in Mutu III cells with increasing amount of cotransfected BCL-6 expression plasmid (0, 0.05, and 0.25 μg). Activity of the wild-type reporter was normalized to 1.0 for each cell type.

Fig 5.

Monoallelic expression of BCL-6 in Ly1 cells. (A) Schematic representation of mutations in the two BCL-6 alleles in Ly1. Each “*” or “X” represents a single mutation. (B) PCR-direct sequencing analysis of the BCL-6 genomic and mRNA sequences. Ramos cell line was used as a germ-line control. Arrowheads indicate position 257 with respect to transcriptional start site. (C) Restriction analysis of the genomic and RT-PCR products corresponding to exon 1. EcoNI digestion shortens the mutated RT-PCR product from 342 to 306 bp, and its corresponding genomic PCR product from 290 to 254 bp. (D) Endogenous BCL-6 in Ly1 cells is resistant to expression of exogenous BCL-6. The experiment was performed as in Fig. 3.

Fig 6.

BCL-6 preferentially binds to the wild-type exon 1 in Ly1 cells. Both Ly1 and the control Ly7 cells were analyzed for association of BCL-6 and acetylated H3 with the exon 1 sequence. Location of the PCR primers is given with respect to exon 1 and the surrounding mutations. Various amounts of recovered genomic DNA as well as a fraction of the total chromatin input were used in PCR reactions with the BCL-6-e1 and BCL-6 i2 primer pairs. IgG, normal rabbit IgG used as control antibody; N3, polyclonal anti-BCL-6 antibody; N.C., negative control PCR reaction with no template.