The U3 region of MCF 247 contains multiple DNA elements predicted to bind Ikaros, a lymphoid-cell-restricted transcriptional regulator. Here, we provide evidence that Ikaros binds to Ikaros-binding sites present in the DR sequences and in sequences proximal to the promoter (Fig. , sites 1 to 5). Moreover, we provide several pieces of evidence that these sites are functional. First, ectopic expression of Ikaros in murine cells that do not normally express this protein elevated the activity of a reporter containing wild-type U3 sequences. Consistent with this, U3 sequences with site-directed mutations in multiple Ikaros-binding sites are transcribed less efficiently than wild-type sequences in T cells that express endogenous Ikaros. In addition, one Ikaros-binding site (site 3; Fig. ), when mutated alone, reduced transcription from the MCF promoter significantly. These studies indicate that Ikaros-binding sites, along with other DNA elements in U3, functionally regulate the virus promoter. Additional evidence demonstrating that the Ikaros-binding sites in U3 are functional is provided by studies with a variant MCF virus containing a point mutation in the third Ikaros-binding site. This variant virus replicated less efficiently than the wild-type virus in thymocytes, suggesting that the mutation in the third Ikaros-binding site reduces transcription from integrated MCF proviruses in vivo. Last, mice infected with this variant MCF virus developed frank leukemia with a delayed onset relative to mice infected with the wild-type virus. We conclude that the Ikaros-binding sites in the U3 sequences of integrated proviruses are functional and contribute to the expression of cellular loci important for T-cell lymphoma induction.
To our knowledge, this is the first demonstration that Ikaros binds predicted Ikaros-binding sites in the U3 sequences of a retrovirus. This was documented by EMSA and competition assays using U3 probes with wild-type and mutant Ikaros-binding sites, as well as specific antibodies. We note that the U3 regions from several human retroviruses and lentiviruses (human T-cell leukemia virus type 1 and human immunodeficiency virus type 1, respectively), other MCF viruses (Moloney, Friend, Rauscher, and MCF 1233 viruses), as well as Cas-Br-E, SL3H, Moloney, and Friend MuLVs, contain predicted Ikaros-binding sites in their U3 regions (43
). In fact, several probes used in this study are identical with the U3 sequences from other MuLVs, including those of an endogenous xenotropic virus (Bxv-1) and other polytropic viruses isolated from lymphomas in AKR/J mice (MCF13, MCFr35, MCF 30-2, and Tikaut) (1
). It is therefore likely that Ikaros binds predicted Ikaros-binding sites in the regulatory sequences of other retroviruses, in addition to the binding observed in MCF 247.
The functional significance of the Ikaros-binding sites in the U3 region of MCF 247 was first tested by transient-gene expression assays. Here, ectopic expression of Ikaros in murine cells that do not normally express this protein increased the activity of a reporter gene under the control of native MCF U3 sequences. Similar studies with T cells (that express endogenous Ikaros) demonstrated that the five Ikaros-binding sites were not equal and that the third site is a key Ikaros-binding site in the U3 region of MCF 247.
Studies with a variant MCF virus containing a point mutation in the third Ikaros-binding site verified that this DNA element plays a role in enhancer function in vivo. Since virion proteins are synthesized from viral mRNAs transcribed from integrated proviral DNAs, the expression of a virus-specific protein (MCF gp70) on thymocytes was used as an indicator of transcription from the integrated provirus and virus replication in vivo. At 5 and 7 weeks p.i., MCF gp70 was less abundant on thymocytes from mice injected with the variant MCF virus than on those from mice injected with wild-type virus; by 10 weeks p.i., the difference in MCF gp70 abundance on thymocytes from these two groups of mice was less apparent. We recognize that viral recombination events, or rearrangements, might have generated viral genomes with an MCF env
sequence and U3 sequences different than those in the injected virus. In fact, we previously documented recombination between an injected MuLV and the endogenous viral sequences carrying the MCF env
). However, this recombinant virus was first detected at 14 weeks p.i. Thus, although formally possible, it is unlikely that a recombinant virus having U3 sequences different from that of the injected virus contributes to the expression of MCF gp70 at early times (5 to 7 weeks) after inoculation of the variant MCF virus. The low levels of MCF gp70 on thymocytes from mice inoculated with the variant virus are consistent with the conclusion that the mutation in the third Ikaros-binding site present hindered virus replication in vivo. This same mutation delayed (by 34 days) the induction of lymphoma by the variant MCF virus. These data provide the first evidence that a mutation in an Ikaros-binding site diminishes the pathogenic potential of an MuLV. Since Ikaros-binding sites are predicted in the U3 sequences of other retroviruses, we anticipate that Ikaros or other Ikaros family members bind these DNA elements and contribute to the enhancer activities of these retroviruses.
Reduced levels of virus replication and the delayed onset and slightly delayed time course of lymphoma induction by the variant MCF virus may reflect the time needed to select an MCF virus that replicates in thymocytes more efficiently than the variant virus. This virus, if generated, may contain a reversion of the point mutation in the third Ikaros-binding site, rearrangements, or other compensatory mutations in the U3 sequences. Our preliminary studies of the U3 sequences expressed in tumors induced by the variant MCF virus indicate that second-site mutations occur more frequently than the reversion of the mutation in site 3 (N. L. DiFronzo, J. O. Benson, and Q. N. Pham, unpublished data). Reversions and second-site mutations in viruses evolving from MuLVs engineered with point mutations in sequences that bind AML-1 (also known as core binding factor) and nuclear factor 1 have been documented; these reversions and second-site mutations frequently increase transcriptional activity of the LTR and increase virus-induced lymphomagenesis (19
). We are currently investigating whether the U3 sequences of viruses expressed in tumors induced by our variant MCF virus evolved in vivo and whether they increase transcription from MCF U3 sequences.
Ikaros-binding sites are present in the regulatory sequences of several lymphoid-cell-restricted genes, including loci encoding CD2, CD4, CD8-α, the interleukin-2 (IL-2) enhancer, IL-2 receptor α, beta interferon, major histocompatibility complex class II, as well as subunits of the T-cell receptor (α, β, and δ) and CD3 (δ, γδ, and
); however, the functional relevance of these Ikaros-binding sites has not been determined experimentally. In this report, we demonstrate that Ikaros-binding sites in the U3 region of an MCF MuLV are functional in T cells. Mutation of one of these sites, in the U3 region of a replication-competent MCF virus, reduced virus replication in vivo and delayed the onset of lymphoma induction. We conclude that this Ikaros-binding site, in the genetic context of its native enhancer and promoter sequences, regulates expression of viral sequences and cellular loci important for transformation.
Ikaros is a hematopoietic-cell-restricted transcriptional regulator required for lymphoid lineage commitment (23
). The molecular mechanism by which Ikaros dictates this process is largely unclear but is most likely related to the ability of Ikaros to either activate or repress transcription of lymphoid-cell-specific cellular loci (12
). These two activities depend on the genetic context of the Ikaros-binding sites, transcription factor-binding sites adjacent to the Ikaros-binding sites, and the cellular proteins physically associated with Ikaros (5
). It is possible that these two factors contribute to the differential effects of mutant Ikaros-binding sites on transcriptional activity from the viral promoter. At the present time, the array of protein binding sites in the MCF 247 U3 sequences that regulate virus replication in thymocytes and lymphoma induction have not been determined. Further studies are necessary to identify these protein-binding sites and to identify proteins that bind U3 sequences and interact with Ikaros to more fully elucidate molecular mechanisms regulating the LTR of integrated proviruses.