Fujitani Y, Kajimoto Y, Yasuda T, Matsuoka T A, Kaneto H, Umayahara Y, Fujita N, Watada H, Miyazaki J I, Yamasaki Y, Hori M
Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita 565-0871, Japan.
Mol Cell Biol. 1999 Dec;19(12):8281-91. doi: 10.1128/MCB.19.12.8281.
Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development.
Pax4是一种含配对结构域（PD）的转录因子，在胰腺β/δ细胞发育中起关键作用。在本研究中，我们对小鼠Pax4的DNA结合和反式激活特性进行了表征。基于PCR的重复筛选轮次导致鉴定出Pax4的PD的最佳DNA结合序列。与Pax家族转录因子中最佳结合序列的保守性一致，Pax4可以结合Pax6的潜在结合位点，Pax6是Pax家族的另一个成员，也参与内分泌胰腺发育。Pax4在HIT-T15细胞中的过表达剂量依赖性地抑制基础转录活性以及Pax6诱导的活性。使用GAL4-Pax4嵌合体进行的详细结构域图谱分析表明，Pax4的C末端区域同时包含激活和抑制结构域。激活结构域在分别含有腺病毒E1A蛋白或E1A样活性的胚胎肾来源的293/293T细胞和胚胎癌来源的F9细胞中具有活性，但在包括胰腺β细胞和α细胞来源的其他细胞中无活性或活性非常弱。实际上，在异源细胞类型中外源过表达13S型E1A可以将激活结构域转化为活性结构域。另一方面，抑制结构域无论细胞类型如何均具有活性。当抑制结构域与异源转录因子PDX-1的反式激活结构域相连时，它可以完全消除PDX-1的反式激活潜力。这些观察结果表明Pax4作为转录抑制因子的主要作用，其功能可能涉及对Pax6功能的竞争性抑制。然而，E1A反应性反式激活结构域的鉴定表明Pax4的功能受到翻译后调控，为调节胰腺发育的机制的复杂性提供了进一步的支持。
