25554839
The novel homeoprotein Prep1 modulates Pbx–Hox protein cooperativity
The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1–Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N‐terminal portions of both proteins. The Prep1–Pbx protein–protein interaction is essential for DNA‐binding activity. Prep1–Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1–Pbx1–HOXB1 complex on a HOXB1‐responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1–Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.
39661371
A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B all
It was previously shown that the chromosome 19 breakpoint of the t(1;19)(q23;p13.3) translocation, found in human pre-B cell acute lymphoblastic leukemias, is within the E2A transcription factor gene on chromosome 19. A cell line with this translocation contains two novel chimeric mRNAs, both with the same 5'E2A sequences but with different lengths of 3' sequence from a previously unrecognized gene dubbed prl, located on chromosome 1. The chimeric RNAs encode a protein that lacks 171 amino acids of E2A, including its DNA binding and dimerization motifs, but have instead a homeobox-related sequence from prl. Therefore, the production of a chimeric E2A-Prl protein may contribute to the acute lymphoblastic phenotype by directly altering the expression of genes normally responsive to the Prl homeoprotein.
7975269
Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor
The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.
27691206
The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials.
The t(1;19) translocation that characterizes 25% of pediatric pre-B cell acute lymphoblastic leukemias (pre-B ALL) produces a chimeric gene, joining 5' sequences that encode a transcriptional activator domain of E2A with 3' sequences that, in part, encode a homeo box domain of a new gene called pbx1. Two E2A-pbx1 transcripts have been cloned. They encode the putative fusion proteins, p85(E2A-Pbx1) and p77(E2A-Pbx1), which differ in Pbx1 sequences alone, containing unique carboxyl termini whose sequences diverge after the Pbx1 homeo box. In this study, an antiserum to Pbx1 was used to investigate the identity and abundance of E2A-Pbx1 fusion proteins in both the pre-B ALL cell line, 697, and in cryopreserved leukemic bone marrow cells, obtained from six children with t(1;19)-positive pre-B ALL. Five species of E2A-Pbx1 proteins were identified in all cells containing t(1;19), two of which were indistinguishable from in vitro-translated p85(E2A-Pbx1) and p77(E2A-Pbx1). To assess the biological properties of p85(E2A-Pbx1) and p77(E2A-Pbx1) in fibroblasts, the cDNAs encoding these proteins were cloned into retroviral vectors, and each was introduced into NIH-3T3 cells. Both p85(E2A-Pbx1) and p77(E2A-Pbx1) are localized in the nucleus, and expression of either resulted in malignant conversion of NIH-3T3 cells as assayed by tumor formation in nude mice. When scored by focus formation, density-independent growth, and growth in agar assays, p77(E2A-Pbx1) was a much more potent transforming protein than was p85(E2A-Pbx1). Because subtle mutations in p85(E2A-Pbx1) converted its transforming activity into that of p77(E2A-Pbx1), we suggest that a sequence within the unique carboxyl terminus of p85(E2A-Pbx1) serves to negatively regulate its biochemical activity.
12617530
E2A-Pbx1, the t(1;19) translocation protein of human pre-B-cell acute lymphocytic leukemia, causes acute myeloid leukemia in mice.
One-quarter of pediatric pre-B-cell leukemias contain the t(1;19) chromosomal translocation, which fuses 5' exons encoding the transactivation domain of the E2A transcription factor gene to 3' exons ecoding the putative DNA-binding region of the unusual homeobox gene, PBX1. To test the leukemic potential of this fused gene, a cDNA encoding its major protein product, p85E2A-Pbx1, was incorporated into a retrovirus construct and introduced into normal mouse marrow progenitors by infection. The cells were used in a bone marrow transplantation protocol to reconstitute the hematopoietic compartments of lethally irradiated recipients. After 3 to 8 months, reconstituted mice developed acute myeloid leukemias that expressed high levels of p85E2A-Pbx1 and were readily transmissible to immunocompetent mice. Most acute myeloid leukemias also grew as granulocytic sarcomas and exhibited some neutrophilic differentiation. These results demonstrate a causative role for p85E2A-Pbx1 in human acute leukemia and indicate that the oncogenic potential of Pbx1 is not limited to pre-B-cell malignancies.
35595166
Human t(1;19)(q23;p13) pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency.
Severe combined immunodeficient (SCID) mice were injected intravenously with 5 x 10(6) primary bone marrow (BM) blasts from newly diagnosed patients with E2A-PBX1 fusion transcript positive t(1;19)(q23;p13) pre-B acute lymphoblastic leukemia (ALL). A marked variation existed in the pattern and extent of leukemic cell engraftment in SCID mice challenged with t(1;19) pre-B ALL blasts. Blasts from some patients caused disseminated leukemia that was detected by histopathology and/or flow cytometry, whereas blasts from other patients produced occult leukemia that was only detected by flow cytometry and/or polymerase-chain reaction. Notably, the ability of primary t(1;19) pre-B ALL blasts to cause disseminated leukemia in SCID mice was associated with poor prognosis. Six of six patients whose blasts caused disseminated leukemia in SCID mice relapsed at a median of 7.8 months (range: 5.7 to 25.2 months). In contrast, the remaining four patients whose blasts did not engraft or only partially engrafted remain in complete remission at 28 to 47 months. A new E2A-PBX-1 fusion transcript positive t(1;19) pre-B ALL cell line (designated LC1;19) with the composite immunophenotype CD7-CD10+CD19+CD45-HLA-DR+C mu+ was established by expanding BM blasts from a SCID mouse, which died of human t(1;19) ALL at 7 weeks after inoculation of primary leukemic blasts from a t(1;19) ALL patient. This cell line caused disseminated and invariably fatal leukemia when greater than 10(4) cells were injected intravenously into SCID mice. Total body irradiation followed by syngeneic BM transplantation (BMT) showed limited efficacy against LC1;19 leukemia in SCID mice. To our knowledge, this study is the first to (1) examine the in vivo growth of primary t(1;19) pre-B ALL blasts in SCID mice and (2) show that leukemic blasts from a majority of newly diagnosed t(1;19) pre-B ALL patients cause disseminated human leukemia in SCID mice. Our results indicate that t(1;19) pre-B ALL is biologically heterogeneous with regard to its in vivo growth pattern in SCID mice, a feature that may be predictive of prognosis. The described LC1;19 SCID mouse model may prove particularly useful for designing more effective treatment strategies against poor-prognosis t(1;19) ALL.
21874222
Pbx1 is converted into a transcriptional activator upon acquiring the N-terminal region of E2A in pre-B-cell acute lymphoblastoid leukemia.
Twenty-five percent of human pediatric pre-B-cell acute lymphoblastic leukemias (ALLs) are characterized by the t(1;19)(q23;p13.3) chromosomal translocation. This translocation joins the 5' region of the E2A gene to the 3' region of the Pbx1 gene. The protein encoded by this chimeric gene contains the N-terminal transcriptional activation domain of E2A fused to the C-terminal region of Pbx1, which contains a putative homeodomain. Here we show that the Pbx1 homeodomain preferentially binds the sequence ATCAATCAA. We further show that promoters containing Pbx1-binding sites are activated by the chimeric E2A-Pbx1 protein but not by Pbx1. These results indicate that the t(1;19) translocation converts a nonactivating DNA-binding protein into a potent transcriptional activator, suggesting an unusual mechanism for oncogenic transformation.
6630683
Fusion with E2A converts the Pbx1 homeodomain protein into a constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation.
E2A-PBX1 is a chimeric gene formed by the t(1;19)(q23;p13.3) chromosomal translocation of pediatric pre-B-cell leukemia. The E2A-Pbx1 fusion protein contains sequences encoding the transactivation domain of E2A joined to a majority of the Pbx1 protein, which contains a novel homeodomain. Earlier, we found that expression of E2A-Pbx1 causes malignant transformation of NIH 3T3 fibroblasts and induces myeloid leukemia in mice. Here we demonstrate that the homeodomains encoded by PBX1, as well as by the highly related PBX2 and PBX3 genes, bind the DNA sequence ATCAATCAA. E2A-Pbx1 strongly activates transcription in vivo through this motif, while Pbx1 does not. This finding suggests that E2A-Pbx1 transforms cells by constitutively activating transcription of genes regulated by Pbx1 or by other members of the Pbx protein family.
768656
Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes
E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.
21003859
Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.
The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.
27516129
Chromosomal translocations involving the E2A gene in acute lymphoblastic leukemia: clinical features and molecular pathogenesis.
0 VER THE PAST DECADE, significant advances have been made in understanding the molecular details of lymphopoiesis and the pathogenesis of lymphoid neoplasia. Major contributions to these advances have been  made through study of Ifl-cell receptor (TCR) genes and the factors that control their ordered recombination and by investigation of nonrandom chromosomal translocations that occur in acute lymphoblastic leukemia (ALL). In addition, the advent of technology that allows investigators to genetically inactivate or “knockout” individual genes by creating homozygous null mice has facilitated efforts to identify proteins whose function is essential for normal development. These disparate lines of investigation have shown that the protein products of the E2A gene are essential for normal lymphopoiesis and can be converted into oncogenic transcription factors by chromosomal translocations. In this review, I will summarize briefly our current understanding of the function of E2A proteins within the lymphoid system, particularly genetic evidence showing that they are absolutely required for normal B-cell development. Attention will  then be turned to discussing two translocations, t(1; 19)(q23;p13) and t(17;19)(421-22;~13), that disrupt E2A. The clinical features of t( 1; 19)’ and t( 17; 19)+ ALLs will be reviewed, particularly those of the former that illustrate the clinical importance of molecular diagnosis. I will also discuss, in detail, the structural features and functional properties of E2A-PBX1 and E2A-HLF, the fusion proteins created by the t(1; 19) and t(17; 19), respectively, and will review the experimental evidence implicating these chimeras in leukemogenesis.
11439205
PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1.
Two new homeobox genes, PBX2 and PBX3, were isolated on the basis of their extensive homology to PBX1, a novel human homeobox gene involved in t(1;19) translocation in acute pre-B-cell leukemias. The predicted Pbx2 and Pbx3 proteins are 92 and 94% identical to Pbx1 over a large region of 266 amino acids within and flanking their homeodomains, but all three proteins diverge significantly near their amino and carboxy termini. Chromosome in situ hybridizations demonstrated that the PBX genes are not clustered but map to separate chromosomal loci: PBX1, 1q23; PBX2, 3q22-23; PBX3, 9q33-34. Expression of PBX2 or PBX3 was not restricted to particular states of differentiation or development, as mRNA transcripts of these genes were detected in most fetal and adult tissues and all cell lines, unlike PBX1, which is not expressed in lymphoid cell lines. Similar to PBX1 RNA, PBX3 RNA is alternatively spliced to yield two translation products with different carboxy termini, a feature not observed for PBX2. Their extensive sequence similarity and widespread expression suggest a generalized, overlapping role for Pbx proteins in most cell types. Differences in their amino and carboxy termini may modulate their activities, mediated in part by differential splicing and, for PBX1, protein fusion following t(1;19) chromosomal translocation.
44721382

 extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene pbx1

Mutations in the Drosophila gene extradenticle (exd) cause homeotic transformations by altering the morphological consequences of homeotic selector gene activity. We have cloned and sequenced exd: it encodes a homeodomain protein with extensive identity (71%) to the human proto-oncoprotein Pbx1. exd is expressed during embryogenesis when the selector homeodomain proteins of the Antennapedia and bithorax complexes establish segmental identity. Maternally expressed exd is uniform and can suppress the segmental transformations of embryos lacking zygotic exd. While zygotic exd expression is also at first uniform, later expression is modulated by the homeotic selector genes. These studies support the view that exd acts with the selector homeodomain proteins as a DNA-binding transcription factor, thereby altering their regulation of downstream target genes.
10821374
The DNA binding specificity of ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein
The Ultrabithorax (Ubx) and Antennapedia (Antp) genes of Drosophila encode homeodomain proteins that have very similar DNA binding specificities in vitro but specify the development of different segmental patterns in vivo. We describe cooperative interactions between Ubx protein and a divergent homeodomain protein, extradenticle (exd), that selectively increases the affinity of Ubx, but not Antp, for a particular DNA target. We also provide evidence that Ubx and exd bind to neighboring sites on this DNA and interact directly to stabilize the DNA-bound form of Ubx. Thus, the ability of different homeotic genes to specify distinct segmental patterns may depend on cooperative interactions with proteins such as exd that selectively modulate their otherwise similar DNA binding specificities.
23525066
Coordinate regulation of downstream genes by extradenticle and the homeotic selector proteins.
Mutations in the Drosophila gene extradenticle (exd), a homologue of the human proto‐oncogene pbx1, cause homeotic transformations by altering the morphological consequences of homeotic selector gene activity. exd has been proposed to act by contributing to the specificity of selector homeodomain proteins for their downstream targets. Here we show that exd is indeed required for the appropriate regulation of at least some of these target genes. Expression patterns of wingless, teashirt and decapentaplegic (dpp) are altered in the embryonic midgut of embryos lacking exd, while the expression of their respective regulators (abd‐A, Antp and Ubx) remains normal. Co‐regulation of dpp by exd and Ubx was investigated in greater detail by examining the expression of reporter constructs in exd embryos. These experiments not only define dpp regulatory regions responsive to exd, but also distinguish two functions of exd in the regulation of dpp. exd acts with Ubx to activate dpp expression in parasegment 7 (PS7), via a minimal visceral mesoderm enhancer, and exd represses dpp expression anterior to PS7. We show that even when Ubx is ubiquitously expressed at high levels in exd embryos, Ubx is incapable of activating dpp enhancer expression. Thus, exd is an indispensable component in target gene regulation by the homeotic selector proteins.
18658549
Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx
Comparison of Hoxb-1 regulatory regions from different vertebrates identified three related sequence motifs critical for rhombomere 4 (r4) expression in the hindbrain. Functional analysis in transgenic mice and Drosophila embryos demonstrated that the conserved elements are involved in a positive autoregulatory loop dependent on labial (lab) family members. Binding of Hoxb-1 to these elements in vitro requires cofactors, and the motifs closely resemble the consensus binding site for pbx1, a homolog of the Drosophila extradenticle (exd) homoedomain protein. In vitro exd/pbx serves as a Hoxb-1 cofactor in cooperative binding and in Drosophila expression mediated by the r4 enhancer is dependent on both lab and exd. This provides in vivo and in vitro evidence that r4 expression involves direct autoregulation dependent on cooperative interactions of Hoxb-1 with exd/pbx proteins as cofactors.
12737794
Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.
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-Pbx1 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.
17568404
Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus
Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, including class I Hox proteins. To assess how Pbx proteins influence Hox DNA-binding specificity, we used a binding-site selection approach to determine high-affinity target sites recognized by various Pbx-Hox homeoprotein complexes. Pbx-Hox heterodimers preferred to bind a bipartite sequence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) and the Hox component contacted the more variable 3' half (TNATNN). Binding sites matching the consensus were also obtained for Pbx1 complexed with HoxA10, which lacks a hexapeptide but requires a conserved tryptophan-containing motif for cooperativity with Pbx. Interactions with Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish single nucleotide alterations in the Hox half site both in vitro and in cellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their preferences for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes in the Hox cluster. These observations demonstrate previously undetected DNA-binding specificity for the amino-terminal arm of the Hox homeodomain and suggest that different binding activities of Pbx-Hox complexes are at least part of the position-specific activities of the Hox genes.
1441459
The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1
The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.
17898159
Cooperative interactions between HOX and PBX proteins mediated by a conserved peptide motif
Homeoprotein products of the Hox/HOM gene family pattern the animal embryo through the transcriptional regulation of target genes. We have previously shown that the labial group protein HOXA-1 has intrinsically weak DNA-binding activity due to residues in the N-terminal arm of its homeodomain (M. L. Phelan, R. Sadoul, and M. S. Featherstone, Mol. Cell. Biol. 14:5066-5075, 1994). This observation, among others, suggests that HOX and HOM proteins require cofactors for stable interactions with DNA. We have demonstrated that a putative HOX cofactor, PBX1A, participates in cooperative DNA binding with HOXA-1 and the Deformed group protein HOXD-4. Three Abdominal-B class HOX proteins failed to cooperate with PBX1A. We mapped the interacting domain of HOXD-4 to the YPWMK pentapeptide motif, a conserved sequence found N terminal to the homeodomain of HOXA-1 and many other homeoproteins but absent from the Abdominal-B class. The naturally occurring fusion of the transcriptional activation domain of E2A with PBX1 creates an oncoprotein implicated in human pre-B-cell leukemias (M. P. Kamps, C. Murre, X.-H. Sun, and D. Baltimore, Cell 60:547-555, 1990; J. Nourse, J. D. Mellentin, N. Galili, J. Wilkinson, E. Starbridge, S. D. Smith, and M. L. Cleary, Cell 60:535-545, 1990). A pentapeptide mutation that abolished cooperative interaction with PBX1A in vitro also abrogated synergistic transcriptional activation with the E2A/PBX oncoprotein. The direct contact of PBX family members by the HOX pentapeptide is likely to play an important role in developmental and oncogenic processes.
16580307
Hox gene products modulate the DNA binding activity of Pbx1 and Pbx2
A new family of homeodomain proteins has recently been identified that includes extradenticle, ceh-20 and three mammalian proteins Pbx1, Pbx2 and Pbx3. We show here that two members of this family, Pbx1 and Pbx2 bind cooperatively to DNA with both Hoxb-7 and Hoxb-8. Engrailed-2 modulates the DNA binding activity of the Pbx proteins to a different target site. E2A-Pbx1, a chimeric Pbx1 gene product involved in pre-B acute lymphoblastoid leukemia, has retained its ability to interact with the Hox proteins. These data show that vertebrate Hox and Pbx gene products have the ability to bind cooperatively to DNA.
25487602
Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex
Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.
13112016
Engrailed and Hox homeodomain proteins contain a related Pbx interaction motif that recognizes a common structure present in Pbx.
Hox gene products have the ability to interact with either extradenticle or pbx gene products to bind cooperatively to DNA. The region in Hox proteins that is required for this interaction is located N‐terminal of the homeodomain and contains a highly conserved hexapeptide. We now show that the engrailed gene products also contain a Pbx interaction motif positioned within a previously conserved region, the EH2 domain. The EH2 domain is located N‐terminal of the homeodomain. Two tryptophan residues present in the Drosophila and murine Engrailed EH2 domain are required for cooperativity with extradenticle and Pbx, respectively. A second conserved domain, EH3, is required as well for cooperativity with Pbx, since deletions or an insertion in this region reduce cooperative DNA binding. Peptides containing the Pbx interaction motif of either Engrailed or Hox are capable of destabilizing Engrailed‐Pbx and Hox‐Pbx cooperative DNA binding. These data indicate that the Pbx interaction motifs present in Hox and engrailed gene products recognize a common structure present in the Pbx family of homeodomain proteins.
41635141
Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins.
For many DNA-binding transcription factors it is often difficult to reconcile their highly specific in vivo functions with their less specific in vitro DNA-binding properties. Cooperative DNA binding with cofactors often provides part of the answer to this paradox and recent studies have demonstrated this to be the case for the homeotic complex (HOX) family of transcription factors. However, the unique problem posed by these highly related and developmentally important transcription factors requires additional twists to the standard solution, which are beginning to become apparent from the characterization of the HOX cofactors encoded by the extradenticle and PBX genes.
30372220
Prep1, a novel functional partner of Pbx proteins
The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo.
8400557
A regulatory element that mediates co‐operation between a PEA3‐AP‐1 element and an AP‐1 site is required for phorbol ester induction of urokinase enhancer activity in HepG2 hepatoma cells.
We have characterized a transcriptional enhancer of the human urokinase‐type plasminogen activator (uPA) gene and found a regulatory element required for co‐operation between a PEA3‐‐AP‐1 element and an AP‐1 site in the enhancer. We designated this regulatory element co‐operation mediator (COM). Both the PEA3‐‐AP‐1 element, the AP‐1 site and the COM are required for efficient phorbol ester induction of transcription from the uPA promoter in the HepG2 hepatoma cell line. We show that the COM is also required for co‐operation between the PEA3‐‐AP‐1 element and a glucocorticoid response element, both in the presence or absence of TPA, indicating that the COM is generally capable of mediating synergism between inducible enhancer elements. The COM contains multiple overlapping binding sites for nuclear proteins, designated uPA enhancer factors 1–4 (UEF‐1‐4). We have identified putative binding sites for UEF‐1, −2 and −3. The UEF‐1 and −3 sites in the uPA enhancer are highly conserved between species. We demonstrate the binding of UEF‐3 to the NIP element, a previously characterized regulatory element in the human interleukin‐3 and stromelysin promoters, suggesting that this factor plays a role in regulation of a variety of genes.
21919259
Purification and Characterization of UEF3, A Novel Factor Involved in the Regulation of the Urokinase and Other AP-1 Controlled Promoters (*)
Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters.
8037337
Functional characterization of COM, a DNA region required for cooperation between AP-1 sites in urokinase gene transcription.
The inducible uPA enhancer contains two phorbol ester responsive elements: the combined PEA3/AP-1A and a downstream AP-1B site. The integrity of all three sites is essential for enhancer activity. The interposed 74 bp long DNA region (COM, Cooperation Mediator) is in turn required for the synergistic action of the PEA3/AP-1 and AP-1 sites. Here we present a characterization of the COM sequence: our results show that COM and COM-binding proteins (UEF, Urokinase Enhancer Factors) may play a structural role in the induction of the uPA enhancer by phorbol-myristate-acetate (TPA). (1) COM has a bipartite structure (uCOM and dCOM) and each half contributes by about 50% to the COM-mediated TPA induction. (2) COM function is strictly dependent on its position, but not on its orientation and neither the entire COM nor individual UEF-binding sites can directly transactivate a test promoter. (3) The requirement for COM in TPA-induction is partly eliminated by the deletion of COM sequence. However, also in the COM-deleted enhancer, integrity of the PEA3/AP-1A and AP-1B sites is essential for enhancer function. We conclude that COM and COM-binding proteins provide a structural surface which facilitates the cooperation between the transactivator proteins bound at the PEA3/AP-1A and AP-1B sites. Sequences homologous to uCOM are found in the promoters of other inducible genes, coding for proteases, cytokines and chemokines: for example, in the promoter of the MIP-1alpha/LD78 chemokine gene, a 15/18 nucleotides identity is found in a region mediating positive and negative functions in TPA induction. Thus, COM-like elements represent a general enhancer function which, although lacking an intrinsic transactivating capacity, modulates the synergism between transcription factors bound to distant sites.
29300056
A single amino acid can determine the DNA binding specificity of homeodomain proteins
Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.
14466528
Extradenticle protein is a selective cofactor for the Drosophila homeotics: role of the homeodomain and YPWM amino acid motif in the interaction.
The Drosophila homeotic selector (HOM) genes encode a family of DNA binding transcription factors that specify developmental fates of different body segments by differentially regulating the activity of downstream target genes. A central question is how the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro. Specificity could be achieved by differential interactions with protein cofactors. The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax, abdominal-A, and perhaps other HOM genes. By using a yeast two-hybrid system, we demonstrate selective interaction of the extradenticle homeodomain protein with certain Ultrabithorax and abdominal-A proteins but not with an Antennapedia protein or a more distant homeodomain protein. Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts. The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms, and this variable region appears to affect the interaction detected in the assay.
30815885
The hexapeptide LFPWMR in Hoxb-8 is required for cooperative DNA binding with Pbx1 and Pbx2 proteins.
The Hox gene products are DNA-binding proteins, containing a homeodomain, which function as a class of master control proteins establishing the body plan in organisms as diverse as Drosophila and vertebrates. Hox proteins have recently been shown to bind cooperatively to DNA with another class of homeodomain proteins that include extradenticle, Pbx1, and Pbx2. Hox gene products contain a highly conserved hexapeptide connected by a linker of variable length to the homeodomain. We show that the hexapeptide and the linker region are required for cooperativity with Pbx1 and Pbx2 proteins. Many of the conserved residues present in the Hoxb-8 hexapeptide are required to modulate the DNA binding of the Pbx proteins. Position of the hexapeptide relative to the homeodomain is important. Although deletions of two and four residues of the linker peptide still show cooperative DNA binding, removal of all six linker residues strongly reduces cooperativity. In addition, an insertion of 10 residues within the linker peptide significantly lowers cooperative DNA binding. These results show that the hexapeptide and the position of the hexapeptide relative to the homeodomain are important determinants to allow cooperative DNA binding involving Hox and Pbx gene products.
46549181
Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins
The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.
20213842
The highest affinity DNA element bound by Pbx complexes in t(1;19) leukemic cells fails to mediate cooperative DNA-binding or cooperative transactivation by E2a-Pbx1 and Class I Hox proteins – evidence for selective targetting of E2a-Pbx1 to a subset of Pbx-recognition elements
Oncoprotein E2a-Pbx1 contains the N-terminal transactivation domains of E2a and the majority of the homeodomain protein, Pbx1. Using recombinant proteins, both Pbx1 and E2a-Pbx1 heterodimerize with Hox proteins on bipartite elements, Pbx1 binding a 5′ TGAT core and Class I Hox proteins binding adjacent 3′ TAAT, TTAT, or TGAT cores. In contrast to these in vitro results, nuclear extracts from E2a-Pbx1-transformed cells assemble an abundant Pbx-containing complex on TGATTGAT that excludes E2a-Pbx1, suggesting that an uncharacterized in vivo partner discriminates between E2a-Pbx1 and Pbx proteins, distinguishing it from Hox proteins. Here, we describe the DNA-binding properties of this complex, and identify TGATTGAC (PCE; Pbx Consensus Element) as its optimal recognition motif. In vitro, the PCE fails to bind heterodimers of Class I Hox proteins plus either Pbx1 or E2a-Pbx1. Likewise, in vivo, the PCE fails to mediate cooperative transactivation by E2a-Pbx1 plus Class I Hox proteins. Thus, the PCE binds a Pbx dimer partner that behaves unlike Class I Hox proteins. Competition analysis indicates that the Pbx-containing complex that binds the PCE also binds the TGATTGAT Pbx-Hox element and binds promoter elements required for tissue-specific expression of a number of cellular genes. Thus, different Pbx partners dictate targetting of Pbx heterodimers to related DNA motifs that differ in the sequence of their 3′ half-sites, and E2a-Pbx1 heterodimerizes with only a subset of Pbx partners, restricting its potential DNA targets.
6236365
Pbx-1 Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox partner at nucleotides 3' to a TAAT motif.
Heterodimers between the Pbx/Exd and Hox/HOM-C classes of homeodomain proteins bind regulatory elements in tissue-specific and developmentally regulated genes. In this work, we characterize the half-site bound by both Pbx1 and Hox proteins on a prototypic element (TGATTAAT) and determine how the orientation of the Hox protein contributes to the DNA binding specificity of Pbx-Hox heterodimers. We demonstrate that the Hox protein binds the 3' TAAT sequence as its recognition core and exhibits sequence-specific binding at positions 3' to the TAAT core. Unfavored sequences at this position, such as two cytosines, abrogate binding to the element. The upstream Pbx1 core sequence, TGAT, must immediately juxtapose the Hox core. This geometry maintains the preference of Hox/HOM-C proteins for a T base at position -1, as T represents the fourth position of the Pbx1 core, and suggests that this T base is bound by both Pbx1 and Hox proteins, Pbx1 binding in the major grove and the Hox protein binding in the minor grove. Pbx1 also exhibits base selectivity 5' to its TGAT recognition sequence.
12145220
Specificity of HOX protein function depends on DNA-protein and protein-protein interactions, both mediated by the homeo domain.
Transcription of human HOX gene promoters in cultured cells is positively and negatively regulated by HOX proteins interacting with specific target sequences. The human HOXD9 protein activates transcription of the HOXD9 promoter by interacting with the HCR sequence and is antagonized by the HOXD8 protein. HOXD8 is not intrinsically a repressor, since it can activate transcription on different targets. Complete or partial HOXD8/HOXD9 homeo domain swapping indicates that the ability to recognize, and activate transcription from, the HCR target in vivo depends on the amino terminus and helix 1 of the homeo domain. The inhibitory activity of HOXD8 is not affected by deletion of the homeo domain helix 2/3 region, whereas it requires the amino terminus/helix 1 region and an additional, effector domain located at the protein amino-terminal end. This activity is therefore DNA-binding independent, and possibly mediated by protein-protein interactions. Affinity chromatography experiments show that the homeo domain amino terminus/helix 1 region is able to mediate direct interactions between HOX proteins in solution. These data indicate that specificity of HOX protein function in vivo depends on both DNA-protein and protein-protein interactions, mediated by the same sub region of the homeo domain.
36851939
A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer
DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type. Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.
8873725
Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.
A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).