TRANSCRIPTIONAL ACTIVATION OF A RETROVIRUS ENHANCER BY CBF (AML1) REQUIRES A 2ND FACTOR - EVIDENCE FOR COOPERATIVITY WITH C-MYB

Transcriptional enhancer sequences within the long terminal repeats (L TRs) of murine leukemia viruses are the primary genetic determinants o f the tissue specificity and potency of the oncogenic potential of the se retroviruses. SL3-3 (SL3) is a murine leukemia virus that induces T -cell lymphomas. The LTR enhancer of this virus contains two binding s ites for the transcription factor CBF (also called AML1 and PEBP2) tha t flank binding sites for c-Myb and the Ets family of factors. Using c otransfection assays in P19 cells, we report here that CBF and c-Myb c ooperatively stimulate transcription from the SL3 LTR. By itself, c-My b had no stimulatory effect on transcription. However, when cotransfec ted with a cDNA encoding one form of the alpha subunit of CBF called C BF alpha 2-451, a level of transactivation higher than that seen with CBF alpha 2-451 alone was detected. The negative regulatory domain nea r the carboxyl terminus of c-Myb did not affect this activity. Electro phoretic mobility shift assays indicated that CBF and c-Myb bind to DN A independently. Therefore, it appears that the cooperative stimulatio n of transcription by these factors occurs at a step in the process of transcription after the two factors are bound to the enhancer. Sequen ces near the carboxyl terminus of CBF alpha 2-451 were important for c ooperativity with c-Myb, consistent with previous reports that this re gion contains an activation domain. However, CBF alpha 2-451 failed to activate transcription from a version of the SL3 LTR in which the enh ancer was replaced with five tandem CBF-binding sites. Thus, it appear s that transcriptional activation of the SL3 enhancer by CBF requires that an appropriate heterologous transcription factor be bound to a ne ighboring site in the regulatory sequences.