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. 2008;3(11):e3823.
doi: 10.1371/journal.pone.0003823. Epub 2008 Nov 27.

The Zinc Finger SET Domain Gene Prdm14 Is Overexpressed in Lymphoblastic Lymphomas With Retroviral Insertions at Evi32

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Free PMC article

The Zinc Finger SET Domain Gene Prdm14 Is Overexpressed in Lymphoblastic Lymphomas With Retroviral Insertions at Evi32

E J Dettman et al. PLoS One. .
Free PMC article

Abstract

Background: AKXD recombinant inbred strains of mice have proven to be very useful in the identification of potential oncogenes and tumor suppressors involved in the development of lymphoid and myeloid malignancies. In these tumors, the hematopoietic insertion of an active AKV murine leukemia virus (MuLV) is associated with the onset of disease. Common sites of retroviral insertion (CIS) identify genes causally associated with the development or initiation of lymphoma.

Methodology: In the present study, we analyzed a previously uncharacterized CIS, Ecotropic Viral Integration Site 32 (Evi32), which is located on mouse chromosome 1. We analyzed candidate genes in the region to identify those involved in Evi32 mediated oncogenesis.

Results: Here we show that proviral insertion at Evi32 correlates with significant overexpression of a putative transcription factor, PR-domain containing 14 (Prdm14). Tumors with insertions at Evi32 are consistently lymphoid in nature. Prdm14 is normally expressed early in embryonic development with the highest expression in undifferentiated embryonic stem (ES) cells. This study implicates Prdm14 as a proto-oncogene involved in lymphoblastic lymphoma formation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Evi32 Viral Insertion Sites.
1a) The genomic locus surrounding Evi32 insertions. Locations of proviral insertions were determined by either VISA PCR or by PCR after identification of Evi32 integrations. Arrows indicate direction of transcription and the orientation of the retroviral integration. The exact orientation and location of the retroviral integration in animal 18-077 was not determined, but it is contained between the HindIII and XbaI digestion sites as determined by Southern blot analysis. Table at top right indicates the distance of each retrovirus from the first exon of Prdm14. 1b) Closeup view of Evi32 integrations. Restriction enzymes indicated were those used in digestion of tumor DNA to show genomic rearrangements at Evi32. Arrow indicates direction of transcription of Prdm14, and the black boxes show the eight exons of Prdm14. Prdm14 gene structure is based on VEGA prediction (OTTMUSG00000017097) obtained from the Ensembl database. RT-PCR amplification indicates that the VEGA prediction is a true Prdm14 transcript. 1c) Southern blots showing retroviral integrations at Evi32. Genomic DNA from indicated tumors was digested with KpnI. Brain serves as somatic tissue control from same animal to show rearrangement of Evi32 locus within the tumor tissue only. Rearranged bands seen in Evi32 tumors are indicated by asterisks and show genomic rearrangements at Evi32. BR = Brain, SP = Spleen, LN = Lymph Node.
Figure 2
Figure 2. Tif2 and Slco5a1 are not misexpressed in Evi32 tumors.
2a) Q RT-PCR was performed on cDNA prepared from total RNA samples from tumors with and without insertions at Evi32 and control tissue obtained from 8 week old C57BL/6J mice. Expression of Slco5a1 was normalized to 18S expression levels and then quantified. We found that the expression of Slco5a1 does not appear to be consistently altered with Evi32 proviral insertions and is expressed at lower levels than seen in normal spleen, thymus, and bone marrow. Expression levels are shown on a log scale to show lower levels of expression. 2b) Expression of Tif2 was quantified in the same manner as Slco5a1. This analysis revealed that Tif2 is expressed at similar or lower levels than normal spleen, thymus, or bone marrow in all Evi32 tumors. Expression levels are shown on a log scale to show lower levels of expression. 2c) Northern blot analysis of Tif2 was performed using total RNA obtained from Evi32 tumor 27-186 and control tissue obtained from 8 week old C57BL/6J mice. The probe to Tif2 was directed towards common exons found in all isoforms. Sizes were estimated using 18S and 26S migration distances. The 27-186 Evi32 tumor showed the greatest expression of Tif2 as determined by Q RT-PCR, but shows no increase in expression by northern analysis. SP = Spleen, LN = Lymph Node, Th = Thymus.
Figure 3
Figure 3. Prdm14 is overexpressed in Evi32 tumors.
3a) Q RT-PCR was performed on cDNA prepared from total RNA samples from AKXD tumors with and without insertions at Evi32 and control tissue obtained from 8 week old C57BL/6J mice. As before, expression levels were normalized to 18S expression then quantified. This analysis revealed that Prdm14 expression was upregulated in all Evi32 tumors. The expression levels of Prdm14 in Evi32 tumors varied between 30 and 50 fold more than in control spleen. 3b) Northern analysis of Prdm14 was performed using total RNA obtained from Evi32 tumors and control tissue obtained from 8 week old C57BL/6J mice. The probe to Prdm14 was nearly the full length coding sequence amplified by PCR. Sizes were estimated using 18S and 26S migration distances. This analysis shows specific upregulation of Prdm14 in all Evi32 tumors. 3c) RT-PCR was performed on Evi32 tumors using a viral 3′ LTR specific primer coupled with a Prdm14 second exon primer. cDNA obtained from the 27-001 produced a prominent band consistent with a viral fusion transcript. 3d) The amplicon from 27-001 was sequenced, and viral elements were found to be linked to the second exon of Prdm14 which contains the beginning of the coding sequence of Prdm14. SP = Spleen, LN = Lymph Node, Th = Thymus.
Figure 4
Figure 4. Prdm14 is expressed most highly in ES cells.
4a) Northern analysis of embryonic tissue compared with the Evi32 tumor 27-186. Gapdh was used as a loading/RNA integrity control. We found expression of the 2.7 kb band in all ES cell samples, but none in any of the other embryonic tissues. The larger 6.5 kb transcript seen in the Evi32 tumor is not found in the ES cells. 4b and c) Quantitative RT-PCR was performed on cDNA obtained from the same tissue from 4a. Prdm14 and Oct-3/4 expression levels were quantified relative to 18S expression levels within each tissue. Expression levels are on a log scale to emphasize lower levels of expression. Results show specific expression of Prdm14 within Oct-3/4 positive ES-cells. All embryonic days are whole embryos unless otherwise indicated. YS = yolk sac.

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