B [5], and is a key regulator of the differentiation and proliferation

B [5], and is a key regulator of the differentiation and proliferation of cells [6], [7], [8], [9]. In common with other members of the family B-Myb contains three functional regions (figure 1B),including a highly conserved, N-terminal DNA-binding domain (DBD) that recognises the ��-Sitosterol ��-D-glucoside consensus sequence PyAACG/TG (the Myb binding site (MBS), [10]). Adjacent to the DNA-binding region is a central transactivation domain (TAD), which shows no significant conservation across Myb proteins, whilst the regulatory C-terminus of the MC-LR site protein contains the highly conserved region (CR) and the negative regulatory domain (NRD, [11]). The activity of B-Myb is tightly regulated by several types of post translational modification including acetylation and phosphorylation [12], [13], [14], [15], [16], [17]. For example, cyclin A/CDK2-mediated phosphorylation of B-Myb at the transition from G1 to S phase dramatically increases its transactivation potential, which is believed to involve the relief of repression by the C-terminal NRD [12], [13], [14], [16], [18]. A number of B-Myb regulated genes have now been identified in which activation of transcription involves the binding of B-Myb to consensus target sites (MBS) in their promoter or enhancer regions, leading to the recruitment of essential partner proteins such as the coactivators p300 and CBP [11], [17], [19], [20], [21], [22], [23], [24], [25], [26]. Previous studies have shown that c-Myb and A-Myb interact with the KIX domain of p300 and CBP via their centralFeatures of the B-Myb TAD-p300 TAZ2 Complextransactivation domain [27], [28], [29]. In contrast, the B-Myb transactivation region (residues 240?71) has been found to interact with the C-terminal E1A-binding region of p300, in particular, residues 1710?891 [15], [17]. The precise molecular basis of the interaction and functional synergy between B-Myb and p300 remains to be determined and is the focus of the work reported here. In this communication we report detailed characterisation of the principal domains involved in B-Mybp300 interactions and of the complex formed, including identification of the binding surface for the B-Myb TAD on the TAZ2 domain of p300.Materials and Methods Expression, Refolding and Purification of p300 TAZThe coding sequence of human p300 (1726?812), corresponding to murine CBP TAZ2 [30], was obtained by PCR amplification and cloned into the NdeI and BamHI sites of pET23a (Novagen). The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21-CodonPlusH (DE3) RP cells (Stratagene) according to the manufacturer’s guidelines. Expression trials revealed that p300 TAZ2 was produced in E. coli as insoluble inclusion bodies. Unlabelled samples of p300 TAZ2 were produced from cells grown on LB medium. Uniformly 15N and 15 N/13C-labelled samples were 1527786 produced from cells grown on minimal medium, as described previously [31], [32]. After induction of p300 TAZ2 for 4 hours at 37uC, cells were harvested by centrifugation prior to lysis in buffer containing 50 mM TrisHCl, 100 mM NaCl, 2 mM Dithiothreitol (DTT) and 0.5 (v/v) Triton X-100, pH 8.0, supplemented with 100 mg/ml Lysozyme (Sigma), 10 mg/ml DNase (Sigma), 5 mM MgCl2, an EDTA-free protease inhibitor tablet (Roche), and 100 mM phenylmethyl sulfonylfluoride (PMSF). Washed inclusion bodies were preparedfrom the cell lysate as described previously [31] and then solubilised in denaturing buffer (6 M-guanidine HCl, 20 mM Tris, 20 mM DTT, 100 mM NaCl buffer, pH 8.5) to.B [5], and is a key regulator of the differentiation and proliferation of cells [6], [7], [8], [9]. In common with other members of the family B-Myb contains three functional regions (figure 1B),including a highly conserved, N-terminal DNA-binding domain (DBD) that recognises the consensus sequence PyAACG/TG (the Myb binding site (MBS), [10]). Adjacent to the DNA-binding region is a central transactivation domain (TAD), which shows no significant conservation across Myb proteins, whilst the regulatory C-terminus of the protein contains the highly conserved region (CR) and the negative regulatory domain (NRD, [11]). The activity of B-Myb is tightly regulated by several types of post translational modification including acetylation and phosphorylation [12], [13], [14], [15], [16], [17]. For example, cyclin A/CDK2-mediated phosphorylation of B-Myb at the transition from G1 to S phase dramatically increases its transactivation potential, which is believed to involve the relief of repression by the C-terminal NRD [12], [13], [14], [16], [18]. A number of B-Myb regulated genes have now been identified in which activation of transcription involves the binding of B-Myb to consensus target sites (MBS) in their promoter or enhancer regions, leading to the recruitment of essential partner proteins such as the coactivators p300 and CBP [11], [17], [19], [20], [21], [22], [23], [24], [25], [26]. Previous studies have shown that c-Myb and A-Myb interact with the KIX domain of p300 and CBP via their centralFeatures of the B-Myb TAD-p300 TAZ2 Complextransactivation domain [27], [28], [29]. In contrast, the B-Myb transactivation region (residues 240?71) has been found to interact with the C-terminal E1A-binding region of p300, in particular, residues 1710?891 [15], [17]. The precise molecular basis of the interaction and functional synergy between B-Myb and p300 remains to be determined and is the focus of the work reported here. In this communication we report detailed characterisation of the principal domains involved in B-Mybp300 interactions and of the complex formed, including identification of the binding surface for the B-Myb TAD on the TAZ2 domain of p300.Materials and Methods Expression, Refolding and Purification of p300 TAZThe coding sequence of human p300 (1726?812), corresponding to murine CBP TAZ2 [30], was obtained by PCR amplification and cloned into the NdeI and BamHI sites of pET23a (Novagen). The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21-CodonPlusH (DE3) RP cells (Stratagene) according to the manufacturer’s guidelines. Expression trials revealed that p300 TAZ2 was produced in E. coli as insoluble inclusion bodies. Unlabelled samples of p300 TAZ2 were produced from cells grown on LB medium. Uniformly 15N and 15 N/13C-labelled samples were 1527786 produced from cells grown on minimal medium, as described previously [31], [32]. After induction of p300 TAZ2 for 4 hours at 37uC, cells were harvested by centrifugation prior to lysis in buffer containing 50 mM TrisHCl, 100 mM NaCl, 2 mM Dithiothreitol (DTT) and 0.5 (v/v) Triton X-100, pH 8.0, supplemented with 100 mg/ml Lysozyme (Sigma), 10 mg/ml DNase (Sigma), 5 mM MgCl2, an EDTA-free protease inhibitor tablet (Roche), and 100 mM phenylmethyl sulfonylfluoride (PMSF). Washed inclusion bodies were preparedfrom the cell lysate as described previously [31] and then solubilised in denaturing buffer (6 M-guanidine HCl, 20 mM Tris, 20 mM DTT, 100 mM NaCl buffer, pH 8.5) to.