Transactivation Domain Of c-Myb Research Articles

Binding-and-Folding Recognition of an Intrinsically Disordered Protein Using Online Learning Molecular Dynamics.

Intrinsically disordered proteins participate in many biological processes by folding upon binding to other proteins. However, coupled folding and binding processes are not well understood from an atomistic point of view. One of the main questions is whether folding occurs prior to or after binding. Here we use a novel, unbiased, high-throughput adaptive sampling approach to reconstruct the binding and folding between the disordered transactivation domain of c-Myb and the KIX domain of the CREB-binding protein. The reconstructed long-term dynamical process highlights the binding of a short stretch of amino acids on c-Myb as a folded α-helix. Leucine residues, especially Leu298-Leu302, establish initial native contacts that prime the binding and folding of the rest of the peptide, with a mixture of conformational selection on the N-terminal region with an induced fit of the C-terminal.

Conformational Preferences of an Intrinsically Disordered Protein Domain: A Case Study for Modern Force Fields.

Molecular simulations of intrinsically disordered proteins (IDPs) are challenging because they require sampling a very large number of relevant conformations, corresponding to a multitude of shallow minima in a flat free energy landscape. However, in the presence of a binding partner, the free energy landscape of an IDP can be dominated by few deep minima. This characteristic imposes high demands on the accuracy of the force field used to describe the molecular interactions. Here, as a model system for an IDP that is unstructured in solution but folds upon binding to a structured interaction partner, the transactivation domain of c-Myb was studied both in the unbound (free) form and when bound to the KIX domain. Six modern biomolecular force fields were systematically tested and compared in terms of their ability to describe the structural ensemble of the IDP. The protein force field/water model combinations included in this study are AMBER ff99SB-disp with its corresponding water model that was derived from TIP4P-D, CHARMM36m with TIP3P, ff15ipq with SPC/Eb, ff99SB*-ILDNP with TIP3P and TIP4P-D, and FB15 with TIP3P-FB water. Comparing the results from REST2-enhanced sampling simulations with experimental CD spectra and secondary chemical shifts reveals that the ff99SB-disp force field can realistically capture the broad and mildly helical structural ensemble of free c-Myb. The structural ensembles yielded by CHARMM36m, ff99SB*-ILDNP together with TIP4P-D water, and FB15 are also mildly helical; however, each of these force fields can be assigned a specific subset of c-Myb residues for which the simulations could not reproduce the experimental secondary chemical shifts. In addition, microsecond-timescale MD simulations of the KIX/c-Myb complex show that most force fields used preserve a stable helix fold of c-Myb in the complex. Still, all force fields predict a KIX/c-Myb complex interface that differs slightly from the structures provided by NMR because several NOE-derived distances between KIX and c-Myb were exceeded in the simulations. Taken together, the ff99SB-disp force field in the first place but also CHARMM36m, ff99SB*-ILDNP together with TIP4P-D water, and FB15 can be suitable choices for future simulation studies of the coupled folding and binding mechanism of the KIX/c-Myb complex and potentially also other IDPs.

Amyloid formation by intrinsically disordered trans-activation domain of cMyb

The cMyb trans-activation domain is one of the model systems to understand the folding and binding mechanisms in intrinsically disordered proteins. cMyb (291–315) TAD (cMyb TAD) upon interaction with KIX plays a crucial role in transcriptional regulation. However, nothing is known regarding its aggregation behaviour on change of buffer conditions or stressed environment. Notably, most of the disease-associated amyloid-forming proteins such as Aβ, Tau, α-synuclein, and amylin are natively unstructured. Nevertheless, to date, very fewer evidence on aggregation behaviours on TAD domains are available. Therefore, this is necessary to investigate the aggregation propensity of intrinsically disordered cMyb TAD domain in isolation. As an essential step in that direction, we have extensively studied the aggregation behaviour of cMyb TAD using the standard approaches for aggregation studies and systematically probed the amyloid conformations. These aggregates are ThT and ANS-positive whose amyloid nature was also confirmed by Far-UV CD spectroscopic studies suggesting that cMyb TAD fibrils are rich in β-sheet secondary structure, transmission electron microscopy revealed the formation of characteristic long branched amyloid fibrils of 6–16 nm diameter, and MTT assay in SH-SY5Y neuroblastoma cells suggest that these aggregates are cytotoxic. This amyloid nature of cMyb TAD may affect its binding with KIX and alter cMyb function (transcriptional regulation) under acidic/stressed conditions.

The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here we found that the transcription factor c-Myb was essential for generating and maintaining stem cells within the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell–like central memory cells than Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity upon adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.

Dual Mechanism of Rag Gene Repression by c-Myb during Pre-B Cell Proliferation.

Developing B lymphocytes undergo clonal expansion following successful immunoglobulin heavy chain gene rearrangement. During this proliferative burst, expression of the Rag genes is transiently repressed to prevent the generation of double-stranded DNA (dsDNA) breaks in cycling large pre-B cells. The Rag genes are then reexpressed in small, resting pre-B cells for immunoglobulin light chain gene rearrangement. We previously identified c-Myb as a repressor of Rag transcription during clonal expansion using Abelson murine leukemia virus-transformed B cells. Nevertheless, the molecular mechanisms by which c-Myb achieved precise spatiotemporal repression of Rag expression remained obscure. Here, we identify two mechanisms by which c-Myb represses Rag transcription. First, c-Myb negatively regulates the expression of the Rag activator Foxo1, an activity dependent on M303 in c-Myb's transactivation domain, and likely the recruitment of corepressors to the Foxo1 locus by c-Myb. Second, c-Myb represses Rag transcription directly by occupying the Erag enhancer and antagonizing Foxo1 binding to a consensus forkhead site in this cis-regulatory element that we show is crucial for Rag expression in Abelson pre-B cell lines. This work provides important mechanistic insight into how spatiotemporal expression of the Rag genes is tightly controlled during B lymphocyte development to prevent mistimed dsDNA breaks and their deleterious consequences.

Effect of Transient Helicity of cMyb TAD on its Binding Affinity to the Kix Domain of CBP/p300

Intrinsically disordered proteins (IDPs) are involved in various protein‐protein interactions and they often experience a coupled folding and binding reaction when they bind to their protein partners. Many IDPs, like the cMyb trans activation domain (cMyb TAD), exhibit transient helical structures in solution that resemble their bound structures. We hypothesize that the levels of transient helicity in the free IDP will affect the binding affinity to their protein partners in a predictable way. Previous studies have shown that increasing the transient helicity of intrinsically disordered p53 tumor suppressor increases the binding affinity to MDM2 and had negative effects on cellular function. We aim to study the effect of changing cMyb TAD transient helicity on its binding affinity to the kix domain of CBP/p300.cMyb is a hematopoietic regulator which is known to play a role in tumorigenesis. Unlike p53 TAD, cMyb TAD has a high residual helicity in solution in the absence of binding partners. Prolines that flank the N and C‐terminus of this helical region were mutated to alanines to determine whether there was a change in helicity that would affect the binding of Kix. Upon mutation, the increase in helicity had almost no effect on the binding affinity of the cMyb‐Kix complex. Further, mutations that reduce the helicity of cMyb‐TAD were designed to test the effect of reducing transient helicity on the binding affinity to Kix. To achieve this goal, conservative mutations of the solvent exposed amino acids in cMyb were made. We are currently measuring the transient helicity and binding affinity of these mutants with the long‐range goal of identifying a general relationship between changes in transient helicity and binding affinity of IDPs.Support or Funding InformationThis research was supported by funding to GWD by National Institutes of Health (2R01CA14124406‐A1 and 1R01GM115556‐01A1). This work was also supported by funding to JC by Wellcome trust (WT 095195MA) and by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship to MDC.

Small-Molecule Disruption of the Myb/p300 Cooperation Targets Acute Myeloid Leukemia Cells.

The transcription factor c-Myb is essential for the proliferation of hematopoietic cells and has been implicated in the development of leukemia and other human cancers. Pharmacologic inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. By using a Myb reporter cell line, we have identified plumbagin and several naphthoquinones as potent low-molecular weight Myb inhibitors. We demonstrate that these compounds inhibit c-Myb by binding to the c-Myb transactivation domain and disrupting the cooperation of c-Myb with the coactivator p300, a major driver of Myb activity. Naphthoquinone-induced inhibition of c-Myb suppresses Myb target gene expression and induces the differentiation of the myeloid leukemia cell line HL60. We demonstrate that murine and human primary acute myeloid leukemia cells are more sensitive to naphthoquinone-induced inhibition of clonogenic proliferation than normal hematopoietic progenitor cells. Overall, our work demonstrates for the first time the potential of naphthoquinones as small-molecule Myb inhibitors that may have therapeutic potential for the treatment of leukemia and other tumors driven by deregulated Myb. Mol Cancer Ther; 15(12); 2905-15. ©2016 AACR.

A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein–protein interactions

The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein–protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein–protein interactions of Myb with regulatory proteins.

Molecular Recognition by Templated Folding of an Intrinsically Disordered Protein

Intrinsically disordered proteins often become structured upon interacting with their partners. The mechanism of this ‘folding upon binding’ process, however, has not been fully characterised yet. Here we present a study of the folding of the intrinsically disordered transactivation domain of c-Myb (c-Myb) upon binding its partner KIX. By determining the structure of the folding transition state for the binding of wild-type and three mutational variants of KIX, we found a remarkable plasticity of the folding pathway of c-Myb. To explain this phenomenon, we show that the folding of c-Myb is templated by the structure of KIX. This adaptive folding behaviour, which occurs by heterogeneous nucleation, differs from the robust homogeneous nucleation typically observed for globular proteins. We suggest that this templated folding mechanism may enable intrinsically disordered proteins to achieve specific and reliable binding with multiple partners while avoiding aberrant interactions.

The mechanism of binding of the KIX domain to the mixed lineage leukemia protein and its allosteric role in the recognition of c-Myb.

The KIX domain is a mediator of the interaction between different transcription factors. This complex function is carried out via two distinct binding sites located on opposite sides of the protein; namely, the 'c-Myb site' and the 'MLL site', named after their characteristic ligands-the transactivation domain of c-Myb and the mixed lineage leukemia protein (MLL). Both these ligands are unstructured in isolation and fold only upon binding, posing the KIX domain as an ideal candidate to explore the binding induced folding reaction of intrinsically unstructured proteins. Here, we complement the recent kinetic description on the interaction between KIX and c-Myb, by characterizing the binding kinetics between KIX and MLL, at different pH and ionic strength conditions. Furthermore, we analyze quantitatively the mechanism of allosteric communication between the topologically distinct c-Myb and MLL sites. The implications of our results are discussed in the light of previous work on other intrinsically unstructured systems.

C-Myb Inhibits Myoblast Fusion

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3′ untranslated region (3′ UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3′ UTR of c-Myb was also important because the expression of c-Myb coding region with its 3′ UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3′ UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.

Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain of cMyb to CBP KIX.

Association rates for interactions between folded proteins have been investigated extensively, allowing the development of computational and theoretical prediction methods. Less is known about association rates for complexes where one or more partner is initially disordered, despite much speculation about how they may compare to those for folded proteins. We have attached a fluorophore to the N-terminus of the 25 amino acid cMyb peptide used previously in NMR and equilibrium studies (termed FITC-cMyb), and used this to monitor the kinetics of its interaction with the KIX protein. We have investigated the ionic strength and temperature dependence of the kinetics, and conclude that the association process is extremely fast, apparently exceeding the rates predicted by formulations applicable to interactions between pairs of folded proteins. This is despite the fact that not all collisions result in complex formation (there is an observable activation energy for the association process). We propose that this is partially a result of the disordered nature of the FITC-cMyb peptide itself.

A folding-after-binding mechanism describes the recognition between the transactivation domain of c-Myb and the KIX domain of the CREB-binding protein

A large body of evidence suggests that a considerable fraction of the human proteome may be at least in part intrinsically unstructured. While disordered, intrinsically unstructured proteins are nevertheless functional and mediate many interactions. Despite their significant role in regulation, however, little is known about the molecular mechanism whereby intrinsically unstructured proteins exert their function. This basic problem is critical to establish the role, if any, of disorder in cellular systems. Here we present kinetic experiments supporting a mechanism of binding-induced-folding when the KIX domain of the CREB-binding protein binds the transactivation domain of c-Myb, an intrinsically unstructured domain. The high-resolution structure of this physiologically important complex was previously determined by NMR spectroscopy. Our data reveal that c-Myb recognizes KIX by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step, i.e. binding precedes folding. On the basis of the pH dependence of the observed combination and dissociation rate constants we propose a plausible mechanism for complex formation. The implications of our results in the light of previous work on intrinsically unstructured systems are discussed.

The Histone Acetyltransferase TIP60 Interacts with c-Myb and Inactivates Its Transcriptional Activity in Human Leukemia

The histone acetyltransferase TIP60 is a coregulator of transcription factors and is implicated in tumorigenesis. In this study, we explored potential regulatory relationships between TIP60 and the c-Myb oncoprotein in hematopoietic cells. We first showed that TIP60 is a c-Myb interacting protein and that the interaction is dependent on the TIP60 acetyltransferase domain and c-Myb transactivation domain. We then found that coexpressing TIP60 decreases the transcriptional activation ability of c-Myb in functional reporter assays. A ChIP assay also revealed that TIP60 binds to the c-Myb target gene c-Myc promoter in a c-Myb-dependent manner. Consistently, knockdown of Tip60 expression by siRNA increased endogenous c-Myc expression. Furthermore, coimmunoprecipitation of Jurkat cell lysates revealed that c-Myb is associated with histone deacetylases HDAC1 and HDAC2, known to interact with TIP60 and repress transcription. Finally, we compared Tip60 expression in six primary AML samples with three normal CD34(+) cell samples using quantitative RT-PCR. Tip60 expression was significantly (∼60%) lower in the AML samples. In summary, these studies demonstrate that TIP60 negatively modulates c-Myb transcriptional activity by recruiting histone deacetylases in human hematopoietic cells, leading us to hypothesize that TIP60 is a normal regulator of c-Myb function and that dysregulated or mutated TIP60 may contribute to c-Myb-driven leukemogenesis.

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V(267)NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check.

The Acetyl-Transferase Tip60 Interacts with c-Myb and Inactivates Its Transcriptional Activity in Human Leukemia.

Abstract 3981Poster Board III-917The acetyl-transferaseTip60 (Tat interacting protein, 60 kDa; also known as K(lysine) acetyltransferase 5:KAT5) is a co-regulator of transcription factors and is implicated in tumorigenesis. The protooncogene c-myb encodes a transcription factor, c-Myb, which is essential for normal hematopoisis and promotes hematologic malignancies. In this study, we explored potential regulatory relationships between Tip60 and c-Myb in hematopoietic cells. We first sought to detect any direct physical interactions by performing co-immunoprecitation (co-IP) assays. These revealed that Tip60 associated with c-Myb in Jurkat (T leukemia) and K562 (CML) cells. In vitro translated, epitope-tagged c-Myb and Tip60 also interacted with each other, suggesting that the Tip60, c-Myb interaction did not require an adaptor protein or co-factor. We then sought to define the interacting protein domains. Deletion analysis studies revealed that the interaction between Tip60 and c-Myb was dependent on the Tip60 MYST acetytransferase domain and transactivation domain of c-Myb. We then determined whether Tip60 could acetylate c-Myb, a post-translational event known to modulate c-Myb activity. Interestingly, an in vitro acetyltransferase assay showed that c-Myb was not a substrate of Tip60, even though Tip60 acetylated itself in the same assay. We then examined the effect of Tip60 on the ability of c-Myb to transcriptionally activate known target genes. In HEK293T cells, co-expressing Tip60 with c-Myb decreased c-Myb's ability to activate a luciferase reporter driven by the cim-1 promoter, a verified c-Myb target, by ∼60% compared to activation in the absence of Tip60. The physiologic significance of this observation was then explored. A chromatin immunoprecipiation (ChIP) assay revealed that Tip60 bound to the c-myc promoter, another known c-Myb target gene, in K562 cells. Furthermore, inactivation of endogenous c-Myb in K562 cells stably expressing an inducible c-Myb DNA binding domain reduced the occupancy of Tip60 in the c-myc promoter, suggesting that Tip60 utilizes c-Myb to bind its preferred site in the c-myc promoter. Using c-Myb, Tip60, and appropriate control siRNAs we achieved specific knockdowns of c-Myb, and Tip60 (∼80-90%, and ∼70-80% respectively compared to controls). Consistent with prior reports, c-myc expression decreased ∼60% when c-Myb was targeted, and ∼50% increased when Tip60 was targeted. A mechanistic explanation was sought to explain this finding. Tip60 is represses transcription when associated with histone deacetylases (HDAC), including HDAC1, HDAC2 and HDAC7. Co-IP of Jurkat cell lysates revealed that c-Myb is associated with HDAC1 and HDAC2. Altogether, these data suggest that Tip60 directly associates with c-Myb, and may inhibit its transcriptional activity by recruiting histone deacetylases(HDAC1 and HDAC2) to the activation complex. Finally, we compared Tip60 expression in 6 primary AML samples, with 3 normal CD34+ cell samples using QRT-PCR. Tip60 expression was significantly (∼60%) lower in the AML samples. In summary, these studies demonstrate that Tip60 modulates c-Myb transcriptional activity in human hematopoietic cells leading us to hypothesize that Tip60 is a normal regulator of c-Myb function and that dysregulated or mutated Tip60 may contribute to c-Myb driven leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

C-Myb Associates with MLL1 through Menin, Augments MLL1's H3K4 Methylation Activity, and Regulates Hoxa9 and Meis1 Gene Expression in Primary Human Leukemia Cells.

Abstract 448The c-myb proto-oncogene was first identified as the cellular homologue of the v-myb oncogene carried by the avian leukemia viruses AMV, and E26. c-myb encodes a transcription factor, c-Myb, that is highly expressed in immature hematopoietic cells. In such primitive cells, c-Myb has been found to exert an important role in lineage fate selection, cell cycle progression, and differentiation of both myeloid, B, and T lymphoid progenitor cells. c-Myb is also highly expressed in many leukemia cells and on this basis has been implicated in leukemic transformation. Despite intensive study, a mechanisms based understanding for c-Myb's myriad effects on blood cell development has yet to be fully achieved though c-Myb's ability to interact with a variety of transcriptionally active co-factors, such as p300, CBP, and FLASH, as well as to modulate its own expression, have all been reported to contribute to its activities. Therefore, we undertook a series of biochemical, molecular, and clinical studies to further address c-Myb's role in leukemic hematopoiesis. Using in vitro translated proteins and nuclear extracts from leukemic cells in immunoprecipitation (IP) assays, we found that c-Myb is associated with MLL1, the SET1 proteins WDR5, RbBp5, and Ash2L, and menin, all of which form a complex with histone methyltransferase (HMT) activity. c-Myb associated with the MLL1 and SET1 proteins through menin, which served as an adapter protein by interacting (as previously shown) with the extreme amino terminus of the MLL1 protein, and, as we show, with a region around the c-Myb transactivation domain (aa 194-325). We demonstrated in vitro with purified proteins and an H3 peptide, that c-Myb contributed to the HMT activity of the MLL1 complex. In leukemia patients being treated with a c-myb targeted antisense oligodeoxynucleotide (ASODN), and in leukemic cell lines, silencing c-myb evoked a significant decrease in H3K4 methylation demonstrating biological relevance of this observation. The decrease in H3K4 methylation is the direct result of silencing c-myb and is not due to changes in cell proliferation, and could not be reproduced by silencing B-myb. Also, we confirmed that c-Myb is a downstream target of HoxA9, and Meis 1, but showed unexpectedly that leukemic blasts derived from the c-myb ASODN treated patients, and c-myb siRNA treated cell lines, decrease c-myb expression also led to a decrease in Hoxa9 and Meis1 expression. This suggested the presence of an autoregulatory feedback loop between c-Myb and HoxA9. This finding too was specific for c-myb and not associated with a block in proliferation or silencing B-myb. Finally, disrupting the c-Myb-MLL1 interaction impairs localization of MLL1 and menin on the Hoxa9 gene promoter, as well as the MLL-ENL induced transformation of normal murine bone marrow cells. In summary, our results bring new insights regarding c-Myb function in human hematopoietic cells, suggest new mechanisms whereby c-Myb may contribute to cell transformation, and suggest new therapeutic targets for the treatment of acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Solution Structure of the KIX Domain of CBP Bound to the Transactivation Domain of c-Myb

The hematopoietic transcription factor c-Myb activates transcription of target genes through direct interactions with the KIX domain of the co-activator CBP. The solution structure of the KIX domain in complex with the activation domain of c-Myb reveals a helical structure very similar to that adopted by KIX in complex with the phosphorylated kinase inducible domain (pKID) of CREB. While pKID contains two helices, αA and αB, which interact with KIX, the structure of bound c-Myb reveals a single bent amphipathic helix that binds in the same hydrophobic groove as the αB helix of pKID. The affinity of c-Myb for KIX is lower than that of pKID, and relies more heavily on optimal interactions of the single helix of c-Myb with residues in the hydrophobic groove. In particular, a deep hydrophobic pocket in KIX accounts for more than half the interactions with c-Myb observed by NMR. A bend in the α-helix of c-Myb enables a critical leucine side-chain to penetrate into this pocket more deeply than the equivalent leucine residue of pKID. The components that mediate the higher affinity of pKID for KIX, i.e. the phosphate group and the αA helix, are absent from c-Myb. Results from isothermal titration calorimetry, together with the structural data, point to a key difference between the two complexes in optimal pH for binding, as a result of differential pH-dependent interactions with histidine residues of KIX. These results explain the structural and thermodynamic basis for the observed constitutive versus inducible activation properties of c-Myb and CREB.

Conditional inhibition of erythroid differentiation by c-Myb/oestrogen receptor fusion proteins

The c- myb proto-oncogene encodes a transcription factor that has been implicated in the regulation of haemopoietic cell differentiation and appears also to be required for cell proliferation in a number of different lineages. Typically, transcription of c- myb is down-regulated during haemopoietic cell differentiation, and it has been found in several erythroid and myeloid cell lines that constitutive c- myb expression, from a transfected plasmid, blocks this differentiation process. To investigate further the activity of c- myb in haemopoietic cell differentiation, we have transfected Friend murine erythroleukaemia (F-MEL) cells with plasmids encoding conditionally active c-Myb/oestrogen receptor (Myb/ER) fusion proteins. Transcriptional activity of the Myb/ER fusion proteins was found to be strictly hormone-dependent, and this property was correlated with the ability of these proteins to inhibit erythroid differentiation. From analysis of a Myb/ER protein that lacks the c-Myb transactivation domain, it was apparent that the C-terminal ER transactivation domain could substitute for that of c-Myb in inhibition of differentiation. Activation of Myb/ER in F-MEL cells had no effect upon the early and transient inhibition of entry into S phase associated with dimethyl sulfoxide (DMSO) induction. Further analyses of α-globin and PU.1 gene transcription suggested that c-Myb is unable to influence gene expression immediately following DMSO-induction and that inhibition of F-MEL cell differentiation must therefore result from the function of c-Myb in the post-commitment period. Nonetheless, c-Myb had effects on the erythroid differentiation programme that were clearly dissociated from its role in cell proliferation. The potential use of this system to identify c-Myb target genes involved in F-MEL cell differentiation is discussed.