Liu Gang, Xia Tian, Chen Xinbin
Department of Cell Biology, The University of Alabama, Birmingham, Alabama 35294, USA.
J Biol Chem. 2003 May 9;278(19):17557-65. doi: 10.1074/jbc.M210696200. Epub 2003 Feb 27.
The p53 transcription factor contains two separate tandem activation domains (AD1 and AD2), a proline-rich domain (PRD), and a C-terminal basic domain (BD). Previously, we have shown that these domains are necessary for transcriptional activity. To further characterize the role of these domains in transactivation, we analyzed the regulation of p21, a well characterized p53 target gene, by various p53 mutants deficient in one or more of these domains. We found that the induction of endogenous p21 is compromised by AD1-deficient p53 (p53(AD1(-))), AD2-deficient p53 (p53(AD2(-))), both AD1- and AD2-deficient p53 (p53(AD1(-)AD2(-))), p53(deltaPRD), which lacks PRD, and p53(deltaBD), which lacks BD. However, p53(AD2(-)), p53(deltaPRD), and p53(deltaBD) are still capable of activating exogenous p21 promoter to an extent comparable with that by wild-type p53. Thus, we performed chromatin immunoprecipitation assay to measure the DNA binding ability of various p53 mutants in vivo. We found that like wild-type p53, these p53 mutants are capable of binding to the p53 response elements in the p21 promoter. In contrast, we found that the extent of acetylated histones on the p21 promoter, especially the proximal promoter, and the amount of interaction with p300/CREB-binding protein, which contain histone acetyltransferase activity, directly correlate with the activity of p53 to induce endogenous p21. Furthermore, we showed that down-regulation of p300/CBP by short interference RNA markedly decreases the ability of p53 to induce endogenous p21. These data lead us to hypothesize that when p53 binds to the responsive element(s) of a target gene, its ability to interact with histone acetyltransferase-containing proteins and subsequently the acetylation of histones bound to the proximal promoter dictate the induction level of a target gene.
p53转录因子包含两个独立的串联激活域（AD1和AD2）、一个富含脯氨酸的结构域（PRD）和一个C端碱性结构域（BD）。此前，我们已经表明这些结构域对于转录活性是必需的。为了进一步阐明这些结构域在反式激活中的作用，我们分析了各种缺失一个或多个这些结构域的p53突变体对p21（一个特征明确的p53靶基因）的调控。我们发现，内源性p21的诱导受到AD1缺陷型p53（p53(AD1(-))）、AD2缺陷型p53（p53(AD2(-))）、AD1和AD2双缺陷型p53（p53(AD1(-)AD2(-))）、缺乏PRD的p53(deltaPRD)以及缺乏BD的p53(deltaBD)的损害。然而，p53(AD2(-))、p53(deltaPRD)和p53(deltaBD)仍然能够在一定程度上激活外源性p21启动子，其程度与野生型p53相当。因此，我们进行了染色质免疫沉淀实验以测量各种p53突变体在体内的DNA结合能力。我们发现，与野生型p53一样，这些p53突变体能够结合p21启动子中的p53反应元件。相反，我们发现p21启动子上，尤其是近端启动子上的组蛋白乙酰化程度以及与含有组蛋白乙酰转移酶活性的p300/CREB结合蛋白的相互作用量，与p53诱导内源性p21的活性直接相关。此外，我们表明通过短干扰RNA下调p300/CBP可显著降低p53诱导内源性p21的能力。这些数据使我们推测，当p53与靶基因的反应元件结合时，其与含组蛋白乙酰转移酶的蛋白相互作用的能力以及随后与近端启动子结合的组蛋白的乙酰化作用决定了靶基因的诱导水平。
