PMC

Lack of BLIMP1 protein expression in most non-GCB DLBCL. Double immunofluorescence staining for IRF4 (red) and BLIMP1 (green) in human tonsil (left) and two representative ABC-DLBCL cases (middle: IRF4⁺/BLIMP1⁺; right: IRF4⁺/BLIMP1⁻).

Distribution and features of BLIMP1 mutations in ABC-DLBCL. (A) Schematic representation of the human BLIMP1 protein with its functional domains. Color-coded symbols depict distinct types of alterations leading to BLIMP1 inactivation as detailed in B. Aci, acidic domain; PR, positive regulatory domain; Pro-rich, proline rich domain.

Mutation of the BLIMP1 intron 2 splice acceptor site and loss of WT allele in a primary DLBCL. (A) Chromatograms of genomic DNA from DLBCL no. 438 (and a control sample). The residual WT sequences are likely a result of normal cells contaminating the biopsy because this case carries a monosomy 6. (B) Gel electrophoresis of RT-PCR products obtained from the indicated samples. A shorter BLIMP1α transcript and absence of the WT allele are observed in case no. 438. (C) Sequencing analysis of the RT-PCR product from no. 438 reveals the in-frame loss of exon 3.

Splice donor site mutation and loss of the second allele in Ly10. (A) Sequencing analysis of genomic DNA from Ly10 and a control sample. The arrow indicates a single bp substitution affecting the splice donor site. (B) Schematic diagram of the BLIMP1 gene and its two transcripts; primers used for RT-PCR amplification (triangles) are positioned below the maps. (C) Aberrant BLIMP1α transcripts in Ly10 (arrow), as compared with the U266 multiple myeloma cell line. GAPDH controls for loading. (D) Sequencing of the RT-PCR–generated product reveals aberrant splicing as a result of retention of intronic sequences. (E) Dual-color FISH analysis was performed as described in . Arrows point to the BLIMP1 signal.

Biallelic inactivation of BLIMP1 by rearrangement and deletion in the Ly3 cell line. (A) BLIMP1 genomic locus: filled and empty boxes represent coding and noncoding exons, respectively. Arrows correspond to alternative transcription initiation sites, and triangles below the map depict the primers used for mutational analysis. Restriction sites for Southern blot analysis are also indicated (X, XmaI; RI, EcoRI; S, SpeI) and aligned to the predicted restriction fragments; a vertical arrow points to the breakpoint region, as determined by restriction enzyme digestion. (B) Southern blot analysis of Ly3 and control DNA (N) using the probes indicated. (C) Dual-color FISH analysis of Ly3 cells hybridized with BLIMP1 probes (green, arrow) and a chromosome 6 centromeric probe (red). (D) Chromatograms of BLIMP1 exon 5 genomic sequences. A 2-bp deletion, introducing a premature stop codon, is present in Ly3.