Knoepfler P S, Kamps M P
Department of Pathology, University of California, San Diego, School of Medicine, La Jolla 92093, USA.
Oncogene. 1997 May 29;14(21):2521-31. doi: 10.1038/sj.onc.1201097.
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.
癌蛋白E2a - Pbx1包含E2a的N端反式激活结构域和大部分同源结构域蛋白Pbx1。利用重组蛋白，Pbx1和E2a - Pbx1均可在双联体元件上与Hox蛋白形成异源二聚体，Pbx1结合5' TGAT核心序列，I类Hox蛋白结合相邻的3' TAAT、TTAT或TGAT核心序列。与这些体外实验结果相反，来自E2a - Pbx1转化细胞的核提取物在TGATTGAT上组装了一种丰富的含Pbx的复合物，该复合物排除了E2a - Pbx1，这表明一种未被鉴定的体内伴侣能够区分E2a - Pbx1和Pbx蛋白，使其与Hox蛋白不同。在此，我们描述了这种复合物的DNA结合特性，并鉴定出TGATTGAC（PCE；Pbx共有元件）为其最佳识别基序。在体外，PCE不能结合I类Hox蛋白与Pbx1或E2a - Pbx1形成的异源二聚体。同样，在体内，PCE不能介导E2a - Pbx1与I类Hox蛋白的协同反式激活。因此，PCE结合的Pbx二聚体伴侣的行为与I类Hox蛋白不同。竞争分析表明，结合PCE的含Pbx的复合物也结合TGATTGAT Pbx - Hox元件，并结合许多细胞基因组织特异性表达所需的启动子元件。因此，不同的Pbx伴侣决定了Pbx异源二聚体靶向与其3'半位点序列不同的相关DNA基序，并且E2a - Pbx1仅与一部分Pbx伴侣形成异源二聚体，限制了其潜在的DNA靶点。
