Denecke B, Bartkowski S, Senkel S, Klein-Hitpass L, Ryffel G U
Institut für Zellbiologie (Tumorforschung), Universität-Gesamthochschule-Essen, Germany.
J Biol Chem. 1993 Aug 25;268(24):18076-82.
The transcription factor LFB1 (HNF1) involved in the expression of liver-specific genes is characterized by a serine/threonine-rich activation domain whose transactivation potential differs between mammals and Xenopus. Exchanging the activation domain between the Xenopus and rat LFB1, we produced chimeric transactivators whose activities are primarily determined by the origin of the activation domain. By replacing the serine/threonine-rich activation domain of LFB1 with the acidic activation domain of VP16, we generated transcription factors that act as dominant positive interfering mutants on endogenous LFB1 in differentiated hepatoma cells. As these LFB1/VP16 chimeras show no self-squelching as observed with wild-type LFB1 and increase the activity of saturating LFB1, we postulate that acidic and serine/threonine-rich activation domains use different targets of the basal transcription machinery. Stable transfection of various LFB1 derivatives, including those containing the VP16 transactivation domain, into the dedifferentiated C2 hepatoma cell resulted in cell clones stably expressing LFB1 function. However, as in none of these clones the chromosomal albumin genes are activated, we conclude that the presence of functional LFB1 may not be sufficient to reactivate liver-specific functions lost in dedifferentiated hepatoma cells.
参与肝脏特异性基因表达的转录因子LFB1（HNF1）的特征在于富含丝氨酸/苏氨酸的激活结构域，其反式激活潜能在哺乳动物和非洲爪蟾之间有所不同。通过交换非洲爪蟾和大鼠LFB1之间的激活结构域，我们构建了嵌合反式激活因子，其活性主要由激活结构域的来源决定。通过用VP16的酸性激活结构域替换LFB1富含丝氨酸/苏氨酸的激活结构域，我们生成了在分化的肝癌细胞中对内源性LFB1起显性正干扰突变作用的转录因子。由于这些LFB1/VP16嵌合体没有表现出野生型LFB1所观察到的自我抑制现象，并且增加了饱和LFB1的活性，我们推测酸性和富含丝氨酸/苏氨酸的激活结构域利用基础转录机制的不同靶点。将各种LFB1衍生物，包括那些含有VP16反式激活结构域的衍生物，稳定转染到去分化的C₂肝癌细胞中，产生了稳定表达LFB1功能的细胞克隆。然而，由于在这些克隆中没有一个染色体白蛋白基因被激活，我们得出结论，功能性LFB1的存在可能不足以重新激活去分化肝癌细胞中丧失的肝脏特异性功能。
