Cloning of genes involved in chromosomal translocations by high-resolution single nucleotide polymorphism genomic microarray - PubMed (original) (raw)

. 2008 Aug 19;105(33):11921-6.

doi: 10.1073/pnas.0711039105. Epub 2008 Aug 12.

Seishi Ogawa, Martin Zimmermann, Birte Niebuhr, Carol Stocking, Masashi Sanada, Kari Hemminki, Go Yamatomo, Yasuhito Nannya, Rolf Koehler, Thomas Flohr, Carl W Miller, Jochen Harbott, Wolf-Dieter Ludwig, Martin Stanulla, Martin Schrappe, Claus R Bartram, H Phillip Koeffler

Affiliations

Cloning of genes involved in chromosomal translocations by high-resolution single nucleotide polymorphism genomic microarray

Norihiko Kawamata et al. Proc Natl Acad Sci U S A. 2008.

Abstract

High-resolution single nucleotide polymorphism genomic microarray (SNP-chip) is a useful tool to define gene dosage levels over the whole genome, allowing precise detection of deletions and duplications/amplifications of chromosomes in cancer cells. We found that this new technology can also identify breakpoints of chromosomes involved in unbalanced translocations, leading to identification of fusion genes. Using this technique, we found that the PAX5 gene was rearranged to a variety of partner genes including ETV6, FOXP1, AUTS2, and C20orf112 in pediatric acute lymphoblastic leukemia (ALL). The 3' end of the PAX5 gene was replaced by the partner gene. The PAX5 fusion products bound to PAX5 recognition sequences as strongly as wild-type PAX5 and suppressed its transcriptional activity in a dominant-negative fashion. In human B cell leukemia cells, binding of wild-type PAX5 to a regulatory region of BLK, one of the direct downstream target genes of PAX5, was diminished by expression of the PAX5-fusion protein, leading to repression of BLK. Expression of PAX5-fusion genes in murine bone marrow cells blocked development of mature B cells. PAX5-fusion proteins may contribute to leukemogenesis by blocking differentiation of hematopoietic cells into mature B cells. SNP-chip is a powerful tool to identify fusion genes in human cancers.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

SNP-chip analysis detected genes involved in unbalanced translocations. (A) SNP-chip analysis can identify breakpoints of translocations when one of the paired translocated chromosomes is either lost or duplicated/amplified. (Left) Chromosomal status. Gene dosages are indicated either above or beneath the chromosomes. (Right) Results of SNP-chip analysis. (Ai) Normal chromosomes; gene dosage is normal. (Aii) Reciprocal translocation; gene dosage is normal. (Aiii) One of the paired translocated chromosomes is lost; gene dosage is lower than normal on the left side of the upper chromosome and the right side of the lower chromosome. Arrow heads indicate the breakpoint of the translocation in each chromosome. (Aiv) One of the paired translocated chromosomes is duplicated; gene dosage is higher than normal on the right side of the upper chromosome and the left side of the lower chromosome. Arrow heads indicate the breakpoint of this translocation in each chromosome. (B) Representative cases with unbalanced translocation of der(21)t(12;21)(p13;q22). (Left) Start sites of duplication at 12p13 involving the ETV6 gene. (Right) Start sites of duplication at 21q22 involving the RUNX1 gene. SNP-chip data of representative cases with dup(12)(p13) and dup(21)(q22) are shown. These abnormalities were validated by FISH and/or RT-PCR (data not shown). Results of SNP-chip data were visualized by CNAG software. Lines above each chromosome show total gene dosage; level 2 indicates diploid (2N) amount of DNA, which is normal. (C) Magnified view of SNP-chip data. (Upper) Start sites of duplications at 12p13 and 21q22 are magnified. Signals of individual probe signals are shown. Vertical lines indicated the positions of start sites of duplications. (Lower) Genes involved in the start sites of duplications.

Fig. 2.

Fig. 2.

PAX5 gene is fused to partner genes. (A) Start sites of deletion at 9p13.2 involving the PAX5 gene. (Left) SNP-chip data of representative cases with 9p13.2 deletions. A vertical arrow indicates the start sites of 9p deletion that involves the PAX5 gene. A horizontal arrow shows the direction of transcription of the PAX5 gene. (Right) Chromosomal abnormalities of partner chromosomes. Arrow heads indicate the start sites of duplication or deletions. Genes involved in the start sites are shown. (B) Result of RT-PCR. The ALL samples suggesting the presence of PAX5 fusion genes by SNP-chip analysis were examined by RT-PCR using the primers of PAX5 and the respective partner genes. (C) Fusion sequences of the PAX5 and partner genes. Joining sequences of fused transcripts are shown from the indicated exon of the fused gene. (D) Schematic structure of wild-type and mutant PAX5. Amino acid positions (aa) of each protein are indicated. PAX5/FOXP1 fusion construct has an early termination codon caused by a frame-shift. PD, paired domain; TA, transcription activation domain; O, octapeptide H, homeodomain-like; I, inhibitory domain. (E) Subcellular fractionation of PAX5-fusion proteins. pcDNA vector encoding wild-type PAX5, PAX5-ETV6, PAX5-FOXP1, PAX5-C20ORF112S, or PAX5-C20ORF112L was transfected into 293T cells. Nuclear and cytoplasmic proteins were separated and electrophoresed in the gel. Localization of PAX5-fusion proteins was examined by PAX5 N-terminal specific antibody. Purity of cytoplasmic protein was examined with anti-GAPDH antibody and purity of nuclear proteins with the anti-PARP antibody. C, cytoplasmic fraction; N, nuclear fraction.

Fig. 3.

Fig. 3.

_PAX5_-fusion proteins suppress transcriptional activity of PAX5 in a dominant-negative fashion and block the growth of B cells. (A) Result of EMSA: Wild-type PAX5 and PAX5 fusion were expressed in 293T cells, and nuclear proteins were purified. The purified nuclear proteins were mixed with radioisotope labeled double-strand oligonucleotide DNA, in either the presence or absence of cold competitor oligonucleotides (5-, 25-, and 50-fold cold competitor probes). Intensity of each shifted band indicating DNA–protein complex was measured and plotted graphically. Intensity of shifted bands in the absence of cold competitor probes was regarded as 1.0. (B) Reporter gene assay. Wild-type and mutant PAX5 were mixed at a various ratios (1:0, 1:0.3, 1:1, 1:3, respectively, 1 = 500 ng of construct) and transfected. Forty-eight hours later, relative activity of firefly luciferase was measured and plotted. Results represent the mean values of the three experiments. CD19, PAX5 luciferase reporter construct having PAX5 binding region of CD19 promoter; PAX5, wild-type PAX5; PAX5/ETV6, PAX5/ETV6 fusion; PAX5/C20L, long form of PAX5/C20orf112 fusion in which PAX5 exon 8 is fused to C20orf112 exon 3; PAX5/C20S, short form of PAX5/C20orf112 fusion in which PAX5 exon 5 is fused to C20orf112 exon 8; PAX5/FOXP1, PAX5/FOXP1 fusion with an early termination codon caused by a frame-shift after the site of fusion. (C) Results of expression of wild-type PAX5 and PAX5-fusion proteins. After cotransfection of equal amounts of vector encoding either wild-type or fusion PAX5 genes into 293T cells, the expression of respective proteins was examined by Western blot. Levels of expression of wild-type PAX5 protein were minimally affected by coexpression of the PAX5-fusion proteins. (D) Semiquantitative RT-PCR of downstream target genes of PAX5. Expression of PAX5 downstream target genes was examined by semiquantitative RT-PCR. Nalm 6, a human B cell ALL cell line expressing endogenous PAX5, was transfected with pMSCV-GFP (Empty), pMSCV-GFP-PAX5-C20orf112S (PAX5-C20S), or pMSCV-GFP-PAX5-C20orf112L (PAX5-C20L). GFP-positive cells were sorted and subject to semiquantitative RT-PCR. Optimal cycle numbers to semiquantify the expression of respective genes are as follows; BLK: 25 cycles; Nedd5; 25 cycles; TCF7L2: 25 cycles; ATP1B1: 25 cycles; β-actin: 22 cycles; CCR2: 25 cycles; CCR8: 30 cycles; IRF8: 30 cycles. (E) Structure of human BLK gene. Structure of BLK and primers used for ChIP assay within the 5′ regulatory region (Promoter-PCR) and 3′ end (Control-PCR) of the BLK gene is schematically shown. PAX5 binding site in the promoter region is indicated. (F) ChIP analysis of the PAX5 binding site in the BLK gene promoter. pMSCV-GFP (empty vector) or pMSCV-GFP-PAX5-C20S (PAX5-C20S) was transfected into human Nalm 6 B cell leukemia cells expressing endogenous PAX5. GFP-positive cells were subject to ChIP assay. The cells were fixed in formaldehyde solution and sonicated by ultrasound. DNA-protein complex was incubated with wild-type PAX5 specific antibody, which detected the C-terminal region of PAX5 but not the PAX5-C20orf112S protein (Upper). As a control, the DNA-protein complex was reacted with isotype nonspecific antibody (Lower). Immnoprecipitated DNA was subjected to PCR to amplify either the BLK promoter region containing PAX5 binding sequence (P) or, as an internal control, the 3′ end of the BLK gene (C). (G) Retrovirus infection experiments. Murine bone marrow cells were collected at 5 days after injection of 5FU. The hematopoietic cells were infected by retrovirus containing pMSCV-GFP empty vector (GFP), pMSCV-GFP-C20orf112L (PAX5/C20L), or pMSCV-GFP-C20orf112S (PAX5/C20S). GFP-positive murine hematopoietic cells were sorted and plated at 5 × 104 cells per plate in methylcellulose containing mSCF, mIL7, and hFL. At 8 days after the plating, the colony numbers were counted (Left; results represent means and SD of three experiments). Cell surface antigens on the GFP-positive cells infected with pMSCV-GFP (GFP) at Day 11 were examined by FACS using antibodies against CD43 and B220 (Upper Right), c-kit and CD11b (Lower Right) antibodies, to confirm the development of B cells.

Similar articles

Cited by

References

    1. Armstrong SA, Look AT. Molecular genetics of acute lymphoblastic leukemia. J Clin Oncol. 2005;23:6306–6315. - PubMed
    1. Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med. 2006;354:166–178. - PubMed
    1. Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med. 2004;350:1535–1548. - PubMed
    1. Nannya Y, et al. A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res. 2005;65:6071–6079. - PubMed
    1. Yamamoto G, et al. Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of affymetrix single-nucleotide-polymorphism genotyping microarrays. Am J Hum Genet. 2007;81:114–126. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources