Identification and characterization of the T-cell-specific enhancer of type B leukemogenic virus

Mouse mammary tumor virus (MMTV) is a retrovirus that causes mammary adenocarcinomas and T-cell lymphomas. T-cell lymphomas induced by MMTV have additional proviral integrations that invariably have alterations in the long terminal repeats (LTRs). Type B leukemogenic virus (TBLV) is a thymotropic strain of MMTV that induces rapidly appearing T-cell tumors in mice. TBLV is highly related to MMTV except that TBLV LTRs have a deletion of negative regulatory elements and a multimerization of sequences flanking the deletion. In this study, the multimerized sequences from the TBLV LTR were identified as a novel T-cell-specific enhancer that can act on the TBLV promoter as well as thymidine kinase and c-myc promoters. Substitution mutagenesis of the enhancer identified a critical region that contains binding sites for RUNX1, NFkB and two unknown factors, NF-A and NF-B. RUNX1, NF-A and NF-B all positively regulate TBLV enhancer activity. However, the role of NF-kB in enhancer function is unclear since it does not appear to contribute to the activity of the TBLV LTR in T cells. Additionally, expression of NF-kB antagonizes the positive effect of RUNX1 expression on the TBLV LTR in non-T cells. NF-A appears to be the major contributor to enhancer function and is a lymphoidrestricted factor. The TBLV enhancer also contains functional glucocorticoid receptor (GR) binding sites; however GR binding is not necessary for enhancer activity in T cells. A c-Myb binding site overlaps the GR binding site and expression of c-Myb in non-T cells activates transcription from the LTR. Characterization of TBLV enhancers in proviruses integrated near c-myc revealed that the number of enhancer elements varied, but the most clonal tumors had proviruses containing four enhancer elements. Reporter gene assays showed that LTRs containing four enhancer elements were less effective at activating transcription from either the TBLV or c-myc promoters. In vivo passage of tumor cells in immunocompetent mice revealed a selection for proviral integrations near c-myc with four enhancer elements. Since c-myc overexpression often leads to apoptosis, these results suggested that selection for clonal growth occurred in tumor cells that had modest c-myc overexpression after TBLV insertion to prevent apoptosis.