Qiu Y, Sharma A, Stein R
Department of Molecular Physiology and Biophysics, Vanderbilt Medical Center, Nashville, Tennessee 37232, USA.
Mol Cell Biol. 1998 May;18(5):2957-64. doi: 10.1128/MCB.18.5.2957.
Pancreatic beta-cell-type-specific and glucose-inducible transcription of the insulin gene is mediated by the basic helix-loop-helix factors that bind to and activate expression from an E-box element within its enhancer. The E-box activator is a heteromeric complex composed of a beta-cell-enriched factor, BETA2/NeuroD, and ubiquitously distributed proteins encoded by the E2A and HEB genes. Previously, we demonstrated that the adenovirus type 5 E1A proteins repressed stimulation by the E-box activator in beta cells. In this study, our objective was to determine how E1A repressed activator function. The results indicate that E1A reduces activation by binding to and sequestering the p300 cellular coactivator protein. Thus, we show that expression of p300 in beta cells can relieve inhibition by E1A, as well as potentiate activation by the endogenous insulin E-box transcription factors. p300 stimulated activation from GAL4 (amino acids 1 to 147) fusion constructs of either BETA2/NeuroD or the E2A-encoded E47 protein. The sequences spanning the activation domains of BETA2/NeuroD (amino acids 156 to 355) and E47 (amino acids 1 to 99 and 325 to 432) were required for this response. The same region of BETA2/NeuroD was shown to be important for binding to p300 in vitro. The sequences of p300 involved in E47 and BETA2/NeuroD association resided between amino acids 1 and 1257 and 1945 and 2377, respectively. A mutation in p300 that abolished binding to BETA2/NeuroD also destroyed the ability of p300 to activate insulin E-box-directed transcription in beta cells. Our results indicate that physical and functional interactions between p300 and the E-box activator factors play an important role in insulin gene transcription.
胰岛素基因的胰腺β细胞类型特异性和葡萄糖诱导性转录是由碱性螺旋-环-螺旋因子介导的，这些因子与增强子内的E盒元件结合并激活其表达。E盒激活剂是一种异源复合物，由富含β细胞的因子BETA2/NeuroD以及由E2A和HEB基因编码的普遍分布的蛋白质组成。先前，我们证明了5型腺病毒E1A蛋白可抑制β细胞中E盒激活剂的刺激作用。在本研究中，我们的目的是确定E1A如何抑制激活剂功能。结果表明，E1A通过结合并隔离p300细胞共激活蛋白来降低激活作用。因此，我们表明β细胞中p300的表达可以缓解E1A的抑制作用，并增强内源性胰岛素E盒转录因子的激活作用。p300刺激了BETA2/NeuroD或E2A编码的E47蛋白的GAL4（氨基酸1至147）融合构建体的激活。此反应需要跨越BETA2/NeuroD（氨基酸156至355）和E47（氨基酸1至99和325至432）激活域的序列。已证明BETA2/NeuroD的同一区域对于体外与p300的结合很重要。参与E47和BETA2/NeuroD结合的p300序列分别位于氨基酸1至1257和1945至2377之间。p300中一个消除与BETA2/NeuroD结合的突变也破坏了p300在β细胞中激活胰岛素E盒定向转录的能力。我们的结果表明，p300与E盒激活因子之间的物理和功能相互作用在胰岛素基因转录中起重要作用。
