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.
Knoepfler
P S
PS
Department of Pathology, University of California, San Diego, School of Medicine, La Jolla 92093, USA.
Kamps
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MP
D001483:Base Sequence; D001665:Binding Sites; D002878:Chromosomes, Human, Pair 1; D002888:Chromosomes, Human, Pair 19; D004247:DNA / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000494:pharmacology; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011233:Precipitin Tests; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015533:Transcriptional Activation; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C489213:HOXA5 protein, human; D018398:Homeodomain Proteins; D015514:Oncogene Proteins, Fusion; D010750:Phosphoproteins; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C083400:PBX1 protein, human; C089847:E2A-Pbx1 fusion protein; D004247:DNA