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...

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We have expressed several variants of core binding factor ␤ (CBF␤)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBF␤ segment, required for interaction with CBF␣, prevented CBF␤-SMMHC from inhibiting CBF DNA binding and cell cycle progression. Deletion of 283 carboxyl-terminal residues from the SMMHC domain, required for multimerization, also inactivated CBF␤-SMMHC. Nuclear expression of CBF␤(⌬2-11)-SMMHC was decreased relative to CBF␤-SMMHC. CBF␤(⌬2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBF␤-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBF␤-SMMHC slows cell cycle progression from G 1 to S phase by inhibiting CBF DNA binding and transactivation.The core binding factor (CBF) 1 family is made up of transcription factors that contain a common CBF␤ subunit and one of three CBF␣ subunits: CBF␣1, AML1 (CBF␣2), or CBF␣3 (1-7). CBF␤ increases the affinity of the CBF␣ subunits for DNA, but does not bind DNA directly (4, 7). The CBF␣ subunits contain a domain required for heterodimerization and DNA binding (3,8).Translocations involving subunits of CBF are common in acute leukemias (9). Inv(16) is present in 10% of acute myeloid leukemias (AMLs) and encodes CBF␤-SMMHC, in which CBF␤ is fused to the tail domain of SMMHC (10). t(8;21) is present in 12% of AMLs and encodes AML1-ETO, which includes the DNA-binding domain of AML1 (11). 25% of pediatric B-lineage acute lymphocytic leukemias contain t(12;21), which encodes TEL-AML1 (12, 13).Each of these "CBF oncoproteins" inhibits CBF activities (14 -20). Also, mice expressing CBF␤-SMMHC or AML1-ETO fail to develop definitive hematopoiesis, just as do mice lacking AML1 or CBF␤ (21-26).CBF activates the expression of several lymphoid and myeloid genes, suggesting that lack of differentiation accounts for the phenotypes of CBF null mice (27-30). We expressed CBF␤-SMMHC from the zinc-responsive MT promoter in 32D cl3 myeloid and Ba/F3 B-lymphoid cells (14). Induction of CBF␤-SMMHC resulted in decreased CBF DNA binding and slowed proliferation during G 1 phase. The differentiation of 32D cl3 cells in response to granulocyte colony-stimulating factor was unaffected. We proposed that initial genetic alterations occur during leukemogenesis that bypass the growth inhibitory effect of CBF␤-SMMHC, potentiating inhibition of differentiation.We have now expressed several CBF␤-SMMHC mutants in Ba/F3 cells. Amino acids 1-165 of the 182-amino acid CBF␤ protein are present in the majority of CBF␤-SMMHC fusion proteins, although a variant containing only residues 1-133 is present in rare patients (31). Amino acids 1-73 and 102-137 are highly conserved between Drosophila Brother and Bigbrother and murine CBF␤ (32). Segment 1-137 is sufficient to strongly increase the affinity of CBF␣ subunits for DNA, whereas segment 1-133 or 1-132 does so o...

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5) and/or C (3) residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH 2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH 2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex. These data support the notion that the NH 2-terminal region of Tax is important for interaction with CREB while the COOH end of Tax most likely is involved in communication with the basal transcriptional machinery.

Human T-lymphotropic virus type 1 Tax interacts specifically with the cellular transcription factor CREB and the viral 21-bp repeat element to form a Tax-CREB-DNA ternary complex which mediates activation of viral mRNA transcription. Analyses of Tax and Tax mutants indicate that, like CREB, Tax incorporates into the ternary complex as a dimer. The ability of Tax to form a dimer is necessary for its interaction with CREB and the 21-bp element. Analyses of several Tax mutants with amino acid substitutions spanning residues 123 to 204 indicate that intersubunit Tax dimerization correlates with its ability to assemble into the ternary complex and activate transcription. Tax also enhances the DNA binding activities of specific bZip domains in vitro. The ability of Tax to enhance DNA binding of bZip proteins can be explained in part by Tax dimerization. This activity alone is not sufficient for transactivation. A dual amino acid substitution mutant of Tax, M47 (L319R, L320S), completely abrogated for activation of the human T-lymphotropic virus type 1 long terminal repeat as a result of a defect in the transactivation domain, continues to stimulate binding of bZip proteins to DNA.