Transcriptional activation of a retrovirus enhancer by CBF (AML1) requires a second factor: evidence for cooperativity with c-Myb

Abstract

Transcriptional enhancer sequences within the long terminal repeats (LTRs) of murine leukemia viruses are the primary genetic determinants of the tissue specificity and potency of the oncogenic potential of these retroviruses. SL3-3 (SL3) is a murine leukemia virus that induces T-cell lymphomas. The LTR enhancer of this virus contains two binding sites for the transcription factor CBF (also called AML1 and PEBP2) that flank binding sites for c-Myb and the Ets family of factors. Using cotransfection assays in P19 cells, we report here that CBF and c-Myb cooperatively stimulate transcription from the SL3 LTR. By itself, c-Myb had no stimulatory effect on transcription. However, when cotransfected with a cDNA encoding one form of the ␣ subunit of CBF called CBF␣2-451, a level of transactivation higher than that seen with CBF␣2-451 alone was detected. The negative regulatory domain near the carboxyl terminus of c-Myb did not affect this activity. Electrophoretic mobility shift assays indicated that CBF and c-Myb bind to DNA independently. Therefore, it appears that the cooperative stimulation of transcription by these factors occurs at a step in the process of transcription after the two factors are bound to the enhancer. Sequences near the carboxyl terminus of CBF␣2-451 were important for cooperativity with c-Myb, consistent with previous reports that this region contains an activation domain. However, CBF␣2-451 failed to activate transcription from a version of the SL3 LTR in which the enhancer was replaced with five tandem CBF-binding sites. Thus, it appears that transcriptional activation of the SL3 enhancer by CBF requires that an appropriate heterologous transcription factor be bound to a neighboring site in the regulatory sequences.

Second-site proviral enhancer alterations in lymphomas induced by enhancer mutants of SL3-3 murine leukemia virus: negative effect of nuclear factor 1 binding site

“…To investigate if the effect of the deletions was to abolish the NF1 binding site, we tested whether or not constructs without deletions but with VOL. 71,1997 SL3-3 ENHANCER VARIANTS FROM LYMPHOMAS 1201 on July 10, 2020 by guest http://jvi.asm.org/ mutated NF1 sites also gave enhanced transcription levels. A mutation of three consecutive base pairs from GCC to ATT in the NF1 site has previously been found to abolish the binding of NF1 to this site in band-shift assays (45).…”

“…Also, we have been unable VOL. 71,1997 SL3-3 ENHANCER VARIANTS FROM LYMPHOMAS 1199 on July 10, 2020 by guest http://jvi.asm.org/ to amplify proviral structures from DNA preparations from uninfected mice (data not shown).…”

“…Most of the enhancer function of SL3-3 is contained in the U3 region within a 72-bp direct repeat followed by a third repetition of 34 bp (21). Characterized binding sites for transcription factors in this repeat region include the helix-loop-helix proteins SEF2-1 (or E2-2) (12) and ALF1 (43,44) the glucocortocoid receptor (GR) (9), nuclear factor 1 (NF1) (45), and the hematopoietic factors Ets1 (40), Myb (71), and AML1 (65,66,72). Of these factors, ALF1, GR, AML1, and c-Myb have been directly shown to activate the transcription of SL3-3 (9,42,71,72).…”

“…AML1 is implicated through chromosomal rearrangements in several forms of acute myeloid leukemia in humans (34,39,46), and a murine AML1 gene is essential for normal hematopoietic development (48). AML1 binds to the enhancer core DNA sequence, found in many T-cell-specific genes and vi-ruses, including all commonly known MLVs (19) and may act in synergy with members of the Myb and Ets transcription factor families (24,62,71). AML1 which we have previously referred to as SL3-3 enhancer factor 1 (SEF1) (65, 66) has two different binding sites which are both repeated once in the SL3-3 enhancer.…”

Increased lymphomagenicity and restored disease specificity of AML1 site (core) mutant SL3-3 murine leukemia virus by a second-site enhancer variant evolved in vivo

“…Each site is thus present two or three times. The interacting factors include the AML1 transcription factor family (45,54), nuclear factor 1 (NF1) (9,29), members of the Ets transcription factor family (28,53), basic helix-loop-helix (bHLH) factor transcription factors (26,27), the glucocorticoid receptor (GR) (6), and the c-Myb transcription factor (28,53). Current thinking holds that the transcription factor binding sites in the SL3-3 repeat region, as in similar regions in other MLVs, match the transcription factor profile of the hematopoietic target cells, enabling the virus to have a high replication rate and to act as a powerful insertional mutagen, thus determining the specificity and strength of the virus as a pathogen.…”