Proviral insertions induce the expression of bone-specific isoforms of PEBP2αA (CBFA1): Evidence for a new myc collaborating oncogene

Abstract

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2␣A (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNAbinding domain and share a novel N terminus different from the previously reported PEBP2␣A products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2␣A and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with MYC

“…The CD2 LCR drives high level expression of Cbfa1 protein in the T-cell compartment and reveals an in vivo complex with Cbfb Expression of the major lymphoma isoform of Cbfa1 targeted to the T-cell compartment was achieved by cloning a 2.3 kb cDNA fragment encoding the Cbfa1-G1 isoform (Stewart et al, 1997) into the polylinker region of the human CD2 minigene vector VA hCD2 (Zhumabekov et al, 1995). Following microinjection of embryos using a 13.5 kb CD2-Cbfa1-G1 restriction fragment (Figure 1a), four lines of mice were generated which showed copy-number dependent expression of the CD2-Cbfa1-G1 transgene.…”

“…Analysis of a range of tissues from G1-500, showed a 3.5 kb transgene transcript present only in thymus, spleen and mesenteric lymph node (Figure 1b), con®rming the targeting speci®city of the transgene. Western blot analysis of G1-500 thymocytes using an anti-C terminal Cbfa1 antibody (Stewart et al, 1997) showed a major protein species of 70 kDa at greatly elevated levels compared to control thymocytes (Figure 1c).…”

“…Upper panel: anti-Cbfa1 C-terminal antibody, lower panel: anti-Cbfb antibody on complexes immunoprecipitated with antiCbfa1 antibody. T47i represents a mouse T-cell line expressing high levels of a number of til-1/Cbfa1 isoforms (Stewart et al, 1997) Cbfa1 perturbs T-cell development and synergises with myc F Vaillant et al during the same period. Necropsy revealed the extensive involvement of most, if not all, lymphoid tissues.…”

“…As we identi®ed the til-1/Cbfa1 gene as a potential myc-collaborating gene (Stewart et al, 1996(Stewart et al, , 1997 we assessed the e ect of crossing CD2-Cbfa1-G1 mice with CD2-myc transgenic animals. CD2-myc mice develop T-cell tumours, primarily involving the thymus, at an incidence of around 30% by 12 months of age depending on the genetic background (Stewart et al, 1993).…”

“…These insertions result in the transcriptional activation of a lymphoma-speci®c isoform of Cbfa1, suggesting that this CBF family member may act as a myc collaborator in lymphoma development. While the coding sequence of Cbfa1 is not interrupted by these insertions, suggesting that a full-length gene product may be responsible, the activated gene was found to encode at least ®ve isoforms, including two with a novel C-terminal domain unrelated to known Runt family proteins (Stewart et al, 1997). In addition, the products expressed as a result of proviral insertion were distinct from previously described Cbfa1 forms by virtue of their distinct 5' exons and N-terminal coding sequence.…”