seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 11, 2000: pp. 377–382 doi: 10.1006/scdb.2000.0182, available online at http://www.idealibrary.com on

CBF—A biophysical perspective

John H. Bushweller

Core binding factor (CBF) is a heterodimeric transcription of all fetal and blood cell lineages in the mammalian factor consisting of a DNA-binding subunit (Runx, also embryo.6, 7 Chromosomal translocations involving referred to as CBFA, AML 1, PEBP2α) and a non- the RUNX1 and CBFB genes have been identified in DNA-binding subunit (CBFB). Biophysical characterization a substantial number of myeloid and lymphocytic of the two proteins and their interactions is providing leukemias.8 The clear importance of CBF in normal a detailed understanding of this important transcription development as well as in leukemia makes their factor at the molecular level. Measurements of the relevant biophysical characterization an essential component binding constants are helping to elucidate the mechanism of an overall effort to understand the functioning of of leukemogenesis associated with altered forms of these CBF both in its normal developmental role and in its proteins. Determination of the 3D structures of CBFB and role as an oncogene. the DNA- and CBFB-binding domain of Runx, referred to as the Runt domain, are providing a structural basis for the functioning of the two proteins of CBF. Stoichiometry and binding interactions

Key words: core binding factor / CBF / Runx / AML1 / leukemia / hematopoiesis / structure Of necessity, any biophysical characterization must begin with establishing the stoichiometry of the c 2000 Academic Press interactions between the proteins of interest as well as the binding constants involved. All of the equilibria involving the two proteins of CBF and DNA are illustrated in Figure 1. Both electromobility gel shift assays1, 9 and more recent sedimentation equilibrium Introduction measurements10 have provided unequivocal proof for the 1 : 1 : 1 stoichiometry of interaction between the Core binding factors (CBFs) are heterodimeric proteins of CBF and DNA. Based on this knowledge, more extensive binding studies have been carried out transcription factors consisting of a DNA-binding 9–11 subunit (Runx) and a non-DNA-binding subunit on the Runt domain of Runx1 and on CBFB. The results on CBFB demonstrated that an N-terminal (CBFB).1–4 All Runx subunits share a functional 141-amino-acid form of CBFB was indistinguishable domain, referred to as the Runt domain, which from the full-length protein in terms of its ability is responsible for the DNA-binding as well as the 11 CBFB-binding activities. As described elsewhere in to bind the Runt domain of Runx1, consistent with the ability of this truncated form to rescue this issue, CBFs are critical for a number of develop- 12 mental processes in Drosophila as well as mammals. definitive hematopoiesis in CBFB knockout cells. The predominant protein resulting from the inv(16) The mammalian RUNX2 gene is required for bone associated with 12–15% of acute myeloid leukemias development5 and the RUNX1 and CBFB genes have fuses the first 165 amino acids of CBFB to the been demonstrated to be essential for the emergence coiled-coil region of smooth muscle myosin. These binding studies show that this fusion retains all of the determinants of CBFB necessary for binding to From the Department of Molecular Physiology and Biological Runx. Subcellular localization studies of the inv(16) Physics, University of Virginia, Charlottesville, VA 22908-0736 USA. 13 E-mail: [email protected] oncoprotein also confirm its ability to bind to Runx. c 2000 Academic Press A recent extensive EMSA and microcalorimetry 1084–9521/00/000377 06/$35.00/0/0 + 377 J. H. Bushweller

tein outside of the Runt domain. This inhibition is relieved upon the binding of CBFB, providing a more definitive and substantive role for the CBFB subunit. Interestingly, the region of Runx implicated in this auto-inhibition is not present in most of the fusion proteins resulting from translocations involving the RUNX1 gene. This implies that these oncoprotein forms of Runx1 will have substantially higher DNA- binding affinities than their wildtype counterparts and may provide an explanation for their associated dysregulation. In addition, these fusion proteins also lack the region necessary for targeting of the protein to the nuclear matrix, a function which has also been Figure 1. Schematic illustration of the four equilibria in- shown to be essential for transcriptional regulation 16 volving the two proteins of CBF and DNA. The proposed by Runx. conformational change in Runx induced by CBFB is indi- cated as a change in the contacts made by Runx with the DNA upon CBFB binding. Three-dimensional structure of CBFB

Recent structural work on the heterodimerization domain of CBFB17, 18 and on the Runt domain of Runx119, 20 have provided a much more detailed picture of the functioning of the two proteins of CBF. The structure of CBFB [Figure 2(a)] revealed it to have a novel α/β fold that consists of two non-autonomously folded regions that are separated by a flexible loop. The N-terminal region starts with α-helices 1 and 2 which lead into β-strand 1. β-strand 1 is followed by a short 310 helix and subse- quently α-helix 3. α-helix 3 is followed by β-strands 2 and 3 with a type 1 β-turn between them. The first three β-strands combine to form a three-stranded Figure 2. (a) Ribbon representation of the solution struc- anti-parallel β-sheet. The N-terminal region is con- ture of CBFB. The helices are colored red and yellow, the nected via a loop region that was shown by NMR β-strands cyan and other segments gray. (b) Map of the relaxation measurements to be flexible in solution. chemical shift perturbation data on a ribbon representation The C-terminal region begins with β-strands 4, 5 of CBFB. Yellow indicates large chemical shift changes upon and 6, which also combine to form a three-stranded binding of Runx1–DNA to CBFB. anti-parallel β-sheet and the final C-terminal α-helix 4. There are limited contacts between the two sheets that make it one continuous β-sheet. study of all the equilibria involving the two proteins of CBF and DNA10 has shown that the CBFB subunit enhances the DNA-binding affinity of the isolated Binding site for Runx on CBFB Runt domain of Runx1 approximately sixfold. All of the studies described to this point were carried out utilizing the Runt domain of Runx, not full-length The mapping of binding sites for ligands, be they protein. Two studies on the binding behavior of proteins, nucleic acids or small molecules, can be longer forms of the Runx protein14, 15 have shown identified readily using an NMR method referred to the full-length proteins to have substantially reduced as chemical shift perturbation analysis. Addition, in affinity for DNA relative to the isolated Runt domain. this case, of a Runx Runt domain–DNA complex to These data strongly suggest the presence of (an) CBFB results in the formation of a ternary complex. inhibitory domain(s) present in the full-length pro- Because the chemical shifts, or frequencies, of the

378 CBF—A biophysical perspective nuclei in an NMR experiment are very sensitive to fold, specifically the s-type Ig fold best exemplified their local environment, comparison of the back- by the structure of the fibronectin repeat element. bone NH chemical shifts of CBFB in the presence Interestingly, this fold has been identified in the and absence of the Runt domain–DNA complex DNA-binding domains of a number of other critical can identify those amide NH moieties that display mammalian transcription factors including NF-κB, substantial perturbations upon binding. All of the NFAT, , STAT-1 and the T-domain. All of the amino acids save proline have an associated amide DNA-binding domains of these proteins share the NH signal in the NMR experiment, thus this provides s-type Ig fold, thus the Runt domain belongs to a fam- a high-resolution map of the perturbations. The ily of structurally related s-type Ig fold, DNA-binding observed changes may result from regions of CBFB in domains. In addition to the overall fold being shared direct contact with the Runt domain–DNA complex among these proteins, a number of these proteins or they may result from conformational changes also display homologous short β-sheet interactions associated with that binding. This has proven to be like those observed in the Runt domain.19 For all of an extremely powerful method for the elucidation of these structurally related proteins, binding to DNA binding sites on proteins and nucleic acids. Such a is mediated by the loop regions that connect the study has been carried out in two laboratories17, 18 for various β-strands. For all the previously characterized the identification of the Runx Runt domain binding members of this family, DNA binding is mediated by site on CBFB, with very similar results. The results of loops located either at the ‘top’ or ‘bottom’ of the one such study are shown in Figure 2(b).17 The asso- fold as depicted in Figure 3, but not by both ends. ciated perturbations map to a very localized region on the structure of CBFB that includes β-strand 3 and the loops leading into and out of β-strand 3 as well Mapping of the DNA-binding site on the Runx1 as β-strands 4 and 5 and their associated connecting Runt domain loop. We have also observed perturbations for two residues at the N-terminal end of the protein. Muta- In order to identify the region of the Runt domain in- genesis of residues in this region of CBFB confirms volved in DNA binding, nuclear Overhauser (NOE) the importance of this region for interaction with spectroscopy was utilized. This type of NMR experi- Runx (Tang and Speck, unpublished results). ment allows one to observe protons that are close to one another in space, less than ca 5 Å in practice. By Three-dimensional structure of the Runx1 Runt utilizing a sample containing fully deuterated Runt domain domain, i.e. no observable protons save the protein amide NH groups, bound to unlabeled DNA, one is Structural studies of the Runt domain of Runx1 able to identify NOEs from the protein NH groups to have been carried out using Runt domain–DNA the DNA. The results of this study19 are shown in Fig- complexes because of the poor behavior of the ure 3(b). In addition, a substantial number of point isolated protein in solution. As shown in Figure 3(a), mutations have been made in the Runt domain by the protein has been shown to be a β-protein with no several labs.9, 21–23 Those that have been shown to af- α-helical content whatsoever, as was predicted from fect either DNA or CBFB binding are also shown in an earlier circular dichroism (CD) study.9 The fold Figure 3(b). As Figure 3(b) shows, DNA binding is of the protein consists of seven clearly discernible mediated by the loop regions of the protein. There β-strands with strands 1, 2 and 5 combining to form is very good concurrence in the location of the ob- one anti-parallel β-sheet and strands 3, 4, 6 and 7 served NOEs and the locations of the point muta- combining to form a second anti-parallel β-sheet. tions affecting DNA binding, confirming this charac- The two sheets are packed against one another at terization. Unlike the other members of the structural a shallow angle to yield a β-sandwich arrangement. family the Runt domain appears to belong to, all the Two short β-sheet interactions were also identified, loops appear to be involved in DNA binding, that is, involving L117/R138-R135/F136 and I168/T169- loops at the ‘top’ and ‘bottom’ of the structure are H78/W79, which, because of their proximity to involved in DNA recognition. The Runt domain may the DNA (see below), have been suggested to be be unique among this group in this respect. This may functionally relevant. The fold identified for the Runt possibly be necessary for the DNA bending that the domain belongs to the classic immunoglobulin (Ig) Runt domain has been shown to effect.24

379 J. H. Bushweller

Figure 3. (a) Ribbon representation of the solution structure of the Runx1 Runt domain. β-strands are colored cyan and other segments are grey. (b) Intermolecular contacts of the Runx1 Runt domain mapped onto a ribbon representation of the structure (in green). The red spheres correspond to the spatial location in the Runt domain of intermolecular protein NH–DNA NOEs observed in a Runx1–DNA complex. Known point mutations that affect DNA binding and CBFB binding are shown as blue and gray spheres, respectively. (c) Map of the chemical shift perturbation data obtained for the binding of CBFB to a Runx1–DNA complex on a ribbon representation of the Runx1 Runt domain (in green). Red indicates large chemical shift changes upon binding of CBFB to the Runx1 Runt domain.

Mapping of the CBFB-binding site on the Runx1 behavior by invoking a conformational change in Runt domain the Runx subunit upon binding to CBFB. Although a clear delineation of this will require structural Chemical shift perturbation mapping studies have, characterization of a ternary complex, which is not as described above for CBFB, been employed to yet available, two pieces of data tend to support the identify the CBFB-binding site on the Runx1 Runt latter mechanism. First, circular dichroism studies of domain.10, 20 The results of one of these studies are the interaction between the Runt domain of Runx1 illustrated in Figure 3(c). Both studies have identified and CBFB show a clear conformational change in one face of the protein, that of the four-stranded one or both proteins upon interaction either on β-sheet, as the location for the binding of CBFB. or off of the DNA.10 Second, one of the chemical The region thus identified is in good agreement with shift perturbation studies10 has identified dramatic the mutagenesis data shown in Figure 3(b) in that chemical shift changes for a specific region of the those point mutations in the Runt domain that affect Runt domain located in close proximity to the DNA CBFB binding are localized to the same region as which was not assigned in the other study.20 The identified by the chemical shift perturbation analysis. magnitude of the observed changes and the dramatic The proximity of the CBFB-binding site on the Runt alterations observed in the Cα chemical shifts,10 domain to regions of the Runt domain involved in which are a strong indicator of backbone confor- DNA binding provides a rationale for the ability mation, strongly support a conformational change of CBFB to alter the DNA-binding affinity of the in the Runt domain associated with this interaction Runt domain. Although the CBFB subunit has been (illustrated schematically in Figure 1). shown to enhance the DNA-binding affinity of the Runx subunit, there was no change observed in the Other structural studies identity of the bases and phosphates contacted in the binary versus the ternary complex.25 This behavior The only other structural characterization of the CBF may be explained by two possible models or some proteins reported thus far has been a crystal structure combination thereof. One could invoke additional of the nuclear matrix targeting signal (NMTS) of contacts being made by CBFB to the DNA, but only Runx1 fused to glutathione-S-transferase.26 The for phosphates or bases that are already in contact results of this study indicated that part of the peptide with Runx. The second would explain the observed

380 CBF—A biophysical perspective adopts a defined tertiary structure described as two 2. Ogawa E, Maruyama M, Kagoshima H, Inuzuka M, Lu J, finger-like loops connected by a U-shaped peptide Satake M, Shigesada K, Ito Y (1993) PEBP2/PEA2 represents a chain. These results provide a meaningful framework new family of homologous to the products of the Drosophila runt and the human AML1 gene. Proc Natl to examine the determinants for nuclear localization Acad Sci USA 90:6869–6863 in the NMTS of Runx proteins. 3. Kagoshima H, Shigesada K, Satake M, Ito M, Miyoshi H, Ohki M, Pepling M, Gergen JP (1993) The Runt-domain identifies a new family of heteromeric DNA-binding transcriptional Future directions regulatory proteins. Trends Genet 9:338–341 4. Wang S, Wang Q, Crute BE, Melinikova IN, Keller SR, Speck NA (1993) Cloning and characterization of subunits of the Regulation of transcription in mammals has been T-cell and murine leukemia virus enhancer core- shown to be a complex process involving the interplay binding factor. Mol Cell Biol 13:3324–3339 and interaction of multiple proteins at a particular 5. Komori T et al. (1997) Targeted disruption of Cbfa1 results site on the DNA. Recent progress in structurally in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764 characterizing a number of ternary complexes with 6. Wang Q, Stacy T, Binder M, Marín-Padilla M, Sharpe AH, two proteins bound to DNA has provided detailed Speck NA (1996) Disruption of the Cbfa2 gene causes necrosis atomic-resolution pictures of how this interplay is and hemorrhaging in the central nervous system and blocks mediated at the molecular level.27 In these cases, the definitive hematopoiesis. Proc Natl Acad Sci USA 93:3444– 3449 interactions with the various partner proteins have 7. Wang Q et al. (1996) The CBFβ subunit is essential for proven to be essential to the in vivo function. CBF CBFα2 (AML1) function in vivo. Cell 87:697–708 has been shown to interact with a number of other 8. Meyers S, Hiebert SW (1995) Indirect and direct disruption proteins including Ets proteins (particularly Ets-1), of transcriptional regulation in cancer, , and AML-1. Crit 28–35 Rev Eukaryot Gene Expr 5:365–383 C/EBP, TLE, PU.1, ALY, Myb, Ear2 and p300. 9. Crute BE, Lewis AF, Wu Z, Bushweller JH, Speck NA In the case of the Runx /Ets-1 interaction, the (1996) Biochemical and biophysical properties of the CBFα2 cooperative nature of the DNA binding of Runx (AML1) DNA-binding domain. J Biol Chem 271:26251–26260 with Ets-1 provides a clear functional role for the 10. Tang Y, Crute BE, Kelley JJ, Huang X, Tan J, Shi J, Hartman 28 KL, Laue TM, Speck NA, Bushweller JH (2000) Biophysical interaction between the two. Future biophysical characterization of interactions between the core binding studies to elucidate the mechanism of this myriad factor α and β subunits and DNA. FEBS Letters 470:167–172 of protein–protein interactions and particularly their 11. Huang X, Crute BE, Sun C, Tang Y, Kelley JJ, Lewis AF, Hart- structural basis will clearly be an area for fertile future man KL, Laue TL, Speck NA, Bushweller JH (1998) Overex- pression, purification, and biophysical characterization of the work on CBF. heterodimerization domain of core-binding factor β subunit. J Biol Chem 273:2480–2487 12. Miller JD, Stacy T, Liu PP, Speck NA (2000) CBFB and CBFB- Acknowledgements SMMHC function in definitive hematopoiesis. Blood (In press) We wish to thank Dr Nancy Speck for many hours of 13. Adya N, Stacy T, Speck NA, Liu PP (1998) The leukemic protein core binding factor B (CBFB)-smooth-muscle myosin fruitful discussions. JHB is supported by grants from heavy chain sequesters CBFA2 into cytoskeletal filaments and the United States Public Health Service R29 AI39536, aggregates. Mol Cell Biol 18:7432–7443 K02 AI10481 and R01 AI42097 from the Institute for 14. Kanno T, Kanno Y, Chen L, Ogawa E, Kim W, Ito Y (1998) Allergy and Infectious Disease. Intrinsic transcriptional activation–inhibition domains of the polyomavirus enhancer binding protein2/core binding factor α subunit revealed in the presence of the β subunit. Mol Cell Note added in proof Biol 18:2444–2454 15. Gu T, Goetz TL, Graves BJ, Speck NA (1999) Auto-inhibition and partner proteins, CBFβ and Ets-1, modulate DNA binding Very recently, a crystal structure of a CBFβ-Runt by CBFα2(AML1). Mol Cell Biol 20:91–103 domain complex has also been reported by Warren 16. Zeng C et al. (1998) Intranuclear targeting of AML/CBFalpha et al. (2000).35 regulatory factors to nuclear matrix-associated transcriptional domains. Proc Natl Acad Sci USA 95:1585–1589 17. Huang X, Peng J, Speck NA, Bushweller JH (1999) Solution structure of core binding factor β and map of the CBFα References binding site. Nat Struct Biol 6:624–627 18. Goger M, Gupta V, Kim W, Shigesada K, Ito Y, Werner MH 1. Ogawa E, Inuzuka M, Maruyama M, Satake M, Naito-Fujimoto (1999) Molecular insights into PEBP2/CBFB-SMMHC associ- M, Ito Y, Shigesada K (1993) Molecular cloning and charac- ated leukemia revealed from the structure of PEBP2/CBFB. terization of PEBP2β, the heterodimeric partner of a novel Nat Struct Biol 6:620–623 Drosophila runt-related DNA binding protein PEBP2α. Virol- 19. Berardi M, Sun C, Zehr M, Abildgard F, Peng J, Speck NA, ogy 194:314–331 Bushweller JH (1999) The Ig fold of the core binding factor

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α Runt domain is a member of a family of structurally and scriptionalregulation. Annu Rev Biophys Biomol Struct functionally related Ig fold DNA binding domains. Structure 28:29–56 7:1247–1256 28. Gu T, Goetz TL, Graves BJ, Speck NA (1999) Auto-inhibition 20. Nagata T, Gupta V, Sorce D, Kim W, Sali A, Chait BT, and partner proteins, CBFβ and Ets-1, modulate DNA binding Shigesada K, Ito Y, Werner M (1999) Immunoglobulin motif by CBFα2(AML1). Mol Cell Biol 20:91–103 DNA recognition and heterodimerization of the PEBP2/CBF 29. Petrovick MS, Hiebert S, Friedman AD, Hetherington CJ, Runt domain. Nat Struct Biol 6:615–619 Tenen DG, Zhang D (1998) Multiple functional domains of 21. Akamatsu Y, Tsukumo S, Kagoshima H, Tsurushita N, Shige- AML1: PU.1 and C/EBPα synergize with different regions of sada K (1997) A simple screening for mutant DNA bind- AML1. Mol Cell Biol 18:3915–3925 ing proteins: application to murine transcription factor 30. Bruhn L, Munnerlyn A, Grosschedl R (1997) ALY, a context- PEBP2α subunit, a founding member of the Runt domain dependent coactivator of LEF-1 and AML-1, is required for protein family. Gene 185:111–117 TCRα function. Genes Dev 11:640–653 22. Kagoshima H, Akamatsu Y, Ito Y, Shigesada K (1996) Func- 31. Levanon D, Goldstein RE, Bernstein Y, Tang H, Gold- tional dissection of the α and β subunits of transcription fac- enberg D, Stifani S, Paroush Z, Groner Y (1998) Tran- tor PEBP2 and the redox susceptibility of its DNA binding ac- scriptional repression by AML1 and LEF-1 is mediated by tivity. J Biol Chem 271:33 074–33 082 the TLE/Groucho corepressors. Proc Natl Acad Sci USA 23. Lenny N, Meyers S, Hiebert SW (1995) Functional domains of 95:11590–11595 the t(8;21) fusion protein, AML-1/ETO. Oncogene 11:1761– 32. Ito Y, Bae S-C (1997) Oncogenes as Transcriptional Regula- 1769 tors, vol 2 (Yaniv M, Ghysdael J, eds) pp. 107–132. Birkhauser, 24. Golling G, Pepling M, Stebbins M, Gergen P (1996) Basel Drosophila homologs of the proto-oncogene prod- 33. Ahn MY, Huang G, Bae S-C, Wee H-J, Kim W-Y, Ito Y uct PEBP2/CBF beta regulate the DNA-binding properties of (1998) Negative regulation of granulocytic differentiation Runt. Mol Cell Biol 16:932–942 in the myeloid precursor cell line 32Dcl3 by ear-2, mam- 25. Wang SQ, Crute BE, Melinikova IN, Keller SR, Speck NA malian homolog of Drosophila seven-up, and a chimeric (1993) Cloning and characterization of subunits of the T- leukemogenic gene, AML1/ETO(MTG8). Proc Natl Acad Sci cell receptor and murine leukemia virus enhancer core USA 95:1812–1817 binding factor. Mol Cell Biol 13:3324–3339 34. Kitabayashi I, Yokoyama A, Shimizu K, Ohki M (1998) Inter- 26. Tang B, Guo B, Javed A, Choi J, Hiebert S, Lian JB, van Wijnen action and functional cooperation of the leukemia-associated AJ, Stein JL, Stein GS, Zhou GW (1999) Crystal structure factors AML1 and p300 in myeloid cell differentiation. EMBO of the nuclear matrix targeting signal of the transcription J 17:2994–3004 factor Acute Myelogenous Leukemia-1/Polyoma Enhancer- 35. Warren AJ, Bravo J, Williams RL, Rabbitts TH (2000) Struc- binding Protein 2αB/Core Binding Factor α2. J Biol Chem tural basis for the heterodimeric interaction between the 274:33 580–33 586 acute leukaemia-associated transcription factors AML1 and 27. Wolberger C (1999) Multiprotein–DNA complexes in tran- CBFβ. EMBO J 19:3004–3015

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