The human T-cell leukemia virus type 1 oncoprotein Tax inhibits the transcriptional activity of c-Myb through competition for the CREB binding protein

Abstract

Tax, the transforming protein of human T-cell leukemia virus type 1 (HTLV-1), is required for strong activation of HTLV-1 transcription. This activation is mediated through interaction with the KIX domain of the cellular coactivator CREB binding protein (CBP). In this study we examined the possibility that the Tax-KIX interaction may mediate effects on cellular gene transcription in vivo, as a growing number of cellular transcription factors have been shown to utilize CBP as a coactivator. We tested the ability of Tax to deregulate the activity of the cellular transcription factor, c-Myb, since both Tax and c-Myb interact with the KIX domain of CBP. Our results show that in vivo, Tax antagonizes the transcriptional activity of c-Myb and, reciprocally, c-Myb antagonizes the transcriptional activity of Tax. Furthermore, c-Myb competes for KIX binding to Tax in vitro, indicating that these two transcription factors bind CBP in a mutually exclusive manner. This novel mechanism of transcriptional interference by Tax may promote globally deregulated cellular gene expression in the HTLV-1-infected cell, possibly leading to leukemogenesis.

FIG. 1

Mutual transcriptional repression by Tax and the cellular transcription factor c-Myb. (A) Tax represses the transcriptional function of c-Myb. HTLV-1-negative Jurkat T cells were transiently cotransfected (with Lipofectamine) with 500 ng of either pMRE-luc (■) (13) or viral CRE-luc (○) (15) reporter plasmids and the indicated amount of the Tax expression plasmid (pIEX [27]). (B) c-Myb represses the transcriptional function of Tax. CV-1 cells (at 60% confluency on 60-mm plates) were transiently cotransfected (with calcium phosphate) with 1 μg of the HTLV-1 promoter reporter pLTR-luc (15) and 1 μg of either the c-Myb (8) or Tax expression plasmid. Cell extracts were assayed for luciferase activity. Reported values represent the means ± standard errors (error bars) from three independent experiments.

FIG. 2

The CBP-binding region of c-Myb is sufficient for Tax repression. (A) Schematic representation of the functional domains of c-Myb. Arrowheads indicate the DNA binding domains of c-Myb. The transcriptional activation domain (TAD) and negative regulatory domain (NRD) are also indicated. aa 185 to 360 includes the CBP binding region of c-Myb (9, 20). (B) Tax represses c-Myb transcription through the CBP binding domain of c-Myb. Jurkat T cells were transiently cotransfected with 200 ng of the p5× GAL4-luc reporter plasmid and the indicated amounts of the Tax expression plasmid. Values for reporter alone (○) or reporter in the presence of 200 ng of cotransfected pGAL-Myb_aa185–360 expression plasmid (■) are indicated. Cell extracts were assayed for luciferase activity. Reported values represent the means ± standard errors (error bars) from three independent experiments.

FIG. 3

The CBP binding region of c-Myb (aa 185 to 360) competes with Tax for KIX binding in vitro. Binding reaction mixtures contained 4 fmol of ³²P-end-labeled viral CRE DNA probe (lane 1) (7) with purified recombinant CREB (0.03 pmol; lanes 2 to 15) (12), with purified recombinant Tax (1 pmol; lanes 3 to 15) (35), and with purified recombinant KIX (1 pmol; lanes 4 to 15) (15). Binding reaction mixtures also contained 2, 4, 12, 20, or 28 pmol of purified GST–c-Myb_aa185–360 (lanes 4 to 9) or GST (lanes 10 to 15), as indicated. Binding reaction mixtures were electrophoresed on a 5% nondenaturing gel, as previously described (7). The positions of the relevant protein-DNA complexes are shown.