The common retroviral insertion locus Dsi1 maps 30 kilobases upstream of the P1 promoter of the murine Runx3/Cbfa3/Aml2 gene

Abstract

The Dsi1 locus was identified as a common integration site for Moloney murine leukemia virus (MLV) in rat thymic lymphomas, but previous efforts to identify a gene affected by these insertions were unsuccessful. We considered the Runx3 gene a potential candidate on the basis of genetic mapping which showed that Dsi1 and Runx3 are closely linked on mouse chromosome 4 and the precedent of the related Runx2 gene, which emerged recently as a Myc-collaborating gene activated by retroviral insertion in thymic lymphomas of CD2-MYC mice. We now report the physical mapping of the Dsi1 locus to a site 30 kb upstream of the distal (P1) promoter of the murine Runx3 gene. Comparison with the syntenic region of human chromosome 1 shows that the next gene is over 250 kb 5' to Runx3, suggesting that Runx3 may be the primary target of retroviral insertions at Dsi1. Screening of CD2-MYC lymphomas for rearrangements at Dsi1 revealed a tumor cell line harboring an MLV provirus at this locus, in the orientation opposite that of Runx3. Proviral insertion was associated with very high levels of expression of Runx3, with a preponderance of transcripts arising at the P1 promoter. These results confirm that Runx3 is a target of retroviral insertions at Dsi1 and indicate that Runx3 can act as an alternative to Runx2 as a Myc-collaborating gene in thymic lymphoma.

FIG. 1.

Genetic linkage map of murine chromosome 4, illustrating close proximity of the Dsi1 locus and Runx3 gene. Also indicated are additional genes and anchor markers mapped in the same region. Distances are in centimorgans from the centromere.

FIG. 2.

Comparative physical map of human RUNX3 and murine Runx3 genes. (A) Physical map of human chromosome 1 spanning 2.7 Mb around the RUNX3 gene, based on public database information (http://genome.ucsc.edu). All of the genes indicated are transcribed in the same orientation as RUNX3. (B) Genomic structure of the human RUNX3 gene. The locations of the distal P1 and proximal P2 promoter regions are indicated. Vertical arrow, P2-associated CpG island. (C) Physical map of murine Runx3 gene. PAC 448-L9 and PAC568-D12 were isolated from the murine RPC121 PAC library. ds1-SR and the Runx3 exon 1 and exon 5 probes are indicated. Only restriction sites mapped with the probes are shown. N* indicates the presence of multiple NotI sites and the inferred position of P2-associated CpG island from draft murine genome sequence (accession number AF169246). Expanded region shows the position of the integrated MLV in T1i. Restriction enzyme abbreviations: SI, SstI; SII, SstII; E, EagI; N, NotI; C, ClaI; R, EcoRI; X, XhoI; B, BamHI. (D) Southern analysis of MLV-infected CD2-MYC lymphomas. Genomic DNA (20 μg; T1i cell line and control [C] nontransgenic thymus) was digested with BamHI (B), SstI (SI), and XhoI (X) and hybridized with the ds1-SR probe. GL, germ line; R, rearranging fragment. HindIII-digested λ DNA was used as molecular size markers (sizes shown in kilobases).

FIG. 3.

Expression of murine Runx3 transcripts. (A) Schematic representation of Runx3 exon structure and derived RNA transcripts. Solid boxes represent exons 1 to 6, and open boxes indicate 5′ and 3′ untranslated sequences. Exon 1- and exon 5-specific probes are indicated by hatched and grey boxes, respectively. (B) Northern blot analysis of 20 μg of total RNA from T1i, T14i T29i, T48i, T82i, T85i, and EL4 cells and from control nontransgenic thymus (Thy) was performed using Runx3 probes, exon 1 (upper) and exon 5 (lower), and GAPDH to control for RNA loading. The blot was hybridized sequentially with the exon 1, exon 5, and GAPDH probes, with stripping between hybridizations. The exon 1 probe detects only those transcripts derived from the P1 promoter, whereas the exon 5 probe detects both P1- and P2-derived transcripts. (C) Graphic representation of expression levels of P1-specific transcripts normalized with respect to GAPDH expression.