Leukemia (1997) 11, 619–623  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

REVIEW Fre2, a proviral integration site of Friend murine leukemia that is closely linked to Fv2 RW Friedrich1, M Veit1, D Eisel1, U Friedrich1, M Pass1,2 and CA Kozak3

1Institute of Medical Virology, Justus Liebig University, Giessen, Germany; and 3Laboratory ot Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Friend (F-MuLV) induces leukemia by Hoatlin et al8 suggested that the Fv2 product can interact integration into the cellular , thereby changing the with the erythropoietin receptor which plays an important role structure or expression of cellular oncogenes. In this report we describe a new F-MuLV integration site Fre2 isolated from in SFFV-induced erythroleukemia, thereby inducing F-SFFV splenic DNA of an erythroleukemic animal. This site was found resistance. to be rearranged in six out of 64 tumors tested; however, in F-MuLV proviruses have been found integrated in Fli1,a five out of these six cases no F-MuLV proviruses could be member of the ets gene family, in at least 90% of all primary detected in the vicinity of the rearrangement sites. The spleen tumors of erythroleukemic animals9 and in erythroid rearrangements represented closely clustered chromosomal cell lines.10 Several additional integration sites in erythroid or breakpoints, presumably chromosomal translocations. Exons myeloid leukemias have been described, including Fli2, Fim1, transcribed into differentially spliced mRNAs of 1.9 and 3.7 kb 11–16 have been found near the breakpoint. Fre2 is closely linked to Fim2/c-fms, Fim3/CB1/Evi1, Fis1, Pim1, c-myc and p53. Fv2, a locus on mouse chromosome 9 involved in erythropoi- In our own studies, it became clear that additional inte- esis. Sequences homologous to Fre2 could not be found in the gration sites for F-MuLV exist in spleen tumors of erythroleu- gene databases. kemic animals. In this article, we describe a new site, Fre2, Keywords: Friend murine leukemia virus; Fre2; Fv2; proviral closely linked to Fv2, with characteristics not seen in other integration site; erythroleukemia retroviral integration sites.

Introduction Results and discussion

Friend murine leukemia virus (F-MuLV) is a nondefective A genomic library in phage ␭ was derived from DNA of a (ecotropic) which can induce a variety of hemato- spleen tumor (tumor No. 25) in a BALB/c mouse. The F-MuLV logic neoplasms, including erythro- and myeloblastic leuke- which had been used for infection contained a bacterial supF mia when injected into newborn mice of appropriate strains. gene as a selectable marker.17 From positive ␭ clones, Virally induced erythroleukemia is a multistage disease, sequences flanking supF-containing proviruses were isolated beginning with an early hemolytic anemia, partially compen- and used as probes for DNA rearrangements in Southern blots sated for by reactive erythropoiesis. Later, the animals become of splenic DNA from erythroleukemic mice. Sites that were severely anemic and show grossly enlarged spleens containing found rearranged in more than one of the tumors were called large numbers of transformed cells that do not differentiate Fre (Friend erythroleukemia; to be published elsewhere). Sev- into erythrocytes. This condition leads to the death of the ani- 1 eral such clones were mapped on mouse chromosomes using mals at an age of about 2–3 months. single copy genomic DNA flanking the viral inserts to identify F-MuLV can also act as a helper virus for the replication- RFLPs (M Pass, FT Sels, A Schulz, D Eisel, U Friedrich, C defective Friend spleen focus-forming virus, which causes Kozak and RW Friedrich, manuscript in preparation). Fre2 was either acute anemia (strain SFFVA) or polycythemia (strain initially mapped to distal chromosome 9 by analysis of an SFFVP) in susceptible newborn or adult mice 3 to 4 weeks intersubspecies cross. Because results suggested close proxim- 2 after infection. Some mouse strains, however, are resistant to ity of Fre2 and the resistance gene, Fv2, we ana- SFFV-induced disease. Several have been held respon- lyzed a second interspecies cross: (NFS/N × M. spretus) × M. sible for Friend virus resistance, including Fv1 to Fv6 and Rfv1 spretus or C58/J.18 This cross could be typed for Fv2 since to Rfv3 (for reviews, see Refs 3–5). Fv2 is of special interest both C58/J and M. spretus carry the recessive resistance allele. in studies of hematopoiesis since it seems to be involved in In this cross, no recombinants were identified in 91 mice the regulation of early erythropoiesis. It was discovered by 6 typed for Fre2 and Fv2 indicating that, at the upper limit of Lilly and described in more detail by Axelrad and coworkers the 95% confidence level, these two genes are no more than (see Ref. 3 for references) as a locus responsible for the resist- 3.2 centimorgans apart. ance of some mouse strains to Friend-SFFV induced disease The F-MuLV flanking sequence isolated from DNA of tumor (allele Fv2r is assumed to be involved in the control of DNA 7 No. 25 was used to screen a genomic library of normal replication associated with early erythropoiesis ). Recently, BALB/c DNA in phage ␭. One such clone (25.5-D) which con- tained a 16.4 kb insert was sequenced. A diagram of the gen- omic structure of this region is shown in Figure 1. The position Correspondence: R Friedrich, Institute of Medical Virology, Frank- of the proviral insert in the DNA of tumor 25 was determined furter Str. 107, D-35392 Giessen, Germany 2Present address: Dept. f. Innere Medizin, Universita¨tsspital Zu¨rich, by PCR and is indicated in this Figure. Ha¨lderliweg 4, CH-8044 Zu¨ rich The flanking sequence (designated probe A in Figure 1) and Received 25 September 1996; accepted 18 January 1997 a sequence derived from the ␭ insert (probe B) were used to F-MuLV integration site Fre2 RW Friedrich et al 620

Figure 1 Genomic structure of Fre2. A genomic library of normal BALB/c spleen DNA in phage ␭ was screened with probe A originally isolated from tumor 25. The restriction enzymes shown were derived after sequencing the insert of one such clone designated 25.5-D. The diagram shows the position of proviral DNA found in tumor 25 and the position of the rearrangements found in tumors 3, 24, 29, 32 and 39. The location of probes used for Southern blot analysis is shown. The lower part of the diagram represents the structure of two exons as derived from the analysis of cDNA clones. Potential initiation and termination codons as well as a potential signal are also given.

screen 63 spleen tumors from mice infected with F-MuLV as only partially to these fragments. This led us to the tentative newborns for DNA rearrangements. By this procedure, five localization of the rearrangement site as shown in Figure 1. additional tumors were found which showed rearranged Fre2 We tried to localize proviral sequences in the vicinity of the DNAs. A Southern blot of tumor DNAs cut with either EcoRI rearrangement sites in tumors 3, 24, 29, 32 and 39 by or EcoRV and hybridized with probe B (see Figure 1) revealed hybridization of viral probes (derived from LTR or that restriction enzyme fragments of DNAs from all tumors sequences) to Southern blots similar to those shown in Figure were of similar sizes (Figure 2). The amount of DNA 2 as well as by PCR with oligonucleotides derived from viral rearranged varies among the tumors. As can be seen from Fig- and cellular sequences. All attempts to find proviral sequences ure 2, only a small fraction of tumor 32 seems to contain in this region were unsuccessful. All tumor DNAs, however, rearranged DNA since most of the hybridizing DNA fragments contained F-MuLV proviruses integrated near Fli1 as expected migrate with the restriction fragments from the control DNA. from our earlier results9 (data not shown). These results sug- In contrast, the intensities of the wild-type EcoRI and EcoRV gest that the observed rearrangement found in these tumors restriction fragments obtained from tumors 29 and 39 (10.4 represents a chromosomal translocation, either a gross and 12.3 kb, respectively) are similar (tumor 39) or even lower rearrangement in the same chromosome or a recombination than the 6 kb EcoRI and EcoRV fragments indicative of the with another chromosome. Fre2 rearrangement. In tumor 3, only little DNA remains at The size of the restriction fragments corresponding to the the position of the wild-type fragment; this could, at least in rearranged DNA is surprisingly similar in all tumors that are part, be due to the degradation of this DNA. Since DNA of positive for this rearrangement. This suggests a rather precise tumor 29 shows only minor degradation, the stronger intensity recombination not only in Fre2 (chromosome 9) but also in of the 6 kb band suggests that either both alleles of Fre2 are the (still unknown) recombination partner. rearranged in most of the tumor cells or that one allele is To localize possible coding regions within the ␭ insert, the deleted. whole insert was hybridized to Northern blots of poly(A+) RNA This was also obvious using restriction enzymes such as from normal mouse tissue. Two RNA species of 1.9 and 3.7 kb HindIII and PstI which generate smaller fragments than those could be found in all mammalian tissues and cell lines tested produced by EcoRI or EcoRV (Figure 3). Smaller fragments (Figure 4). Southern blot hybridizations with probes from vari- than expected of 480 and 560 bp were found in HindIII ous regions of the Fre2 locus did not show cross hybridization digests of tumors 3, 29 and 39. Fragments of 440 and 530 bp to any additional restriction fragments. Since, however, all could be seen in PstI digests of DNAs from the same tumors. Northern blots revealed two RNA species, these two Since only a small fraction of tumor 32 contains DNA must represent differentially spliced mRNAs of the same gene. rearrangements in Fre2, the small HindIII and PstI fragments As presented elsewhere, the expression level varied in differ- can barely be seen. The low intensity of these small fragments ent tissues and was highest in testes and lowest in brain in the Southern blots furthermore suggests that probe C bound tissue.31 As seen in Northern blots shown in Figure 4, the rela- F-MuLV integration site Fre2 RW Friedrich et al 621 tion or affiliation with a specific gene family could be found. Preliminary data suggest that the small Fre2 mRNA consists essentially of the large exon, whereas the large mRNA of 3.7 kb carries additional sequences at its 3Ј and/or 5Ј end. The provirus integration found in only a single tumor in this site and the low number of only five Fre2 rearrangements in 63 additional tumors without concomitant viral integration in the vicinity of the breakpoint demonstrates that this site is not required for erythroleukemia. It could rather fulfill an auxiliary function as it was described by Bear et al21 for the tumor pro- gression loci or as has been described for a number of loci identified in human tumors (see Refs 22 and 23 for examples). To our knowledge, this is the first observation of a virally induced tumor undergoing specific cellular DNA rearrange- ment without retroviral integration at the rearrangement site. However, it has been found in some cases that oncogenes can either be activated by integration of or by chromo- somal translocations. Myc is an example of an oncogene that can be disrupted by proviruses in some tumors and can be rearranged in other tumors not caused by a retrovirus (reviewed in Ref. 24). Fli1, originally found by analysis of inte- gration sites of F-MuLV,9,10,25 commonly shows chromosomal translocations in Ewing’s sarcoma and in human primitive neuroectodermal tumors, neither of which are known to be caused by .26 Both, the close linkage of Fre2 to Fv2 and the observation that both loci seem to be involved in virally induced erythro- leukemia, is intriguing. However, additional studies will be necessary to determine whether Fre2 and Fv2 are identical. A Lys/Ala domain (55% lysine and alanine within 65 amino acids) present in the large exon (Figure 1) is reminiscent of similar regions found in histones and some other nuclear pro- teins where such domains are involved in DNA binding.27 No Figure 2 DNA rearrangements in spleen tumors of erythroleu- direct evidence for the subcellular localization or function of kemic F-MuLV-infected mice. Southern blots of DNAs from several Fre2 is yet available, however. spleen tumors as well as DNA of normal control tissue after digestion To understand the biological function of the rearrangement either with EcoRI or EcoRV. The blots were hybridized with probe B of Fre2 in some erythroleukemias it will be necessary to ident- (see Figure 1). Arrows denote fragment lengths calculated from the ify also the chromosomal partner and the second gene position of marker DNA. Southern blots of DNA from tumors 3, 24, 29, 32 and 39 show rearrangements, whereas DNA from tumor 123 involved in the Fre2 translocations. To this end, we are and from control tissue (lane C) show only the normal allele. presently cloning the breakpoint in Fre2, an effort that is seri- ously hampered by the presence of large regions of repetitive sequences near the breakpoint. tive amount of the two different mRNAs is similar in hemato- To the best of our knowledge, an association of specific poietic mammalian cells (such as the erythroleukemia cell line rearrangements with erythroleukemias has not been demon- HCD57,19 the lymphoid S49 cells, the myeloid cells SP2/0, or strated in mice or men. No rearrangements in region 3p21 of the hybridoma KT3) or in fibroblasts (such as 3T3), and is not the human genome that is homologous to the region of the affected by the presence (HCD57, 3T3 + FS, 113, 117, 153) or mouse chromosome where Fre2/Fv2 are located could be absence of Friend-MuLV (3T3, S49, SP2/0, KT3). Quantitative found in the rare cases of human erythroleukemias. It remains determination of Fre2 mRNA, however, shows that all cell to be determined whether the rearrangements that have been lines of hematopoietic origin contain significantly more Fre2 found in this region (see Refs 28–30 for examples) involve mRNA than 3T3 fibroblasts (Figure 5). Whether this also holds homologous human genes. true for other cells of non-hematopoietic origin remains to be determined. The Figure also shows that the human cell line HepG2,20 originating from a human hepatoma, also expresses mRNAs of the same size, whereas no mRNA of this size could be detected with our probe in Sf9 insect cells. The ␭ insert was also used to screen cDNA libraries for Acknowledgements clones positive for Fre2. Several clones were found and sequenced (Figure 1). So far, two open reading frames have been found in which the derived amino acid sequence did We greatly acknowledge the expert technical assistance of not show any sequence homology to other sequences in the Karin Schultheiss, Irmi Faber-Franek, and M Charlene Adam- data banks. The putative contains a high number of son, and thank Bruce Boschek for help with the manuscript. lysine and alanine residues coded for by a region in the C- This work was supported by a grant from the Deutsche For- terminal half of the large exon (see Figure 1). No other schungsgemeinschaft (Fr 432/5). sequence motifs that might provide a hint of a possible func- F-MuLV integration site Fre2 RW Friedrich et al 622

Figure 3 Similar restriction fragment sizes in all positive spleen tumors. DNAs from a negative tumor (110) as well as from control tissue (C) and DNAs from tumors 3, 29, 32 and 39 were digested with restriction enzymes producing rather small fragments.

Figure 5 Quantitative determination of Fre2 mRNAs in various cell lines. Expression rate is given in percent of ␤-actin expression. Figure 4 Expression of two different Fre2 mRNAs in various cell RNA was filtered in a dot blot apparatus onto a membrane and lines. Ten micrograms total RNA isolated from normal and tumor cell hybridized with the Fre2 cDNA probe described in Figure 4. An ident- lines were subjected to electrophoresis on a 1.5% agarose gel contain- ical dot blot was hybridized with the ␤-actin probe and the intensities 32 ing 0.4 M formaldehyde. After blotting, P-labeled cDNA rep- of the spots, detected by phosphoimaging, were compared using 32 ␤ resenting the large exon (Figure 1) and a P-labeled -actin probe imaging software. The data represent results of three independent were mixed and used for hybridization. Cell lines used: HCD57 experiments. 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