Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E mu-myc transgenic mice

To search for genes that can collaborate with myc in lymphomagenesis, we exploited retroviral insertional mutagenesis in E mu-myc transgenic mice. Moloney murine leukemia virus accelerated development of B lymphoid tumors. Three quarters contained a provirus within the known pim-1 or pim-2 loci, new loci bmi-1 and emi-1, or combinations of these. bmi-1 insertions predominated, occurring in half the tumors, and resulted in elevated bmi-1 mRNA levels. Significantly, the bmi-1 gene, which is expressed in diverse normal cells, encodes a Cys/His metal-binding motif (C3HC4) that resembles those in several DNA-binding proteins and defines a new category of zinc finger gene. Thus, myc-induced lymphomagenesis can entail the concerted action of several genes, including the presumptive nuclear regulator bmi-1.     

Long-range mapping of Mis-2, a common provirus integration site identified in murine leukemia virus-induced thymomas and located 160 kilobase pairs downstream of Myb.

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.     

Nucleotide sequence of bup, an upstream gene in the bmi-1 proviral insertion locus.

The ability of Moloney murine leukemia virus to accelerate lymphomagenesis in E mu-myc transgenic mice is frequently associated with proviral integration within a locus denoted bmi-1. This locus contains not only the bmi-1 gene implicated as a collaborator with myc in lymphomagenesis but also just upstream an unknown gene denoted bup. The nucleotide sequence reported here for bup cDNA and flanking genomic sequences reveals that this widely expressed gene comprises at least 7 exons and potentially encodes a polypeptide of 195 amino acid residues. Computer searches with this polypeptide sequence revealed no close homolog in the databases, nor any conserved motifs, and it is unrelated to the product of the mel-13 gene, which lies just upstream from the bmi-1 homolog mel-18.     

Frequent disruption of the Nf1 gene by a novel murine AIDS virus-related provirus in BXH-2 murine myeloid lymphomas.

Evi-2, a common site of viral integration in BXH-2 myeloid lymphomas, is located within a large intron of the Nf1 tumor suppressor gene. Viral integration at Evi-2 appears to induce disease by disrupting normal Nf1 expression. During our attempts to characterize the nature of the proviruses located at Evi-2, we found that approximately half of the proviruses were defective nonecotropic proviruses (A. M. Buchberg, H. G. Bedigian, N. A. Jenkins, and N. G. Copeland, Mol. Cell. Biol. 10:4658-4666, 1990). This was surprising, since most proviruses characterized at other BXH-2 common integration sites are full-length ecotropic viruses. In the studies described here, we found that this defective provirus carries two large deletions, one in pol and one in env, and is structurally related to another murine retrovirus, the murine AIDS retrovirus. By using oligonucleotide probes specific for this defective provirus, designated MRV, we showed that MRV-related proviruses are carried as endogenous germ line proviruses in most inbred strains. In addition, we identified the endogenous MRV provirus that gives rise to the defective proviruses identified at Evi-2. We present a model that accounts for the positive selection of MRV proviruses at Evi-2, which may allow selective identification of common viral integration sites harboring tumor suppressor genes.     

Preferential selection of human T-cell leukemia virus type 1 provirus lacking the 5' long terminal repeat during oncogenesis

In adult T-cell leukemia (ATL) cells, a defective human T-cell leukemia virus type 1 (HTLV-1) provirus lacking the 5' long terminal repeat (LTR), designated type 2 defective provirus, is frequently observed. To investigate the mechanism underlying the generation of the defective provirus, we sequenced HTLV-1 provirus integration sites from cases of ATL. In HTLV-1 proviruses retaining both LTRs, 6-bp repeat sequences were adjacent to the 5' and 3' LTRs. In 8 of 12 cases with type 2 defective provirus, 6-bp repeats were identified at both ends. In five of these cases, a short repeat was bound to CA dinucleotides of the pol and env genes at the 5' end, suggesting that these type 2 defective proviruses were formed before integration. In four cases lacking the 6-bp repeat, short (6- to 26-bp) deletions in the host genome were identified, indicating that these defective proviruses were generated after integration. Quantification indicated frequencies of type 2 defective provirus of less than 3.9% for two carriers, which are much lower than those seen for ATL cases (27.8%). In type 2 defective proviruses, the second exons of the tax, rex, and p30 genes were frequently deleted, leaving Tax unable to activate NF-kappaB and CREB pathways. The HTLV-1 bZIP factor gene, located on the minus strand, is expressed in ATL cells with this defective provirus, and its coding sequences are intact, suggesting its significance in oncogenesis.     

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes.

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue-specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto-oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non-acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto-oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting E mu-pim-1 and E mu-myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of a novel proto-oncogene, Frat1, contributes to progression of mouse T-cell lymphomas.

Acceleration of lymphomagenesis in oncogene-bearing transgenic mice by slow-transforming retroviruses has proven a valuable tool in identifying cooperating oncogenes. We have modified this protocol to search for genes that can collaborate effectively with the transgene in later stages of tumor development. Propagation of tumors induced by Moloney murine leukemia virus (M-MuLV) in E mu-Pim1 or H2-K-myc transgenic mice by transplantation to syngeneic hosts permitted proviral tagging of 'progression' genes. Molecular cloning of common proviral insertion sites that were detected preferentially in transplanted tumors led to the identification of a novel gene, designated Frat1. The initial selection for integrations near Frat1 occurs in primary tumor cells that have already acquired proviruses in other common insertion sites, yielding primary lymphomas that contain only a minor fraction of tumor cells with an activated Frat1 allele. Transplantation of such primary lymphomas allows for a further expansion of tumor cell clones carrying a proviral insertion near Frat1, resulting in detectable Frat1 rearrangements in 17% of the transplanted E mu-Pim1 tumors and 30% of the transplanted H2-K-myc tumors, respectively. We have cloned and sequenced both the mouse Frat1 gene and its human counterpart. The proteins encoded by Frat1 and FRAT1 are highly homologous and their functions are thus far unknown. Tumor cell lines with high expression of Myc and Pim1 acquired an additional selective advantage in vivo upon infection with a Frat1-IRES-lacZ retrovirus, thus underscoring the role of Frat1 in tumor progression, and the ability of Frat1 to collaborate with Pim1 and Myc in lymphomagenesis.     

Evidence for the Asiatic origin of endogenous AKR-type murine leukemia proviruses.

A restriction endonuclease cleavage map of the genome of AKV, the endogenous, ecotropic leukemia virus of AKR mice, has been derived. By using this map and analyzing DNA from congenic mice, we have defined four DNA fragments diagnostic for AKV proviruses. Analysis of DNAs from 10 strains of American laboratory mice revealed that all strains carrying inducible, ecotropic murine leukemia viruses yielded DNAs which contained the four DNA fragments diagnostic for AKV. Virus-negative strains lacked these fragments in their DNA. Screening DNA from 23 additional mice revealed that, among these mice, only mice from Asia gave rise to the DNA fragments diagnostic of an AKV provirus. We conclude that all of the endogenous ecotropic murine leukemia proviruses in American laboratory mice are closely related since they share a common set of restriction endonuclease cleavage sites. These proviruses appear to derive from the East Asian ancestors of these mouse strains. Analysis of DNA from six selected mice with an additional restriction endonuclease showed that greater than 97% of the nucleotide sequences in each provirus are contigous and that these endogenous proviruses are indistinguishable from proviruses introduced by exogenous infection.     

Massive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites during Primary Infection

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.     

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.     

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.     

Immunologic competence of B cells subjected to constitutive c-myc oncogene expression in immunoglobulin heavy chain enhancer myc transgenic mice

B cell activation is associated with a marked transient rise in expression of the c-myc proto-onco-gene. A unique opportunity to examine the effects of constitutive c-myc expression upon B cell function is provided by transgenic mice in which the c-myc oncogene is regulated by the enhancer (E mu) from the immunoglobulin locus (E mu-myc mice). We have examined the immunologic competence of B cells from E mu-myc mice both in vitro and in vivo. Upon stimulation, many E mu-myc B cells can proliferate to form clones most of which contain antibody-forming cells. However, the frequency of responsive B cells from E mu-myc donors is only about 30% that of B cells from normal littermates. Thus, enforced myc expression is not sufficient to block the differentiation of all B cells, but a much larger fraction of the immunoglobulin-bearing cells from E mu-myc mice are incompetent. Upon immunization, E mu-myc mice mounted specific antibody responses, although some responses were delayed. Isotype switching can occur, since we observed hemolytic plaques of both IgM and IgG type and detected specific antibody of both classes in the serum. Moreover, the serum from nonimmunized E mu-myc mice contained normal levels of both IgM and IgG. Thus constitutive expression of the c-myc gene appears to retard B cell differentiation, but does not grossly impair immunologic function in the intact animal.