Transcription factor AP-4 participates in activation of bovine leukemia virus long terminal repeat by p34 Tax.

Three 21bp repeats can be found in the bovine leukemia virus long terminal repeat, which are crucial for the LTR directed gene expression by the trans activator protein Tax. Previous studies demonstrated that the major target of the Tax directed activation are the CRE-like elements in the center of these repeats. In this work we report that another motif of the 21bp repeats is also required for the Tax activation. Gel retardation--with the wild type or mutant 21bp repeats--revealed that cellular factors from HeLa cells were specifically bound to the center (CRE-like element) and the 3'' region of the repeats, which contains a CAGCTG consensus AP-4 binding site. In vivo analysis using the synthetic 21bp repeats indicated that beyond the consensus CRE-like motif, the AP-4 site is also essential for Tax activation. To determine the role of AP-4 in BLV Tax trans activation, we used the AP-4 cDNA in antisense transient assays. In the in vivo experiments the antisense AP-4 RNA resulted in strongly decreased Tax activation. On the basis of these results we conclude that AP-4 is a good candidate of cellular factors involved in BLV Tax trans activation.     

Characterization of a delayed early serum response region.

The proliferin (PLF) gene promoter provides a relatively simple model system for the study of growth-regulated gene expression in mouse cells. The promoter elements required for this serum-induced regulation have been identified and include an AP-1 site as well as an adjacent element comprised of three imperfect repeats that are similar in sequence to the simian virus 40 (SV40) Sph motif. Distinct protein complexes bound independently to the AP-1 and Sph elements, and both of these juxtaposed sites could be occupied simultaneously. Furthermore, serum stimulation of mouse fibroblasts resulted in similar increases in protein binding to the AP-1 and Sph elements. Consistent with this increase in AP-1 and Sph binding activity, the PLF AP-1 and Sph elements were independently able to confer serum responsiveness to a minimal promoter, and together these two elements acted synergistically in response to serum. Although several members of the AP-1 family were able to activate the PLF gene promoter in transient cotransfection experiments, the predominant AP-1 components interacting with the PLF gene promoter in serum-stimulated cells were Fra-1, JunB, and JunD. Analysis of the Sph element revealed that mutation of Sph repeats I or III abolished serum responsiveness of the PLF gene promoter, and mutation of Sph repeat III decreased protein binding to this element. Although the Sph element is similar in sequence to the SV40 element, the PLF Sph-binding factor is distinct from TEF-1, the factor that binds to the SV40 Sph motif.     

Multiple cDNA clones encoding nuclear proteins that bind to the tax-dependent enhancer of HTLV-1: all contain a leucine zipper structure and basic amino acid domain.

A trans-activator protein, p40tax, of human T cell leukemia virus type 1 (HTLV-1) activates its own promoter and cellular promoters of IL-2, IL-2 receptor alpha and GM-CSF genes. We isolated three cDNA clones encoding cellular proteins that bind to the p40tax-dependent enhancer of HTLV-1 by screening a lambda gt11 cDNA library of an HTLV-1 infected cell line. All three proteins, TREB5, TREB7 and TREB36, contained a leucine zipper structure and basic amino acid domain, which are conserved in FOS, JUN and CREB, and also had multiple potential phosphorylation sites. The proteins expressed in Escherichia coli bound to the p40tax-dependent enhancer of the 21 bp sequence, but not to an inactive mutant carrying a mutation in the CRE region. In DNase I footprint analysis, all three proteins protected the 21 bp sequences in the LTR; however, the patterns were not identical to each other. TREB7 and TREB36 protected all three repeats of the 21 bp, but TREB5 protected only the second repeat. TREB7 and TREB36 protected the 5' and middle portions of the 21 bp which are essential for p40tax-mediated trans-activation, whereas TREB5 and CREB1 protected a narrower part of the middle region of the second 21 bp repeat containing the CRE consensus sequence. These structural features and DNA binding properties suggest that TREB proteins are members of a CREB protein family and that some of them (i.e., TREB7 and TREB36) may be involved in p40tax-mediated trans-activation.