Function of CBFbeta/Bro proteins.

Mammalian core binding factor beta (CBFbeta) and Drosophila Brother (Bro) and Big-brother (Bgb) proteins are transcription factors that dimerize with mammalian Runx and Drosophila Runt and Lozenge proteins and augment their DNA binding affinity and transcriptional potency. CBFbeta is essential for development and sustenance of definitive hematopoiesis during mouse embryogenesis. Bro and Bgb are required for Runt/Lozenge functions in Drosophila development. CBFbeta contributes to leukemogenesis since the CBFB gene is specifically and consistently mutated by a chromosome 16 inversion found in patients with acute myeloid leukemia subtype M4Eo. The ubiquitous expression pattern of the CBFB gene suggests that it may play important roles in many other organ systems.

Zebrafish homolog of the leukemia gene CBFB: its expression during embryogenesis and its relationship to scl and gata-1 in hematopoiesis.

Mammalian CBFB encodes a transcription factor (CBF beta) that in combination with CBF alpha 2 binds to specific DNA sequences and regulates expression of a number of hematopoietic genes. CBFB is associated with human leukemias through a chromosome 16 inversion and is essential for definitive hematopoiesis during mouse embryo development. We have isolated a zebrafish cbfb complementary DNA (cDNA) clone from a zebrafish kidney cDNA library. This cbfb is highly homologous to human and mouse CBFB/Cbfb genes at both the DNA and protein level. In biochemical analyses, cbfbeta binds to human CBF alpha 2 and enhances its DNA binding. During zebrafish development, cbfb is expressed in the lateral plate mesoderm at tail bud stage and in the intermediate cell mass (ICM, the location of embryonic hematopoiesis) between the 21- to 26-somite stages. The cbfb is also expressed in Rohon-Beard cells, cranial nerve ganglia, hindbrain, retina, branchial arches, jaw, and fin buds. Expression of cbfb is decreased or absent in the ICM and Rohon-Beard cells in some hematopoietic mutants and is unaffected in others. We have also analyzed the expression of scl and gata-1 in the same hematopoietic mutants to ascertain the relative order of these transcription factors to cbfb in zebrafish hematopoiesis. Our results indicate that cbfb is expressed in early hematopoietic progenitors and that its expression pattern in the hematopoietic mutants is similar to that of scl. (Blood. 2000;96:4178-4184)

The leukemic protein core binding factor beta (CBFbeta)-smooth-muscle myosin heavy chain sequesters CBFalpha2 into cytoskeletal filaments and aggregates.

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 beta (CBFbeta) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFbeta-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFalpha1 to 3) and a non-DNA binding subunit (CBFbeta). 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 CBFbeta-SMMHC, CBFbeta, and CBFalpha2 (also known as AML1 or PEBP2alphaB). When expressed individually, CBFalpha2 was located in the nuclei of transfected cells, whereas CBFbeta was distributed throughout the cell. On the other hand, CBFbeta-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFalpha2 was able to drive localization of CBFbeta into the nucleus in a dose-dependent manner. In contrast, CBFalpha2 colocalized with CBFbeta-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFalpha-interacting domain within CBFbeta-SMMHC abolished this CBFalpha2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFbeta-SMMHC when coexpressed with CBFalpha2. CBFalpha2 sequestration by CBFbeta-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFbeta-SMMHC plays a dominant negative role by sequestering CBFalpha2 into cytoskeletal filaments and aggregates, thereby disrupting CBFalpha2-mediated regulation of gene expression.

The core binding factor (CBF) alpha interaction domain and the smooth muscle myosin heavy chain (SMMHC) segment of CBFbeta-SMMHC are both required to slow cell proliferation.

We have expressed several variants of core binding factor beta (CBFbeta)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBFbeta segment, required for interaction with CBFalpha, prevented CBFbeta-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 CBFbeta-SMMHC. Nuclear expression of CBFbeta(Delta2-11)-SMMHC was decreased relative to CBFbeta-SMMHC. CBFbeta(Delta2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBFbeta-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBFbeta-SMMHC slows cell cycle progression from G1 to S phase by inhibiting CBF DNA binding and transactivation.

Interaction of the human T-lymphotropic virus type 1 Tax dimer with CREB and the viral 21-base-pair repeat.

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.

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.

Distinct regions in human T-cell lymphotropic virus type I tax mediate interactions with activator protein CREB and basal transcription factors.

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-kappa 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 NH2 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-kappa 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 NH2 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB.

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 HTLV-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-leucine 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-Ala-Arg at positions 282-284 (AAR284), immediately upstream of the highly 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 AAR284 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.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

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.

Cellular factors involved in transcription and Tax-mediated trans-activation directed by the TGACGT motifs in human T-cell leukemia virus type I promoter.

Human T-cell leukemia virus type I (HTLV-I) encodes a 40-kDa nuclear protein, Tax, which stimulates transcription from three 21-base pair (bp) repeats in its U3 region. Tax trans-activation is mediated via cellular factors that interact with the TGACGT motifs in the 21-bp repeats. Gel mobility shift assay and UV cross-linking analysis show that two proteins of 52 and 46 kDa in size bind the 21-bp repeat specifically. Base substitutions in the TGACGT motif which abolished Tax trans-activation abrogated factor binding whereas the repeats containing mutations that did not affect Tax trans-activation supported factor binding as the wild-type repeat. The 52- and 46-kDa factors are present in human T-cell lines Jurkat and MT4 (HTLV-I transformed) and in HeLa cells but are undetectable in a human placental cell line JEG-3, which gave a reduced level of trans-activation. JEG-3 extracts contain a distinct DNA binding activity that shows analogous sequence requirements as the 52- and 46-kDa proteins in interacting with the various 21-bp repeats. c-Jun and CREB (cAMP-responsive element binding factor) can stimulate transcription from HTLV-I long terminal repeats in JEG-3 cells. At least two copies of the 21-bp repeats are required for optimal trans-activation by c-Jun and CREB. Most single point mutations in the TGACGT motif that abolished Tax trans-activation, however, did not affect c-Jun- or CREB-directed transcriptional enhancement. These data indicate that many transcription factors including c-Jun and CREB exert stimulatory effects on HTLV-I transcription although they do not directly respond to Tax. The 52- and 46-kDa cellular proteins most likely are involved directly in Tax-mediated trans-activation, and they are tentatively named Tax activation factors I and II, respectively.