Myb-interacting Protein, ATBF1, Represses Transcriptional Activity of Myb Oncoprotein*

Petr Kaspar, Marta Dvo&rbreve;áková, Jarmila Králová, Petr Pajer, Zbynek Kozmik, and Michal Dvo&rbreve;ákDagger

From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo 2, 166 37 Prague 6, Czech Republic

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein. Deletion mutagenesis revealed amino acids 2484-2520 in human ATBF1 and 279-300 in v-Myb as regions required for in vitro binding of both proteins. Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity. Both ATBF1 2484-2520 and Myb 279-300 regions were required for the inhibitory effect. Finally, the chicken ATBF1 was identified, showing high degree of amino acid sequence homology with human and murine proteins. Our data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279-300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The v-myb oncogene carried by the chicken retrovirus AMV1 is a truncated and mutated version of its cellular progenitor c-myb proto-oncogene. The c-myb gene is expressed mainly in hematopoietic cells, where it is involved in maintaining their immature proliferative stages, while the viral v-myb transforms myelomonocytic cells in vitro and causes acute monoblastic leukemia in virus-infected chicks (1-5). Both c-myb and v-myb have been useful in studying the development of hematopoietic cells, and much work has been devoted to analyzing their properties and regulation.

Myb proteins are transcription factors with a unique N-terminal DNA-binding domain (DBD), a central transactivation domain (TA) and a C-terminal negative regulatory domain containing the leucine zipper structure (LZ) (6-13). The DBD and TA domains are crucial for biological activities of Myb proteins (11, 12, 14). Recently, the LZ motif has been reported to be required for proliferation of v-Myb-transformed monoblasts and for in vivo leukemogenicity of v-Myb (15). In addition to leucine zipper, other parts of v-Myb C terminus were also found to be important for the biological activities of the oncogene.2 All these domains are engaged in interactions with other proteins. Several interactions of Myb DBD and TA have been implicated in regulation of Myb functions in growth and differentiation. The interaction of c-Myb DBD with the EVES motifs of both p100 transcriptional coactivator and c-Myb itself down-regulates c-Myb activity (16). Similarly, the direct binding of c-Maf transcriptional activator to c-Myb DBD was found to repress Myb transcriptional activity and to inhibit Myb- and Ets1-dependent activation of CD13/APN transcription (17). The Myb DBD was also found to interact with the retinoic acid receptor. This interaction appears to inhibit retinoic acid-dependent transactivation (18). On the other hand, the interaction of C/EBPbeta with v-Myb DBD positively affects Myb activity and results in synergistic activation of the mim-1 promoter by both proteins (19). Binding of Cyp-40 (20) and HSF3 (21) further document the complex regulation of Myb DBD by protein-protein interaction. The Myb TA was found to interact with CBP (CREB-binding protein). This interaction results in activation of Myb transcriptional properties (22, 23). Recently, several proteins, including p67 and p160 (24, 25) and p26/28 (15), were also found to associate with Myb LZ. No biological activity has been assigned to these interactions so far.

In an attempt to detect and identify additional protein partners of v-Myb C terminus that could potentially affect biological activities of v-Myb, the v-Myb sequence downstream from transactivation domain was used in this work as a bait in a yeast two-hybrid screen applied to a human cDNA library. These experiments led to the isolation of ATBF1.

ATBF1 gene was first identified in human hepatoma cells. Its protein product binds to and represses the activity of an AT-rich enhancer element of the human alpha -fetoprotein gene (26). ATBF1, a vertebrate homologue of Drosophila ZfH 2 protein, is a 306-kDa protein containing 4 homeodomains, 17 zinc finger motifs, and a number of segments potentially involved in transcriptional regulation. More recently, its longer form, termed ATBF1A, was isolated (27).

In this work, it is shown that ATBF1 binds specifically to Myb proteins both in vitro and in vivo. Binding of ATBF1 to v-Myb can repress transcriptional activity of the oncoprotein in chicken fibroblasts. In this interaction, v-Myb amino acids 279-300 are involved and represent a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated. We also describe the detection of chicken ATBF1 and point to the very high ATBF1 conservation between mammalian and avian species.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cells and Viruses-- Sf9 insect cells grown in SF 900 II (Life Technologies, Inc.) medium were used to produce v-Myb and c-Myb from recombinant baculovirus genomes (28). Chicken embryo fibroblasts (CEF) were grown in Dulbecco's modified Eagle's medium supplemented with 8% fetal calf serum, 2% chicken serum, and antibiotics.

Plasmids-- The bait molecule pB-137 was constructed by inserting the v-myb SalI-XbaI fragment (nucleotides 714-1163) in frame with GAL4 DNA-binding domain in pGBT9 (CLONTECH). In pB-85 bait, the shorter v-myb fragment (nucleotides 714-971) was used. The numbering of v-myb starts at the first coding nucleotide of the gag gene in AMV v-myb (29).

To express various forms of ATBF1 fragment (encoded by the two-hybrid system-selected RX-28 plasmid) as GST fusion proteins in Escherichia coli, the following plasmids were prepared. Plasmid GST-AT438 was constructed by inserting EcoRI-HindIII fragment of ATBF1 from RX-28 into EcoRI-Tth111I site of pGEX-3X (Amersham Pharmacia Biotech) after converting HindIII and Tth111I into blunt ends by Klenow fragment. To make GST-AT175, GST-AT438 was used as a template in PCR reaction using the following primers: 5'-CGTGGGATCCACGGGAATTC-3' and 5'-CTGGCTCTTCAGGGAGTTTC-3'. The amplified ATBF1 fragment was digested with BamHI and inserted into pGEX-3X digested with BamHI and SmaI; GST-AT139 and GST-AT81 were made by inserting BamHI-SmaI and BamHI-StuI fragments of GST-AT175 into BamHI-EcoRI site of pGEX-3X after converting EcoRI site into blunt end; in-frame deletions of BamHI-StuI fragment in GST-AT139 and GST-AT175 created GST-AT58 and GST-AT94, respectively. BamHI and StuI ends were partially filled to recreate BamHI site and maintain the reading frame.

To express various v-myb proteins in TnT (Promega), the wild type v-myb EcoRI-XbaI fragment (nucleotides 360-1163) and its in-frame deletions, namely Delta 721-750, Delta 721-834, Delta 835-900, and Delta 904-933, were constructed in pGEM-4Z.

For transient expression in CEFs, the full-length wild type (wt) or mutant AMV v-myb genes were inserted into pneo vector (30), creating pneo-wt v-myb, pneo-Delta 835-900 v-myb, and pneo-L3,4A v-myb (15), respectively. The pneo-Delta v-myb vector containing no myb sequences was used as a control. Cytomegalovirus expression vector pKW-2T was used to express various ATBF1 constructs. To generate pKW-AT, an 8.6-kilobase HindIII fragment of full-length ATBF1 from pCATBF1A (kindly donated by T. Morinaga; Ref. 31) was used. In pKW-Delta MISAT, the Myb-interacting sequence (MIS) spanning amino acids 2426-2516 was deleted. In pKW-ATHA, three tandemly arranged hemagglutinin epitopes (HA) were attached in frame to the 3' terminus of ATBF1. To measure transcriptional activities, four CAT reporter plasmids were used; 3xMRE-CAT contained three wild type mim-1a Myb recognition elements (15); the SN0.5-CAT contained SmaI-NotI fragment of the chicken c-myb promoter (nucleotides -481 to -15 in Ref. 32) in front of TATA box derived from herpes simplex virus thymidine kinase gene; the ATBF1 recognition element (AFE)-containing reporters, namely AFE SN0.5-CAT and AFEm SN0.5-CAT, contained the wild type or mutated AFE (31), respectively, upstream from SN0.5 sequence. RSV-beta Gal plasmid has been described (15). All constructs were checked by restriction mapping or by sequencing.

Yeast Two-hybrid Cloning-- Yeast two-hybrid screening was performed using the MATCHMAKER two-hybrid system (CLONTECH). The yeast strain HF7c was transformed with transcriptionally inert pB-85 construct using lithium acetate method. Cells that grew on synthetic medium lacking tryptophan were transformed with a HeLa cDNA library directionally cloned into EcoRI-XhoI site of GAL4 activation domain vector pGAD GH. Approximately 1 × 106 yeast transformants were plated on 20 15-cm plates containing synthetic medium lacking tryptophan, leucine, and histidine, and including 10 mM 3-aminotriazol. Plates were incubated at 30 °C for 5 days. Two hundred growing individual Leu+, Trp+, His+ colonies were picked and purified on the same selection medium. Fifty well growing purified colonies were transferred to the filter paper and assayed for beta -galactosidase. Five colonies were positive. Plasmids containing human cDNAs were isolated, and cDNA inserts were sequenced. ATBF1-containing plasmid (RX-28) was co-transformed with the bait construct or control plasmids (see "Results") into SFY526 cells to verify the specificity of two-hybrid assay.

In Vitro Binding Assays-- Logarithmically growing bacterial cultures (10 ml) harboring different GST-ATBF1 constructs were induced for 1 h with 1 mM isopropyl-beta -D-thiogalactoside, sonicated briefly on ice in 1 ml of 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% Nonidet P-40 (Fluka), and 50 µg/ml aprotinin (Sigma), and soluble fraction was incubated with 25 µl of glutathione-agarose beads (Amersham Pharmacia Biotech) for 10 min at 4 °C. Agarose beads were washed four times with the above lysis buffer and then incubated in 200 µl of lysis buffer with insect cell lysates containing v-Myb or c-Myb. Lysates were prepared by sonication of 1 × 104 insect cells in lysis buffer, and soluble fraction was used. Alternatively, 20,000 cpm of 35S-labeled in vitro synthesized (TnT) Myb proteins were used. After rocking at 4 °C for 2 h, beads were washed four times with lysis buffer and bound proteins were analyzed by SDS-PAGE followed by autoradiography or Western blotting.

Co-transfection Transactivation Assay and Preparation of Nuclear Extracts-- Transient transfections were performed by a calcium phosphate precipitation method (33). Four µg of v-Myb-expressing plasmids, 2 µg of reporter plasmids, 5 µg of ATBF1 expression plasmids, 1.5 µg of internal control RSV-beta Gal plasmid, and pBluescript carrier DNA up to 25 µg were co-transfected into approximately 7 × 105 CEF/10-cm Petri dish. Empty expression vectors pKW-2T or pneo-Delta v-myb were used as controls. Forty-eight hours later, cells were harvested and nuclear or whole cell extracts were prepared. CAT activity was determined in whole cell extracts by CAT enzyme-linked immunosorbent assay (Roche Molecular Biochemicals), normalized for beta -galactosidase activity, and plotted as -fold activation. The -fold activation represents CAT values obtained with v-Myb expression vectors divided by CAT values obtained with pneo-Delta v-myb vector. For co-immunoprecipitation experiments, nuclear extracts of transfected cells were prepared as described (34). In these experiments, pKW-ATHA was used.

Immunoprecipitation and Western Blotting-- Nuclear proteins were extracted from 2 × 106 CEF transiently expressing v-Myb and ATBF1-HA using salt and detergent buffer essentially as described (34). A mouse monoclonal anti-HA antibody was used to immunoprecipitate ATBF1-HA, according to the published protocol (35). The antigen-antibody complexes were collected on a protein A-Sepharose beads (Amersham Pharmacia Biotech), washed, and analyzed on 10% SDS-PAGE. For Western blots, the fractionated proteins were transferred onto HybondTM ECLTM nitrocellulose membrane (Amersham Pharmacia Biotech) and v-Myb was detected using myb-specific polyclonal antibody. The blots were developed using ECL reagents (Amersham Pharmacia Biotech).

Reverse Transcriptase-PCR of Chicken ATBF1 Fragment-- Two µg of total RNA prepared (36) from the whole chicken embryo was reverse transcribed using Superscript II reverse transcriptase (Life Technologies, Inc.), and cDNA was used to amplify a chicken ATBF1 fragment. Degenerated PCR primers were derived from homeodomain HD2 (5'-ACN JGN TTY ACN GAY TAY CAR-3') and zinc finger motif Z12 (5'-RCA YTT YTT RCA XXX RTA RTT-3') that are well conserved in Drosophila ZfH-2 and mammalian ATBF1. PCR was performed as follows: 5 min at 94 °C; 1 min at 94 °C, 2 min at 55 °C, 1 min at 72 °C for 35 cycles, and then 1 µl of the PCR reaction was used for reamplification with HD3 (5'-GTN CAR GTN TGG TTY CAR AAY-3') and Z12 primers. The final PCR product (138 base pairs) was cloned into pCRTMII vector (Invitrogen) to yield pCR-chATBF1, sequenced, and used as a probe for RNase protection assay.

RNase Protection Assays-- pCR-chATBF1 was linearized by XhoI and transcribed by SP6 polymerase (Roche Molecular Biochemicals) in the presence of [alpha -32P]GTP to produce radioactive 257-base pair fragment protecting 138 nucleotides of ATBF1 mRNA. As a referential probe, chicken actin was used. RNA probes were purified by polyacrylamide gel electrophoresis and hybridized to 10 µg of total cellular RNA in 40 mM PIPES, pH 6.4, 80% formamide, 0.4 M NaCl, and 1 mM EDTA at 65 °C overnight. After digestion with 5 µg/ml RNase A and 50 units/ml RNase T1 at 37 °C for 1 h, samples were analyzed in 6% polyacrylamide-urea gel and detected by autoradiography.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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v-Myb and ATBF1 C Termini Interact in the Yeast Two-hybrid System-- In an attempt to isolate a protein(s) interacting with v-Myb C terminus, the MATCHMAKER yeast two-hybrid system was used. In this system, the bait molecule pB-137 containing v-myb nucleotides 714-1163 fused in frame to GAL4 DBD and carrying the gene for TRP1 bound to regulatory regions of HIS3 and lacZ reporter genes in HF7c cells auxotrophic for Trp, Leu, and His. The plasmids containing cDNA (HeLa) library fused to GAL4 TA and carrying the gene for LEU2 were introduced into bait-containing cells and transformants were selected on Trp-, Leu-, and His-lacking plates. Interaction of GAL4 TA/cDNA encoded protein with the bait protein resulted in HIS3 transcription and growth of transformants on selective medium. Growing colonies were subsequently screened for the expression of the second lacZ reporter gene. The pB-137 construct alone, however, activated both HIS3 and lacZ genes in HF7c cells. Since v-Myb leucine zipper region has previously been shown to activate transcription in yeast (37), the C-terminal part of LZ was deleted from pB-137 yielding transcriptionally inactive pB-85 (v-myb nucleotides 714-971 fused to GAL4 DBD). Upon introduction of GAL4 TA/cDNAs into pB-85 containing cells, 1 × 106 transformants were obtained, 50 of which reproducibly grew well in the absence of histidine. Among them five colonies were also positive for lacZ expression. cDNA-containing plasmids were isolated from them and sequenced. One clone (RX-28) contained 440 C-terminal codons (amino acids 2345-2783) of human ATBF1 (Ref. 26; Fig. 1A). Four other positive clones carried inserts not homologous to any known sequence. To verify the interaction, the RX-28 plasmid was introduced into HF7c cells containing pB-85. In this case, all the transformed colonies exhibited both HIS+ and LacZ+ phenotype. Finally, instead of HF7c also SFY526 yeast strain was transfected with pB-85, pGBT9 (containing only GAL4 DBD), or pLAM (encodes human lamin C/GAL 4 DBD). LacZ expression appeared only when pB-85 and RX-28 were cotransfected, suggesting that Myb and ATBF1 fragments interact in yeast cells.


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Fig. 1.   Binding of c-Myb and v-Myb to ATBF1 in vitro. A, structure of GST-ATBF1 fusion proteins used for the binding assay. The potential functional domains of ATBF1 are depicted (top): rectangles HD1-HD4, homeodomains; closed rectangles, zinc finger motifs; PQ rectangles, segments rich in proline and glutamine. Structures of six types of GST-ATBF1 fusion proteins with various deletions are shown below. The results of binding assays are indicated at right. B, binding of c-Myb and v-Myb to ATBF1. Glutathione-agarose beads containing GST (lanes 2 and 5) or GST-AT 438 (lanes 3 and 6) were mixed with protein lysate from insect cells expressing c-Myb (lanes 2 and 3) or v-Myb (lanes 5 and 6). Bound proteins were analyzed on 10% SDS-PAGE, followed by Western blotting. Lanes 1 and 4 show the input of c-Myb and v-Myb used for binding assay. C, binding of v-Myb to GST-ATBF1 fusion proteins. Agarose beads containing each of the fusion protein or control GST were mixed with baculovirus expressed v-Myb, as in B. D, analysis of GST-ATBF1 fusion proteins. Protein lysates from 0.5 ml of bacterial culture expressing various GST-ATBF1 fusion proteins or GST were mixed with agarose beads. Bound proteins were analyzed on 10% SDS-PAGE, followed by silver staining.

ATBF1 C Terminus Interacts with Full-length v-Myb and c-Myb in Vitro-- To confirm the v-Myb-ATBF1 interaction observed in yeast cells, the ATBF1 sequence from RX-28 was expressed in bacteria as GST fusion protein from GST-AT 438 vector (Fig. 1A) and bound to glutathione-agarose column. Ly-sates of Sf9 cells synthesizing either v-Myb or c-Myb were applied to the column, and retained proteins were analyzed by the Western blot procedure. As shown in Fig. 1B, both v-Myb and c-Myb proteins bound efficiently to GST-AT 438 (lanes 3 and 6), but not to GST alone (lanes 2 and 5). Smaller-sized proteins detected in lane 1 represent degradation products of c-Myb. Protein molecules larger than p48 v-Myb seen in lanes 4, 5, and 6 represent irreversible reaction products of v-Myb, often generated during prolonged manipulation with v-Myb preparations.

ATBF1 Amino Acids 2484-2520 Are Required for Myb Binding-- In order to delineate the ATBF1 interaction domain, six deletion mutants fused to GST were prepared. The GST-AT 175, GST-AT 139, GST-AT 81, GST-AT 94, and GST-AT 58 constructs (schematically represented in Fig. 1A) were expressed in bacteria (Fig. 1D) and used for the binding assay with v-Myb as above. Fig. 1C shows that only the constructs GST-AT 438, GST-AT 175, and GST-AT 94 bind Myb oncoprotein. Similar results were obtained with c-Myb (not shown). Thus, the ATBF1 amino acids in GST-AT 94 represent the minimal interaction domain where the C-terminal 37-amino acid region (amino acids 2484-2520) referred to as ATBF1-MIS is necessary for binding the full-length Myb proteins in vitro. This proline-rich region covers the C-terminal part of the third ATBF1 PQ box.

Two Myb C-terminal Segments Are Involved in ATBF1 Binding-- In order to define the Myb sequences engaged in ATBF1 binding, several v-Myb deletion mutants were prepared. As shown in Fig. 2A, v-Myb devoid of DNA-binding domain with short in-frame deletions covering the bait fragment was used. The [35S]methionine-labeled Myb proteins (Fig. 2B, lanes 1-6) were mixed with glutathione-agarose-bound GST-AT 438. Labeled proteins retained on the agarose were revealed by electrophoresis (Fig. 2B, lanes 7-12). Smaller sized forms of Myb proteins were probably initiated at an internal ATG codon located 96 nucleotides downstream from the first ATG. None of the Myb proteins bound to the control GST column (data not shown). It has been observed that deletion of Myb amino acids 279-300 encoded by nucleotides 835-900 severely reduced binding to ATBF1 (lane 9). Identical results were obtained with agarose-bound GST-AT 175 (not shown). Since all the Myb mutants contain the LZ region active in protein-protein interactions (15, 24, 25), the potential contribution of LZ to ATBF1 binding was assessed using the L3,4A v-Myb mutant. This mutation was shown to reduce binding of v-Myb LZ to other proteins (15, 24). In our experiments mutation of Myb LZ, leucines 3 and 4 (Leu325 and Leu332) to alanines resulted in a significant decrease of binding to GST-AT 438 (lane 11) as well as to GST-AT 175 (not shown). Thus, the data indicate that there are two adjacent sequences in Myb, namely the amino acids 279-300 present in the bait molecule and also leucines L3 and L4 of the zipper domain, required for efficient binding of ATBF1 in vitro.


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Fig. 2.   Domains of Myb required for binding to ATBF1. A, structure of Myb mutants used for binding assay. Top, various domains of v-Myb are indicated: DBD, DNA binding domain; TA, transactivation domain; PP, multiple phosphorylation sites; LZ, leucine heptad repeat (leucine zipper). Shaded box represents a portion of Myb used in the bait construct. The structures of Myb mutants are shown below. Blank spaces and numbers at left represent deleted nucleotides. Mutations introduced within the leucine zipper in L3,4A construct are indicated by asterisks. The results of binding assays are indicated at right. B, binding of Myb mutants to ATBF1. Various forms of Myb shown above each lane were synthesized in vitro. 35S-Labeled proteins were mixed with agarose beads containing GST-AT 438 (lanes 7-12). Bound proteins were resolved on 12% SDS-PAGE, followed by autoradiography. Lanes 1-6 show the input of proteins used for binding assay.

Detection of Chicken ATBF1-- Previous experiments revealed the interaction between chicken Myb and human ATBF1 proteins. To substantiate further functional studies with these proteins, we searched for the existence, evolutionary conservation, and expression of ATBF1 homologue in chicken cells. Southern blot analysis of the chicken chromosomal DNA detected a set of restriction fragments that hybridized with the human ATBF1 probe at high stringency (Fig. 3A). Degenerated PCR primers, derived from ATBF1 sequence highly conserved in man and Drosophila, were than used for amplification of an ATBF1 fragment from the chicken embryo cDNA. The isolated cDNA fragment displayed very high homology with the human sequence. Additional upstream and downstream sequences were obtained by 5'- and 3'-rapid amplification of cDNA ends and aligned with the human and murine ATBF1 sequences. The chicken fragment shown in Fig. 3B displayed 97% identity of amino acid and 84% conservation of nucleotide sequences (data not shown). Based on the sequence comparison of other chicken ATBF1 cDNA fragments covering approximately one third of mRNA, it can be assumed that the evolutionary conservation of human and chicken ATBF1 proteins is approximately 95%.3


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Fig. 3.   Detection of chicken ATBF1. A, Southern blot analysis of human and chicken DNA. Genomic DNA prepared from chicken spleen and human HeLa cells were digested with EcoRI (lanes 1 and 2) or HindIII (lanes 3 and 4) and separated on a 1% agarose gel, blotted onto a nylon membrane, and hybridized with 32P-labeled ATBF1 cDNA probe spanning nucleotides 7260-7690. B, alignment of amino acid sequences of chicken (ch), mouse (m), and human (h) ATBF1. Diverse amino acids are printed in bold type. Homeodomain HD2 and zinc finger motif Z12 are boxed. The sequence from which the probe for RNase protection was derived is underlined. C, expression of ATBF1 mRNA in adult chicken tissues and chicken cells. RNase protection analyses were conducted using 32P-labeled 138-base pair RNA probe (underlined in B) and 10 µg of total cellular RNA isolated from indicated chicken tissues and cells. As a referential probe, the chicken actin was used.

ATBF1 Is Expressed in Many Chicken Tissues-- RNase protection analysis was conducted to characterize the distribution of ATBF1 mRNA in various chicken tissues, cultured v-Myb transformed primary monoblasts (AMV blasts), and CEF. As shown in Fig. 3C, ATBF1 transcripts were detected in all samples examined. The threshold levels were found in CEF, while the highest expression was detected in spleen and brain.

These results document the existence of chicken ATBF1 and substantiate functional studies with human ATBF1 and chicken v-Myb. Moreover, they show that chicken fibroblasts, which do not express endogenous Myb and synthesize only background amounts of ATBF1 mRNA, are suitable for Myb-ATBF1 co-expression experiments.

Full-length ATBF1 Forms in Vivo Complexes with v-Myb and Inhibits v-Myb Transcriptional Activity-- To document the direct binding of transiently expressed full-length ATBF1 and v-Myb proteins in CEF, a co-immunoprecipitation experiment was performed using a hemagglutinin epitope-tagged ATBF1. CEF nuclear proteins were immunoprecipitated with monoclonal anti-HA epitope antibody, and immunocomplexes were resolved by SDS-PAGE. Western blots were then developed with Myb-specific polyclonal antibody. As shown in Fig. 4A, v-Myb was detectable in anti-HA immunoprecipitates (lane 1). In the control experiment, where the identical procedure was applied to cells transfected only with v-Myb, no signal was obtained (lane 2).


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Fig. 4.   ATBF1-Myb interaction in chicken embryo fibroblasts. A, intracellular association of ATBF1 with v-Myb. Nuclei were isolated from cells co-transfected with v-Myb and ATBF1-HA expression plasmids (lane 1) or v-Myb expression plasmid alone (lane 2). Detergent-extracted proteins were immunoprecipitated with anti-HA antibody. Immunoprecipitates were separated on 10% SDS-PAGE and analyzed by Western blotting using v-Myb specific antibody. B, repression of v-Myb-dependent transcription activation by ATBF1. Plasmids expressing either no ATBF1 (pKW-2T), or ATBF1 deletion mutant (ATBF1-Delta MIS) or full-length ATBF1 (ATBF1) were cotransfected with reporter and Myb expression plasmids as indicated above each column diagram.

In order to assess whether ATBF1 can modulate transcriptional activity of v-Myb, both cDNAs were expressed in CEF in the presence of 3xMRE-CAT, the mim-1 MRE-based reporter. In these experiments, deletion mutants ATBF1-Delta MIS, Delta 835-900 v-Myb and point mutant L3,4A v-Myb were also used to characterize the effect of protein regions required for ATBF1-Myb binding in vitro. Both ATBF1 and ATBF1-Delta MIS displayed a general inhibitory effect, as they reduced the basal activity of the reporter as well as the expression of internal control plasmids having lacZ gene placed under RSV-long terminal repeat or cytomegalovirus promoters. ATBF1 proteins also inhibited v-Myb-dependent activation of 3xMRE-CAT reporter. The full-length ATBF1 appeared to be a stronger inhibitor of v-Myb than ATBF1-Delta MIS, but both CAT values were so close to the background that it was hard to draw a reliable conclusion using them. Therefore, SN0.5-CAT reporter containing a chicken c-myb promoter fragment (SN0.5) was used. Although there is no consensus MRE in the SN0.5, v-Myb binds to it in the electrophoretic mobility shift assay and activates the downstream reporter gene (CAT) by an order of magnitude stronger than multimerized mim-1 MRE sequences.4 With this reporter it was possible to demonstrate that full-length ATBF1 was a more efficient inhibitor than ATBF1-Delta MIS, as it reduced v-Myb activity to 65% of that observed in the presence of ATBF1-Delta MIS (Fig. 4B, a). Insertion of ATBF1 binding sequence (AFE) upstream from the SN0.5 fragment (AFE SN0.5-CAT reporter) resulted in a further reduction of v-Myb activity by full-length ATBF1 to 25% of that obtained with ATBF1-Delta MIS (Fig. 4B, b). This effect appeared to be specifically dependent on ATBF1 binding site, since the point mutation in AFE (AFEm), known to abrogate ATBF1 binding (see AFE[IV] element in Ref. 32), eliminated the increment of inhibition observed with AFE SN0.5-CAT (Fig. 4B-c). The conclusion that ATBF1 inhibits v-Myb via direct interaction has been further supported by experiments with Delta 835-900 v-Myb, the mutant lacking ATBF1 binding region found in pull-down experiments (see Fig. 2B, lane 9). This v-Myb mutant was inhibited to the same extent with both the ATBF1 and ATBF1-Delta MIS, independently of the presence of MIS (Fig. 4B, d). The L3,4A mutant of the v-Myb leucine zipper was inhibited by ATBF1 and ATBF1-Delta MIS similarly as the wt v-Myb, suggesting that Myb LZ may not participate in down-regulation of Myb transcriptional activity (Fig. 4B, e). In conclusion, the data show that ATBF1 can exert transcriptional repression of v-Myb, which is dependent on the presence of interacting amino acid sequences 279-300 (encoded by nucleotides 835-900) and 2484-2520 (encoded by nucleotides 7581-7689 in Ref. 26) in v-Myb and ATBF1, respectively.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The aim of this work was to identify a protein(s) interacting with v-Myb C terminus, as this part of the oncoprotein is involved in a subset of v-Myb activities, namely in the in vitro growth of transformed monoblasts as well as in the dynamics of the leukemic process in chicks (15).2 It was impossible, however, to use the entire v-Myb C terminus as a bait in the two-hybrid system due to the presence of the leucine zipper motif, which behaves as transcription activating domain in yeast (37). Deletion of C-terminal portion of leucine zipper (including L3, L4, and L5) provided a transcriptionally inactive bait pB-85, which was then used to search for proteins interacting with Myb sequences upstream from the leucine L3. Screening of a HeLa cDNA library resulted in the isolation of the 1.2-kilobase fragment encoding C terminus of the complex homeodomain/zinc finger protein, ATBF1 (26), which bound efficiently not only the bait fragment, but also full-length v-Myb and c-Myb proteins in vitro. In co-transfection transactivation assays, ATBF1 behaved as both the general and v-Myb specific repressor of transcription. In ATBF1, a proline-rich sequence represented by amino acids 2484-2520 was found to be crucial for interaction with Myb proteins and was therefore designated as MIS. This sequence was required for in vitro and in vivo interaction and brought about the ATBF1 repressive effect on v-Myb transactivation. No other functional data on the MIS-containing part of ATBF1 protein are available to date.

On the other hand, two v-Myb sequences were required for an efficient in vitro binding of ATBF1: the 22-amino acid region 279-300 (encoded by nucleotides 835-900) and leucines L3 and L4 of v-Myb LZ (not present in the bait molecule). Only amino acids 279-300, however, appeared to mediate the ATBF1 inhibitory effect on v-Myb transactivation, since their deletion eliminated the Myb-specific repression effect of ATBF1. No function has been assigned to this part of v-Myb yet. Our data indicate, however, that it contributes to the transcriptional activity of the oncoprotein, since its deletion reduced v-Myb transactivation to one third (as shown in Fig. 4B, d). In biological assays, the Delta 835-900 v-Myb mutant (in which amino acids 279-300 are missing) displayed reduced transformation and leukemia formation abilities compared with wild type v-Myb.2 Thus, v-Myb amino acids 279-300 can be viewed as a part of v-Myb transactivation domain that might activate transcription machinery through interaction with its factor(s). Binding of ATBF1 to Myb could prevent such interaction and result in down-regulation of v-Myb transcriptional activity similarly as deletion of this region. In agreement with that, ATBF1 did not show specific repression of the Delta 835-900 v-Myb mutant. On the contrary, leucines L3 and L4 of v-Myb LZ do not mediate ATBF1 inhibition of v-Myb transactivation, since mutation of leucines to alanines (that eliminated Myb-ATBF1 binding in vitro) had no effect on ATBF1 inhibitory activity. This mutation of leucines, which partially inactivates the growth promoting and leukemogenic abilities of v-Myb (15), does not inhibit but rather activates transcriptional activity of v-Myb (Ref. 15; Fig. 4B, e). Thus, v-Myb LZ leucines do not seem to take part in v-Myb interaction with the transcription machinery and could be involved in a regulatory mechanism not directly connected with transcription. If this is the case, ATBF1 could modulate also this Myb activity. Alternatively, leucines of v-Myb LZ would only stabilize the in vitro binding of ATBF1. Further work is necessary to address this question.

In addition to v-Myb specific inhibitory activity mediated by amino acids 2484-2520 and 279-300 in ATBF1 and v-Myb, respectively, ATBF1 displayed a general inhibitory effect on expression of different reporters. This supports a view of ATBF1 as a transcriptional inhibitor with a domain(s) of general effect and domains specific for particular tissue-specific transcription factors.

Recently, a related member of the ZfH family, ZEB, the vertebrate homologue of Drosophila ZfH-1, was shown to block transcriptional activity of c-Myb (38). Since our results suggest that c-Myb is likely to be affected by ATBF1 as well, the ZfH/ATBF1 family and Myb proteins might cooperate in regulation of development of specific tissues. The very high evolutionary conservation of the chicken ATBF1 and its high expression in spleen and brain tissues, where also c-Myb is expressed, support such a view.

    ACKNOWLEDGEMENT

We thank Dr. C. Vlcek for a part of sequencing work and Dr. S. Takácová for editorial assistance. We are indebted to Dr. P. Bart&udot;nek for the help with preparation of graphic files.

    FOOTNOTES

* This work was supported by Commission of the European Communities Grant CIPA-CT93-0143), Academy of Sciences of Czech Republic Grant A5052805, Grant Agency of the Czech Republic Grants 204/97/1146 (to M. D.) and 204/96/0014 (to Z. K.), and Howard Hughes Medical Institute Grant 75195-540401 (to M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF104331.

Dagger International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Fax: 420-2-24310955; E-mail: mdvorak{at}img.cas.cz.

2 M. Dvo&rbreve;áková, unpublished observations.

3 P. Pajer, unpublished data.

4 M. Dvo&rbreve;ák, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: AMV, avian myeloblastosis virus; DBD, DNA-binding domain; TA, transactivation domain; LZ, leucine zipper structure; ZfH, zinc finger homeodomain protein; CEF, chicken embryo fibroblasts; GST, glutathione S-transferase; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase; MRE, Myb-responsive element; AFE, ATBF1 recognition element; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; MIS, ATBF1-Myb-interacting sequence; RSV, Rous sarcoma virus; wt, wild type; PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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