Cloning of an Alpha-TFEB fusion in renal tumors harboring the t(6;11)(p21;q13) chromosome translocation - PubMed (original) (raw)

. 2003 May 13;100(10):6051-6.

doi: 10.1073/pnas.0931430100. Epub 2003 Apr 28.

Bae-Li Hsi, Jason D Arroyo, Sara O Vargas, Y Albert Yeh, Gabriela Motyckova, Patricia Valencia, Antonio R Perez-Atayde, Pedram Argani, Marc Ladanyi, Jonathan A Fletcher, David E Fisher

Affiliations

Cloning of an Alpha-TFEB fusion in renal tumors harboring the t(6;11)(p21;q13) chromosome translocation

Ian J Davis et al. Proc Natl Acad Sci U S A. 2003.

Abstract

MITF, TFE3, TFEB, and TFEC comprise a transcription factor family (MiT) that regulates key developmental pathways in several cell lineages. Like MYC, MiT members are basic helix-loop-helix-leucine zipper transcription factors. MiT members share virtually perfect homology in their DNA binding domains and bind a common DNA motif. Translocations of TFE3 occur in specific subsets of human renal cell carcinomas and in alveolar soft part sarcomas. Although multiple translocation partners are fused to TFE3, each translocation product retains TFE3's basic helix-loop-helix leucine zipper. We have identified the genes fused by the chromosomal translocation t(6;11)(p21.1;q13), characteristic of another subset of renal neoplasms. In two primary tumors we found that Alpha, an intronless gene, rearranges with the first intron of TFEB, just upstream of TFEB's initiation ATG, preserving the entire TFEB coding sequence. Fluorescence in situ hybridization confirmed the involvement of both TFEB and Alpha in this translocation. Although the Alpha promoter drives expression of this fusion gene, the Alpha gene does not contribute to the ORF. Whereas TFE3 is typically fused to partner proteins in subsets of renal tumors, we found that wild-type, unfused TFE3 stimulates clonogenic growth in a cell-based assay, suggesting that dysregulated expression, rather than altered function of TFEB or TFE3 fusions, may confer neoplastic properties, a mechanism reminiscent of MYC activation by promoter substitution in Burkitt's lymphoma. Alpha-TFEB is thus identified as a fusion gene in a subset of pediatric renal neoplasms.

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Figures

Figure 1

Figure 1

Light microscopic features of t(6;11) carcinoma. (A) At low magnification, the tumor consisted of epithelioid cells arranged in a nested alveolar or acinar pattern (H&E, original magnification, ×5). (B) Higher magnification showed cells with voluminous clear and granular eosinophilic cytoplasm. Acinar lumens often contained clusters of degenerating cells with pyknotic nuclei. Individual cells were round to polygonal with abundant cytoplasm. Acinar structures were separated by thin vascular channels (H&E, original magnification, ×60). (C) Immunostaining showed rare focal positivity for HMB-45 (H&E, original magnification, ×40).

Figure 2

Figure 2

(A) cDNA prepared from the index primary renal neoplasm was subjected to 5′ RACE with three antisense primers. Agarose gel electrophoresis reveals RACE products. Predicted PCR products derived from native TFEB message migrate with a smaller molecular weight than the novel PCR products (bands outlined with dotted line). (B) Primary renal tumor genomic DNA as well as cloned RACE product were subjected to PCR with an upstream primer derived from the novel non-TFEB sequence and a downstream primer derived from TFEB. Both DNA samples yielded identically sized PCR products.

Figure 3

Figure 3

(A) 6p21.1 translocation breakpoint evaluated in t(6;11)(p21.1;q13) metaphase cell by dual-color FISH. BACs telomeric (red) and centromeric (green) to TFEB are separated by the translocation. Yellow arrow indicates overlapping signal from nontranslocated chromosome. (B) 11q13 translocation breakpoint evaluation by using BACs telomeric (red) and centromeric (green) to the Alpha locus. Rearrangement of this locus is shown in each of four interphase tumor cells. (C) Colocalization of TFEB centromeric BAC (green) and Alpha telomeric BAC (red) in tumor interphase cell. Overlapping green and red FISH signals appear yellow when merged.

Figure 4

Figure 4

(A) Schematic representation of the t(6;11)(p21.1;q13) indicating translocations of the Alpha gene on chromosome 11 into intron 1 of TFEB preserving the entire coding region of TFEB. (B) Sequence of the Alpha-TFEB junctions in the two tumors identifying the translocation breakpoints in TFEB intron 1. In-frame initiation and termination codons are bold. Chromosome 6 sequence is italicized. Nucleotides of unclear origin at the breakpoints are underlined.

Figure 5

Figure 5

(A) Northern blot analysis of primary tumor poly(A)+ RNA probed with TFEB sequence derived from 5′ RACE hybridizes to the Alpha-TFEB fusion. A faint smaller band may correspond to endogenous TFEB message. Primary tumor mRNA hybridized with Alpha sequence derived from the 5′ RACE demonstrates two mRNA species corresponding to the molecular weight of Alpha message and Alpha-TFEB message. In a control cell line (CCS292), hybridization with Alpha reveals an mRNA species with its predicted molecular weight. (B) Multiple tissue Northern blot analysis (prenormalized to GAPDH) for TFEB and Alpha expression demonstrating expression of both genes in all tissues examined.

Figure 6

Figure 6

Constitutive expression of TFE3 promotes clonogenic growth. B16 melanoma cells were cotransfected with plasmid containing a puromycin resistance gene (Puro) together with either dominant-negative TCF (dn TCF) or dominant-negative TCF and either MITF or TFE3. All transfections contained equivalent amounts of vector (or vector + insert) DNAs in addition to constant puromycin resistance plasmid. After ≈10 days of puromycin selection, plates were washed, fixed, and stained with crystal violet.

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