INTRODUCTION

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The complex interplay between transcription factors bound to DNA provides enormous opportunity for regulation of gene expression. Not surprisingly, combinatorial control that utilizes multiple transcription factors is the rule for most eukaryotic enhancers. Recent findings implicate auto-regulation as an integral feature of these protein partnerships. There are regions within proteins that negatively regulate DNA binding or protein-protein interactions, presumably through intramolecular interactions (24). Positive regulation, as mediated by the creation of multiprotein complexes, can inactivate auto-inhibition. The molecular pathways for assembling these multiprotein complexes are beginning to emerge from systems in which both biochemical and structural approaches are aggressively undertaken.

The DNA-binding  subunits of the core-binding factors (CBFs) represent a model system of combinatorial control, as they display auto-inhibition that is rescinded through interactions with two different partner proteins. One partner is CBF, a subunit that binds CBF subunits and stimulates DNA-binding activity without itself binding DNA (56, 85). CBF subunits also interact with members of the ets family of DNA-binding proteins to form ternary complexes on DNA (19, 33, 41, 78, 86). These different classes of partnerships provide an opportunity to develop a mechanistic model for regulating DNA binding by both intra- and intermolecular interactions.

The CBFs comprise a small family of proteins involved in multiple developmental pathways in vertebrates and invertebrates (75). DNA-binding CBF subunits in mammals are encoded by three genes (CBFA1, CBFA2 (AML1), and CBFA3), and the non-DNA-binding CBF subunit is encoded by the CBFB gene (4, 5, 36, 39, 48, 56, 57, 85). CBFA1 is required for bone development in mammals (34, 60). CBFA2 (AML1) and CBFB are essential for the emergence of definitive hematopoietic progenitors and stem cells in the mammalian embryo (52, 53, 58, 68, 82, 83). The Drosophila CBFA homolog runt functions in three developmental pathways: sex determination, segmentation, and neurogenesis (16, 17, 28, 67). The Drosophila gene lozenge, which also encodes a DNA-binding  subunit, plays a role in developmental pathways involving the eye, antenna, and tarsal claws and in the development of crystal cells, a blood cell lineage (13, 64, 77).

The ets proteins constitute a larger family of transcription factors that share a common DNA-binding domain, termed the ETS domain (25, 71). There are over 50 ets genes identified throughout metazoa, including over 20 paralogs in the human genome. Studies of vertebrate, Caenorhabditis elegans, and Drosophila ets proteins demonstrate roles in cell growth, differentiation, and transformation. For example PointedP2 (PntP2), a proposed ortholog of mammalian Ets-1 and Ets-2, is essential for R7 photoreceptor development in Drosophila and is the nuclear target of phosphorylation in the signal transduction pathway originating from the Sevenless receptor (2, 11, 59). In hematopoiesis, the ets protein PU.1 is required for B-cell and macrophage development (42, 70). Ets-1 is required for natural killer cell development (6), while both Ets-1 and Fli-1 are required for maintaining normal numbers of T cells (9, 44, 50). Both ets and CBF genes (FLI1, ERG, TEL, CBFA2, and CBFB) are frequent targets of chromosomal translocations in human leukemias (63); thus, dysregulation of ets or CBF function appears to be an underlying cause of hematopoietic transformation. One translocation, t(12;21), the most frequent chromosomal rearrangement in pediatric acute lymphocytic leukemia (43, 66, 72), actually fuses the ets gene TEL to CBFA2 (23, 65). Other ets and CBF genes (FLI-1, Pu.1, CBFA1, and ets-1) are preferential proviral insertion sites in leukemias and lymphomas induced by retroviruses (7, 49, 76) or oncogenes captured by acutely transforming retroviruses that cause leukemia (35, 54).

Many cell types in vertebrates express multiple ets genes, leading to a requirement for regulatory pathways that can dictate specificity of action of a particular ets protein. A common pathway to such specificity is partnerships with other transcription factors. Two well-characterized examples are the requisite interaction between serum response factor and one of the ets proteins Elk-1 and SAP-1 (14, 38) and the partnership between the ets protein PU.1 and the insulin response factor-related protein Pip (10). Biochemical and genetic analyses suggest that certain ets and CBF proteins also form partnerships. In Drosophila, both PntP2 and Lozenge are required for R7 cell development; PntP2 receives the signal from the Sevenless receptor, while Lozenge is required for the competency of R7 precursor cells to respond to the Sevenless signal (11, 13, 59). In vertebrates, Ets-1, Ets-2, PU.1, and GABP have been implicated as putative partners for the CBF proteins in regulating transcription of genes expressed in T, B, and myeloid cells (18, 19, 33, 41, 62, 78, 86). Ets-1 and CBF proteins were shown to bind cooperatively to the T-cell receptor - and -chain enhancers, and synergistically activate transcription from the T-cell receptor -chain enhancer in vivo and in vitro (19, 33, 41, 78, 86). The minimal B-cell-specific enhancer from the immunoglobulin µ-chain gene consists of binding sites for PU.1, CBF, and Ets-1 (or a related ets protein) (18). PU.1 and CBF2 cooperatively activate transcription from the macrophage colony-stimulating factor promoter in myeloid cells (62). The osteopontin gene, which encodes a major noncollagenous bone matrix protein, contains a promoter responsive to both the CBF1 protein and Ets-1 (69).

In this study, we used rigorous quantitative analyses to approach the issues of building multiprotein complexes. This methodology provides a framework for mechanistic investigations of both intra- and intermolecular regulation, including key insights for analyzing the structural basis of cooperativity between CBF2 and two of its partners, CBF and Ets-1. The CBF proteins share a 128-amino-acid region of homology, named the Runt domain after the founding member of the CBF family (31). The Runt domain constitutes the DNA-binding domain of the CBF proteins and the heterodimerization domain for CBF (31, 45, 57). Here we show that the full-length CBF2 protein exhibits auto-inhibition, and we identify sequences C terminal to the Runt domain of CBF2 that inhibit both DNA binding and heterodimerization with the CBF subunit. The C-terminal inhibitory sequences in CBF2, however, do not repress binding of the - heterodimer to DNA. The second partnership that we characterize is that between CBF2 and Ets-1. The sequences within CBF2 that modulate its interaction with Ets-1 map to the N-terminal 214 amino acids, whereas the C-terminal auto-inhibitory sequences in CBF2 are not required. Finally, we demonstrate that cooperative binding of CBF2 with Ets-1 is not augmented by the CBF subunit. A model that integrates these phenomena is presented.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of CBF2(451) and truncated derivatives. We created a modified pVL1392 baculovirus transfer vector containing a Kozak sequence followed by sequences encoding a hexahistidine (H₆) tag, two FLAG epitopes [(FLAG)₂], and coding sequences for full-length CBF2 [CBF2(451)] (75) or its truncated derivatives. A PCR primer complementary to the H₆ codons in the bacterial expression plasmid pQE30 (Qiagen), with a Kozak sequence and a BglII site at the 5' end (5'-TTAGATCTCCGCCATGGGAGGATCGCATCACCATC-3' was used in conjunction with a reverse primer (5'-CATTACTGGATCTATCAACAGG-3') to amplify the H₆ tag from pQE30. The PCR product was digested with BglII and BamHI and subcloned into the pBK-CMV vector (Stratagene) between the BglII (converted from a SpeI site) and BamHI sites. Complementary DNA encoding full-length CBF2(451) (with an in-frame BamHI site preceding the ATG start codon) was subcloned in frame with the H₆ tag, between the BamHI site and a KpnI site in the pBK-CMV polylinker. The resulting plasmid was partially digested with BamHI, and complementary oligonucleotides encoding the FLAG epitope (5'-GATCTATGGACTACAAAGACGATGACGATAAGG-3' and 3'-ATACCTGATGTTTCTGCTACTGCTATTCCCTAG-5') were subcloned into the BamHI site.

Partial purification of CBF2(451). All purification steps were performed at 4°C. Sf9 cells were harvested by centrifugation at 1,000 × g, and crude nuclei were prepared by hypotonic lysis (15). Nuclei were resuspended in 5 packed cell volumes of 6 M guanidine HCl-10 mM sodium phosphate (pH 8.0)-0.1% Triton X-100-10% glycerol (buffer A) and stirred for 1 h. The nuclear debris was pelleted (25,000 × g, 15 min), and the supernatant from 1.5 × 10⁹ Sf9 cells was incubated with 2 to 3 ml of Ni-nitrilotriacetic acid (NTA) resin (Qiagen) for 1 h with continuous agitation. The protein was renatured on the Ni-NTA column by the following batch washes (5 min each, followed by centrifugation at 200 × g for 5 min): two washes with 30 ml of buffer A; three washes with 50 ml of 8 M urea-10 mM sodium phosphate (pH 8.0)-150 mM NaCl-0.1% Triton X-100-10% glycerol (buffer B); three washes with 50 ml of 8 M urea- 10 mM sodium phosphate (pH 7.4)-150 mM NaCl-0.1% Triton X-100-10% glycerol (buffer C); three washes with 50 ml of 1 M urea-10 mM sodium phosphate (pH 7.4)-300 mM NaCl-0.1% Triton X-100-10% glycerol (buffer D); and three washes with 50 ml of 10 mM sodium phosphate (pH 7.4)-300 mM NaCl-0.1% Triton X-100-10% glycerol (buffer E). The resin was then resuspended in 10 ml of 10 mM sodium phosphate (pH 7.4)-150 mM NaCl-0.1% Triton X-100-10% glycerol (buffer F) and poured into a column (5 ml). H₆(FLAG)₂-CBF2(451) was eluted from the Ni-NTA resin with 20 ml of buffer F containing 200 mM imidazole. Protein fractions were frozen and stored at 70°C.

Native purification of truncated CBF2 proteins. Crude nuclei from infected Sf9 cells were prepared by hypotonic lysis and extracted with 20 ml of 10 mM sodium phosphate (pH 7.8)-500 mM NaCl-10% glycerol (buffer G) for 30 min at 4°C. The nuclei were pelleted (25,000 × g, 20 min), and the supernatant was collected and incubated with 2 ml of Ni-NTA resin for 1 h with continuous agitation. The resin was washed once with 20 ml of 10 mM sodium phosphate (pH 7.8)-500 mM NaCl-0.1% Triton X-100-10% glycerol (buffer H), poured into a column (5 ml), and washed with 20 ml of buffer H plus 15 mM imidazole. H₆(FLAG)₂-CBF2 proteins were eluted with 10 ml of 10 mM sodium phosphate-150 mM NaCl-0.1% Triton X-100-10% glycerol (buffer I) plus 200 mM imidazole. Peak fractions from the Ni-NTA column were loaded directly onto an anti-FLAG M2 monoclonal antibody column (1 ml; Sigma), and the flowthrough fraction was readsorbed three times. The column was washed with 50 ml of buffer I, and the H₆(FLAG)₂-CBF2 proteins were eluted from the anti-FLAG column with 0.33 mM FLAG peptide in 6 ml of buffer I as instructed by the manufacturer. Protein fractions were frozen and stored at 70°C. The concentrations of CBF2 active for DNA binding were determined as described previously (12, 29).

N280

Synthetic oligonucleotides. A high-affinity site (81, 84) was used to measure the binding affinity of CBF to DNA. An ets/CBF composite oligonucleotide (SC1/core) derived from the murine leukemia virus (MLV) enhancer was used to measure cooperative DNA binding. SC1/core contains a high-affinity ets site (55) juxtaposed to a core-binding site:

The spacing of the native MLV enhancer is retained in SC1/core. The higher affinity of the SC1 site facilitated more accurate quantification.

EMSA. Equilibrium constants of CBF2 and Ets-1 were determined by electrophoretic mobility shift assays (EMSA) using conditions described previously (12, 29). When protein titrations were used, the concentrations were in a range that resulted in approximately 0 to 100% binding. For proteins that were added in saturating amounts, the concentrations were at least 10-fold above the K_D (equilibrium dissociation constant) of the protein for its specific site (CBF2 and Ets-1; 2 × 10⁸ M), ensuring >90% DNA occupancy. In all assays, the DNA concentrations were at least 10-fold below the estimated K_D of either CBF2 or Ets-1 (10¹¹ M), ensuring that the total protein [P_t] was an accurate estimate of free protein [P]. For most of the binding reactions, the protein(s) and DNA were added simultaneously and incubated on ice for 20 min. To measure the apparent affinity of CBF2 in the presence of Ets-1, CBF2 and DNA were preincubated for 20 min on ice. Saturating amounts of Ets-1 were added following the incubation, and all of the reactions were incubated for an additional 20 min. In most cases, DNA and protein-DNA complexes were resolved on 6% native polyacrylamide gels. Eight percent acrylamide gels were used for measuring cooperative DNA binding with CBF2 fragments smaller than CBF2(1-331) and for measuring the K_D of CBF for CBF2-DNA complexes. Following electrophoresis, the gels were dried and the radioactivity was quantified by the volume integration of individual bands by phosphorimaging (Molecular Dynamics ImageQuant).

Measurement of K_D. For assays containing only a single binding species, CBF2 or Ets-1, K_Ds were measured as described previously (29). In brief, the fraction of free DNA, [D]/[D_t], was determined by measuring the ratio of the free DNA signal analyzed at each protein concentration to the DNA signal in a control lane containing no protein. The fraction of DNA in complex with protein, [PD]/[D_t], was derived from the relationship [PD]/[D_t] = 1  [D]/[D_t]. Multiple experiments were performed with the same range of protein concentrations to provide a mean and standard error of each data point. Data were fit to the rearranged mass action equation, [PD]/[D_t] = 1/(1 + K_D/[P]), using nonlinear least squares analyses (Kaleidagraph; Synergy Software) to derive K_D values with standard error.

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	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Purification of CBF2 proteins. The CBF2 proteins were produced by using a baculovirus expression system and partially purified by His and FLAG tag affinity chromatography (Fig. 1). Full-length CBF2(451), due to its tight association to the nuclear matrix (32, 87), was obtained from insect cell extracts under denaturing conditions and refolded on the Ni-NTA column (Fig. 1A). Limited quantities of partially purified material were obtained by this method, and no further purification was possible without loss of activity. A series of C-terminal truncations in CBF2(451) starting at amino acid 331 were engineered (Fig. 2B). These truncated proteins were purified to homogeneity from soluble nuclear extracts by sequential affinity chromatography on Ni-NTA and anti-FLAG antibody columns (Fig. 1A). The concentrations of active full-length and truncated CBF2 proteins were determined by DNA titrations. Representative examples of the purification and activity determination are shown in Fig. 1.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Expression and purification of CBF2. (A) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel displaying fractions from each step of the purification for CBF2(451) and two truncated derivatives, CBF2(1-331) and CBF2(1-214). Lanes: M, molecular weight markers; NE, unfractionated nuclear extract; Ni-NTA, eluate from the Ni-NTA column; FLAG, eluate from the anti-FLAG monoclonal antibody column. Arrows indicate expected position of the CBF2 bands. (B) Activities of CBF2 proteins quantified by DNA titration in an EMSA. Concentrations (molar) of protein-DNA complex [PD] versus total input DNA [Dt] are plotted.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Modulation of CBF2 DNA binding by C-terminal sequences. (A) Equilibrium DNA binding studies of full-length CBF2(451) and CBF2(41-214) were performed by EMSA and used to generate DNA binding curves. Data from at least three experiments provide mean and standard error for each data point. KD values were obtained by curve fitting as described in Materials and Methods. (B) Summary of equilibrium dissociation constants for truncated CBF2. The black rectangle in the schematic diagram of CBF2 represents the DNA-binding Runt domain. The gray and stippled boxes represent the H6 and FLAG tags, respectively. Relative affinity was calculated as the ratio of mutant affinity to the affinity of CBF2(451). (C) Summary of equilibrium dissociation constants for CBF2 proteins tagged at amino acid 312 with H6 (gray box).

Sequences C terminal to the Runt domain in CBF2(451) inhibit DNA binding. Quantitative DNA binding assays detected a significant difference between the affinity of full-length CBF2(451) and the isolated DNA-binding Runt domain, CBF2(41-214). Figure 2A presents protein titrations performed on a high-affinity core site. Full-length CBF2(451) displays a 69-fold-lower affinity for DNA than CBF2(41-214). Sequences in CBF2(451) that inhibit DNA binding were mapped by analyzing the affinity of sequentially truncated proteins (Fig. 2B). A C-terminal truncation to amino acid 214 [CBF2(1-214)] derepressed DNA binding significantly (34-fold). Further truncation from amino acid 214 to 190 had no added effect. All truncated CBF2 proteins containing additional C-terminal sequences between amino acids 214 and 451 exhibited lower DNA-binding affinity than CBF2(1-214). However, none of the truncated proteins bound DNA as poorly as CBF2(451). These results map the C-terminal inhibitory sequences over a large region between amino acids 214 and 451, and they suggest that there are multiple inhibitory elements distributed throughout this large region. Alternatively, the inhibitory sequences are distant from each other in the primary structure but located on a single surface of the folded protein.

C-terminal sequences in CBF2 modulate heterodimerization with CBF. CBF increases the affinity of the CBF subunits for DNA. In quantitative analyses, a sixfold increase in DNA-binding affinity of a Runt domain fragment, CBF2(41-214), was observed in the presence of the CBF subunit (Crute et al., submitted). The auto-inhibition phenomenon raises the question of whether the inhibitory sequences that affect DNA binding also influence binding of the CBF2-CBF heterodimer to DNA or modulate heterodimerization of the CBF2 and CBF subunits. To address these questions, we analyzed DNA binding of inhibited and activated forms of CBF2 in the presence and absence of CBF. CBF2(1-331) was chosen as the inhibited species, as it is the largest CBF2 protein fragment that we could purify to homogeneity. The binding properties of CBF2(1-331) were compared to those of the isolated Runt domain CBF2(41-214), which represents the uninhibited species. CBF2(1-214), another uninhibited species, was also analyzed to assess the impact of sequences N terminal to the Runt domain on interactions with CBF on and off the DNA.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Thermodynamic box describing interactions between CBF2, CBF, and DNA. (A) Schematic diagram of the potential interactions between CBF2 (), CBF (), and DNA. The modeled bend in DNA induced by the Runt domain is suggested by both circular permutation analysis and circular dichroism spectroscopy (22; Crute et al., submitted). (B) Equilibrium dissociation constants (K2) of CBF2(41-214), CBF2(1-214), and CBF2(1-331) for DNA. Data from at least three experiments are presented. Standard errors are 1.1 × 1012 M, 2.1 × 1012 M, and 7.1 × 1012 M, respectively. (C) Equilibrium dissociation constants (K4) of CBF2-CBF heterodimers for DNA. Standard errors are 3.9 × 1013 M for CBF2(1-214) and 1.8 × 1013 M for CBF2(1-331). (D) Equilibrium dissociation constants (K3) of CBF for CBF2-DNA complexes. Data represent at least three experiments. Standard errors are 3.2 × 109 M, 1.5 × 109 M, and 3.5 × 109 M for CBF2(41-214), CBF2(1-214), and CBF2(1-331), respectively. (E) Summary of equilibrium dissociation constants K1, K2, K3, and K4. K4 for CBF2(41-214) was not determined directly but calculated from K2K3 = K1K4. K1 for CBF2(41-214) was determined independently (Crute et al., submitted).

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Ets-1 enhances CBF2 DNA binding. CBF2 also functions in association with Ets-1 (33, 86). To compare this partnership to that of the CBF2-CBF heterodimer, quantitative EMSAs were used to investigate DNA binding cooperativity. We chose a composite binding site that contains a high-affinity ets binding site (SC1) (55) juxtaposed to a CBF binding site similar to that found in the Moloney MLV enhancer (74). The spacing between the two sites retains the configuration within the Moloney MLV enhancer. The binding affinity of each protein alone on this engineered composite site, termed SC1/core, was determined by protein titrations with a constant, limited amount of DNA (10¹² M). The K_D of CBF2(1-331) for the SC1/core site was 3.0 × 10⁹ M, and the K_D of Ets-1 was 8.5 × 10¹⁰ M (Fig. 4A and B).

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Ets-1 and CBF2 bind DNA cooperatively. (A) EMSA of equilibrium DNA binding studies of CBF2(1-331) titrated onto DNA alone or in the presence of Ets-1 (left) or Ets-1 titrated onto DNA alone and in the presence of CBF2(1-331) (right). (B) Equilibrium DNA binding curves for CBF2(1-331) (left) and Ets-1 (right); data from panel A. Symbols: , binary protein = DNA complexes; , ternary complexes. Equilibrium DNA binding curves display [PD/[Dt] as the mean (±standard error) of at least two independent experiments. (C) Thermodynamic box depicting potential interactions between Ets-1, CBF2, and DNA. Equilibrium dissociation constants were obtained from panels A and B. KD values and standard error were obtained from the curve fit of means as described in Materials and Methods.

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Specific regions of CBF2 are involved in cooperative DNA binding with Ets-1. Sequences required for cooperative interactions with Ets-1 were mapped by testing deletion mutants of CBF2. Protein titrations of CBF2 were performed under saturating conditions for Ets-1 (Fig. 5). Removal of CBF2 sequences C terminal to amino acid 214 did not affect cooperative binding with Ets-1 (Fig. 5B). Thus, the intramolecular inhibitory sequences in the C terminus of CBF2(1-331) do not appear to be required for cooperative binding. No cooperative binding was observed between Ets-1 and the isolated CBF2 Runt domain, CBF2(41-214) (Fig. 5C). Removal of amino acids 190-214 to create CBF2(1-190) also disrupted cooperative DNA binding with Ets-1 (Fig. 5D). Again, reciprocal cooperativity was obtained when Ets-1 was titrated onto DNA saturated with the truncated proteins CBF2(1-331) and CBF2(1-214) but not with CBF2(41-214) and CBF2(1-190) (data not shown). Thus, sequences N terminal to amino acid 41 and between amino acids 190 and 214 in CBF2 contribute to cooperative binding with Ets-1.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Sequences in CBF2 required for cooperative DNA binding with Ets-1. Equilibrium DNA binding studies were performed by EMSA with truncated CBF2 proteins in the absence (open circles) or presence (closed circles) of Ets-1 (A to D). Equilibrium DNA binding curves display [PD]/[Dt] as the mean (±standard error) of at least two independent experiments. (E) Summary of equilibrium dissociation constants derived from binding curves in panels A to D. KD values and standard error were obtained from the curve fit of means as described in Materials and Methods.

CBF and Ets-1 do not synergistically stimulate CBF2 DNA binding. Our findings implicate both CBF and Ets-1 as partners for CBF2. A remaining question is whether these two proteins can work together to enhance CBF2 DNA binding. To facilitate the visualization of complexes containing all three proteins on DNA, we used an Ets-1 deletion mutant, Ets-1^N280, that has a molecular mass of 18 kDa. The accompanying report (20) demonstrates that Ets-1^N280 retains all sequences required for cooperative binding with CBF2(1-331). Protein-DNA complexes containing Ets-1^N280 alone, CBF2 alone, CBF2-CBF, CBF2-Ets-1^N280, and CBF2-CBF-Ets-1^N280 can be clearly distinguished by EMSA (Fig. 6A). We titrated the CBF2(1-331)-CBF heterodimer onto DNA alone and onto DNA saturated with Ets-1^N280 (Fig. 6A and B). Ets1^N280 did not further augment binding of CBF2-CBF to DNA (Fig. 6B and C). In a reciprocal experiment, we titrated Ets-1^N280 onto DNA saturated with CBF2(1-331) in the presence or absence of CBF (Fig. 6). The presence of CBF did not further augment cooperative DNA binding between CBF2 and Ets-1^N280. In other words, no synergistic activation was observed in the presence of both CBF2 partner proteins. In an important control, comparable levels of CBF (in the absence of CBF2) did not affect the affinity of Ets-1^N280 for DNA (data not shown). We conclude that Ets-1^N280 and CBF cannot stimulate DNA binding by CBF2(1-331) in a synergistic or even an additive fashion on this composite site.

View larger version (55K): [in this window] [in a new window]   FIG. 6.   DNA-binding enhancement by Ets-1 and CBF is neither additive nor synergistic. (A) EMSA of equilibrium DNA binding studies of CBF2(1-331) titrated onto DNA saturated with Ets-1N280 in the presence of CBF protein (left) and of Ets-1N280 titrated onto DNA saturated with the CBF2-CBF heterodimer (right). Control lanes to the left of each panel document the position of each of the protein-DNA complexes. (B) Equilibrium DNA binding curves for CBF2(1-331) (left) and Ets-1N280 (right). The identity of each curve is indicated in panel C. (C) Summary of equilibrium dissociation constants. Relative binding affinities (fold enhancement) compare KD values for multiprotein-DNA complexes to those obtained from DNA binding studies of Ets-1 and CBF2 in isolation. KD values are presented as the mean (±standard error) of at least two independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

CBF2-CBF partnership. We quantitatively analyzed DNA binding by CBF2 and modulation of this activity by intramolecular inhibitory sequences and by two protein partners, CBF and Ets-1. CBF2 DNA binding is inhibited by at least two independent domains. The first domain is the DNA-binding Runt domain itself. The CBF subunit stimulates DNA binding by the Runt domain sixfold. We have proposed that the Runt domain assumes an inhibited conformation that is alleviated by association with the CBF subunit (Crute et al., submitted). Indeed, circular dichroism spectroscopy reveals that association of the Runt domain and CBF, either in solution or on the DNA, is accompanied by a conformational change in one or both proteins (Crute et al., submitted). Our working hypothesis is that CBF "locks in" a high-affinity DNA-binding conformation of the Runt domain (Fig. 7A and B). The structural basis for this phenomenon awaits determination of the Runt domain and CBF structures, which are under way (8, 21, 27, 51).

View larger version (31K): [in this window] [in a new window]   FIG. 7.   Models for interactions between CBF2, CBF, and Ets-1. (A) The Runt domain (RD) is in equilibrium between a high- and low-affinity DNA-binding conformation. (B) Heterodimerization with CBF () locks the Runt domain into its high-affinity DNA-binding conformation, shifting the DNA-binding equilibrium to the right. (C) C-terminal inhibitory sequences in CBF2 further shift the equilibrium of the Runt domain toward its low-affinity DNA-binding conformation and mask the CBF heterodimerization surface. Association of the C-terminal inhibitory sequences to the Runt domain is destabilized when CBF2 is bound to DNA. Dissociation of the inhibitory sequences unmasks the CBF binding surface on the Runt domain. (D) The high-affinity DNA-binding conformation of the Runt domain is stabilized by the CBF subunit. Association of the C-terminal inhibitory sequences to the Runt domain is also directly inhibited by the CBF subunit, which masks the interaction site. The DNA-binding affinity of this complex is the same as that of the Runt domain-CBF complex in panel B. (E) Binding of CBF2 to DNA exposes the Ets-1 interaction surface, which includes (but is not restricted to) sequences N terminal to the Runt domain. Tethering of Ets-1 to CBF2 on the DNA increases the likelihood of a productive binding event, resulting in increased affinity. Ets-1 does not mask the Runt domain surface to which CBF and the C-terminal inhibitory domain bind. Conformational changes in the Ets-1 protein itself are not depicted in this diagram (see the accompanying report [20]).

View larger version (13K): [in this window] [in a new window]   FIG. 8.   Summary of CBF2(451) functional domains. Shown are boundaries of the DNA-binding and heterodimerization domains as defined by Kagoshima et al. (30). Autoinhibition of both DNA binding and heterodimerization maps to the C-terminal half of the protein. RD, Runt domain.

Partnership with Ets-1. Cooperative DNA binding between CBF2(1-331) and Ets-1 provides another example whereby auto-inhibition is rescinded through protein-protein interactions. Ets-1 increases the affinity of CBF2(1-331) for DNA approximately sevenfold. Enhancement of CBF2(1-331) DNA binding by Ets-1 required preincubating CBF2(1-331) with DNA prior to addition of Ets-1. This order-of-addition effect strongly suggests a conformational change in CBF2 or that the DNA is necessary for cooperative DNA binding. The accompanying report (20) demonstrates that cooperative binding between Ets-1 and CBF2(1-331) also occurs on nicked DNA templates, indicating that cooperativity is unlikely to be mediated by through-DNA effects. Taken together, the data suggest that a DNA-induced conformational change in the CBF2(1-331) protein is required for cooperative DNA binding with Ets-1 to occur. We hypothesize that this conformational change must precede the entry of Ets-1 into the ternary complex to enable the most stable complex to form.