Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox Proteins Ching-Pin Chang, 1 Wei-Fang Shen, 2 Sofia Rozenfeld, 2 H. Jeffrey Lawrence, 2 Corey Largman, 2 and Michael L. Cleary 1,3 ~Laboratory of Experimental Oncology, Department of Pathology, Stanford University Medical Center, Stanford, California 94305 USA; 2Department of Medicine, San Francisco Veterans Affairs Medical Center, and University of California, San Francisco, California 94121 USA The human proto-oncogene PBX1 codes for a homolog of Drosophila extradenticle, a divergent homeo domain protein that modulates the developmental and DNA-binding specificity of select HOM proteins. We demonstrate that wild-type Pbx proteins and chimeric E2a-Pbxl oncoproteins cooperatively bind a consensus DNA probe with HoxB4, B6, and B7 of the Antennapedia class of Hox/HOM proteins. Specificity of Hox-Pbx interactions was suggested by the inability of Pbx proteins to cooperatively bind the synthetic DNA target with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes. [Key Words: Pbx; Hox; E2a-Pbxl; homeo domain proteins; proto-oncogene] Received December 15, 1994; revised version accepted February 17, 1995. Hox proteins are members of a large family of vertebrate basis of DNA-protein interactions alone, as best illus- transcription factors that share a similar DNA-binding trated by studies of fushi tarazu (ftz) showing that high motif known as the homeo domain (Scott et al. 1989). affinity homeo domain-DNA interactions are neither Hox proteins have many features in common with sufficient nor necessary for its biological activity (Fitz- Drosophila homeotic selector (HOM) proteins including patrick et al. 1992; Ananthan et al. 1993; Schier and primary sequence, chromosomal organization of their re- Gehring 1993). spective genes, and regionally and developmentally re- Several studies suggest that the functional specificity stricted expression profiles (Akam 1989; McGinnis and of homeo domain proteins is determined by the com- Krumlauf 1992). Some Hox proteins have also been bined effects of various individual interactions both shown to function similarly in vivo to their Drosophila within and outside of the homeo domain. Such interac- counterparts. For both Hox and HOM proteins, the ho- tions have been shown experimentally to influence the meo domain has been shown to mediate DNA binding in DNA-binding specificity of homeo domain proteins. For vitro and play an important role in the ability of these instance, combinatorial interactions between MCM1, proteins to regulate transcription through specific target MATal and MATa2 determine the ability of al and a2 sequences (Desplan et al. 1988; Hoey and Levine 1988; to recognize specific operators that mark genes regulat- Mtiller et al. 1988; Gehring et al. 1994a). The homeo ing mating type in yeast (Johnson 1992). Homeo domain domain has also been shown to make important contri- proteins may influence the DNA binding of nonhomeo butions to the functional specificity of HOM proteins domain proteins such as serum response factor (SRF), a (Kuziora and McGinnis 1989, 1991; Dessain et al. 1992). mammalian protein with similarity to MCM1 (Gruene- However, recent data suggest that homeo domain pro- berg et al. 1992). Protein-protein interactions may also teins do not achieve their functional specificity on the influence the transcriptional properties of homeo do- main proteins as shown for HoxD8 inhibition of HoxD9 3Corresponding author. activation, an effect dependent on contacts within helix GENES & DEVELOPMENT 9:663-674 91995 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/95 $5.00 663 Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Chang et al. 1 of their homeo domains (Zappavigna et al. 1994). Stud- comparable conditions (Johnson et al. 1995) and could be ies of the POU and Oct 1 proteins (Stern et al. 1989; stably coexpressed with Pbxl in yeast cells. The homeo Ingraham et al. 1990; Voss et al. 1991; Treacy et al. 1992; domain and flanking amino acids of HoxB7 were fused to Verrijzer et al. 1992; Xue et al. 1993), vHNF1 (Rey-Cam- the GAL4 DNA-binding domain in yeast vector pAS2 pos et al. 1991), and Paired class homeo proteins (Wilson (construct pAS-ypHDB7) and various portions of Pbxl et al. 1993)provide additional evidence that cooperative were fused to the GAL4 activation domain contained in interactions play an important role in regulating their pACT2 (Fig. 1 ). Yeast cells cotransformed with pACT- DNA-binding and functional properties. Pbxlb and pAS-ypHDB7 showed activation of lacZ re- Previous studies have identified a distinctive subfam- porter gene expression under the control of the GAL4 ily of atypical homeo domain proteins that are potential UAS, whereas no f}-gal activity was observed in cells candidates for interactions with Hox/HOM proteins. transformed with each of the plasmids individually (Fig. These include the mammalian Pbx proteins, Drosophila 1; data not shown). The interaction between Pbxl and extradenticle (exd), and Caenorhabditis elegans ceh-20 HoxB7 required the homeo domain of Pbxl as its dele- (Monica et al. 1991; B/irglin and Ruvkun 1992; Rauskolb tion abrogated activation of ~-gal expression (Fig. 1). A et al. 1993), notable for their extensive similarities both minimal portion of Pbxl consisting of 88 amino acids within and outside of their homeo domains. Pbxl is a spanning the homeo domain and immediate carboxy-ter- proto-oncogene that was originally discovered at the site minal flanking amino acids was sufficient for interaction of t(1;19) chromosomal translocations in pre-B cell acute with HoxB7. These observations suggested the potential leukemias (Kamps et al. 1990; Nourse et al. 1990). In for interaction between Pbxl and HoxB7 proteins medi- Drosophila, genetic studies suggest that the Pbx ho- ated, at least in part, by the homeo domain of Pbxl. molog exd serves a unique role in segment identity through parallel interactions with homeotic selector pro- teins such as ultrabithorax (Ubx) and antennapedia In vitro interaction of Pbx and Hox proteins is (Antp) (Peifer and Weischaus 1990; Rauskolb and Weis- stimulated by the presence of Pbx-binding site DNA chaus 1994). Recent studies support this role by demon- The potential interaction of Pbxl with HoxB7 was in- strating cooperative interactions between exd and select vestigated further by immunoprecipitation analyses of in HOM proteins (or engrailed) in DNA-binding assays vitro-synthesized proteins. These experiments employed (Chan et al. 1994; VanDijk and Murre 1994) and in a wild-type Pbxla with an epitope tag at its amino termi- yeast two-hybrid system (Johnson et al. 1995). These nus. In vitro translated Pbxla-flag and HoxB7 were studies are consistent with the thesis that biological mixed and subjected to immunoprecipitation analysis specificity of homeotic selector proteins may be with antibodies against the epitope tag. In parallel, the achieved, at least in part, through cooperative interac- translocation-derived E2a-Pbx l a fusion protein was also tions that affect the manner in which they recognize and tested for potential interaction with HoxB7 with an anti- regulate downstream subordinate genes. E2a monoclonal. Precipitation of Pbxla or E2a-Pbxla In this report we demonstrate that the DNA-binding from in vitro translates showed little or no coprecipi- affinity of Pbx proteins is markedly stimulated through tated HoxB7 (Fig. 2). However, when an oligonucleotide interactions with a subset of Hox proteins under condi- (5'-CGAATTGATTGATGCACTAATTGGAG-3') con- tions where they demonstrate little or no inherent DNA- taining consensus Hox (Pellerin et al. 1994) and Pbx binding affinity alone. Cooperative DNA binding re- (VanDijk et al. 1993; LeBrun and Cleary 1994; Lu et al. quired the homeo domains and specific flanking amino acids in both the Hox and Pbx proteins. The conserved YPWM amino acid motif upstream of the homeo domain in many Hox proteins was essential for cooperativity, pACT-Pbx Constructs J~-gal Activity thereby establishing a role for this highly conserved el- ement that may define a subset of Hox proteins capable pACT of cooperative interactions with Pbx proteins. These pACT-Pbxlb I'"~1. L ~ t347 4- studies support a model in which Pbx and Hox proteins pACT-PbxAC r-~l, ,89.1 ~ 1319 4"- act in parallel as heterotypic complexes to regulate ex- pACT-P bx 1a3.N r'--I .............. 222 ~ + pression of specific subordinate genes. pACT-Pbxl HD ~ .............. 22,2r~-1309 4- pACT-PbxN r-'~l..89.1 i TM Results pACT-PbxlaC r-'q ....................