T he heterodimeric CBF transcription factor genes (CBFA2 (also known as AML1) and CBFB) are the most common translocation targets in human acute myeloid leukaemia (AML), accounting for 30% of AML cases 1 . Approximately onehalf are attributable to a chromosome 16 inversion, inv(16)(p13; q22), found con-

The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor ␤ (CBF␤) and the smoothmuscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBF␤-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBF␣1 to 3) and a non-DNA binding subunit (CBF␤). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBF␤-SMMHC, CBF␤, and CBF␣2 (also known as AML1 or PEBP2␣B). When expressed individually, CBF␣2 was located in the nuclei of transfected cells, whereas CBF␤ was distributed throughout the cell. On the other hand, CBF␤-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBF␣2 was able to drive localization of CBF␤ into the nucleus in a dosedependent manner. In contrast, CBF␣2 colocalized with CBF␤-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBF␣-interacting domain within CBF␤-SMMHC abolished this CBF␣2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBF␤-SMMHC when coexpressed with CBF␣2. CBF␣2 sequestration by CBF␤-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBF␤-SMMHC plays a dominant negative role by sequestering CBF␣2 into cytoskeletal filaments and aggregates, thereby disrupting CBF␣2-mediated regulation of gene expression.

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB.In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G-or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G-and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.Human T-cell lymphotropic virus type I (HTLV-I) is the prototype of a group of retroviruses (including HTLV-I, HTLV-II, simian T-cell leukemia virus, and bovine leukemia virus [BLV]) that produce regulatory proteins to modulate viral mRNA synthesis and utilization (6). The 40-kDa nuclear protein, Tax, encoded by the 3' region of the HTLV-I genome is a transcriptional activator (6). Tax does not bind DNA directly (13,21). Like many other viral transactivators such as herpes simplex virus VP16 and adenovirus Ela (for a review, see reference 20), Tax forms multiprotein complexes with host cell transcription factors to gain control of the cellular mRNA synthetic machinery for viral replication (1,18,(27)(28)(29). The ability of Tax to alter gene expression in HTLV-I-infected cells appears to be causally linked to HTLV-I pathogenesis, which is manifested clinically as adult T-cell leukemia (5, 16, 24) and tropical spastic paraparesis/HTLV-I-associated myelopathy (2,12,17,22).Tax activates viral transcription from three 21-bp repeats in the U3 region of the HTLV-I long terminal repeat (3,23,25,26). Mutational analyses indicated that two copies of the repeat and the CRE (cyclic AMP [cAMP]-responsive-element)-like sequences, TGACG, in the repeats are crucial for transactivation by Tax (3,13,23,25,26 Tax action (10,19). This is best shown by the inability of HTLV-I Tax to transactivate thre...

The transactivator protein of human T-lymphotropic virus type I (HTLV-I), Tax, forms multiprotein complexes with the ubiquitous transcription factor CREB and the CREB/ATF-1 heterodimer. The interaction between Tax and CREB is highly specific and results in increased binding of the Tax/CREB complexes to the HTLY-I 21-bp repeats. Despite the extensive sequence similarities between CREB and ATF-1, Tax interacts with ATF-1 only marginally. Compared with CREB, Tax/CREB exhibits greatly increased DNA recognition specificity and preferentially assembles on a consensus binding site, GGGGG(T/A)TGACG(T/C)(A/C)TA(T/C)C-CCCC, homologous to the HTLV-I 21-bp repeats. Here we report that Tax affects CREB binding to the Tax-inducible DNA elements by interacting with the basic-eucine zipper (bZip) domain of CREB. We show by domain switching that the basic region in CREB bZip can confer on c-Jun and ATF-1 leucine zippers the ability to interact with Tax in vitro. Mutational analyses further demonstrate that the amino acid residues of CREB critical for Tax/CREB interaction are Ala-AlaArg at positions 282-284 (AAR"), immediate upstream of the higly conserved DNA-binding domain (R/K)XX(R/K) N(R/K)XAAXX(S/C)RX(R/K)(K/R) characteristic of all bZip proteins. Specific amino acid substitutions in AAR2" of CREB weakened or abolished Tax/CREB interaction, whereas reciprocal changes in ATF-1 allowed It to interact with Tax. These results support a model in which the specific interaction between Tax and the AAR284 residues near the DNA-binding domain of CREB results in a multiprotein complex with altered DNA recognition property. This protein complex assembles selectively on the viral Tax-responsive 21-bp repeats to augment transcription.Tax is a 40-kDa nuclear protein encoded by the 3' region of the genome of human T-lymphotropic virus type I (HTLV-I). It stimulates HTLV-I transcription via three imperfect 21-bp repeats in the proviral long terminal repeat (1-3). cAMP response element (CRE)-like motifs in the repeats are crucial for Tax transactivation (3). We previously detected three cellular protein factors in Jurkat T cells and HeLa cells that bound specifically to the CRE in the HTLV-I 21-bp repeats. Two ofthese factors interacted with Tax directly (4,5). These three cellular factors were identified to be CREB homodimer, CREB/ATF-1 heterodimer, and ATF-1 homodimer (5). Tax interacts directly with the CREB subunits in CREB homodimer and CREB/ATF-1 heterodimer and stabilizes their binding to the HTLV-I 21-bp repeats (5). Both CREB and ATF-1 are members of the basic-leucine zipper (bZip) family of transcription factors and are highly similar in primary amino acid sequences (6-8). The interaction between Tax and CREB is highly specific. Despite the extensive amino acid similarities shared between ATF-1 and CREB, Tax interacts marginally with ATF-1 (5). By selecting for preferred Tax/CREB or CREB binding sites in vitro, the Tax/ CREB complex was found to exhibit greatly altered DNA recognition specificity compared with CREB (9). The...