Control of Prostate-Specific Gene Expression

该启动子已成为转基因小鼠前列腺上皮细胞靶向基因的首选启动子，越来越多地用于构建靶向前列腺癌治疗基因的基因治疗结构，并作为研究雄激素受体作用的实验手段。因此，PB启动子是研究前列腺特异性基因表达调控机制的理想模型。通过检测前列腺癌和非前列腺癌细胞中PB启动子的31个碱基对连接子突变和转基因小鼠中的6个PB结构，我们已经证实，当突变时，两个元件仅在前列腺细胞系中降低生物活性，并且是转基因小鼠前列腺特异性表达所必需的。这两种DNA元素， 命名为TS1和TS2(TS为组织特异性)，显示了相似的序列，相同的EMSA模式和功能互换。此外，TS-1位于雄激素受体结合位点-1(ARBS-1)的上游，与ARBS-1相邻，TS-2位于ARBS-2的下游与ARBS-2相邻。我们已经鉴定了一个转录因子(Tf)为HNF3tx，它是一个52kD的叉头家族成员。叉头家族的转录因子在节段形成、器官发育和细胞分化中起着重要作用。此外，我们的数据表明，HNF3tx作为AR功能的共激活子发挥作用。尽管已经确定了40多个AR的协同调节因子，但HNF3pha和叉头家族的任何其他成员都没有参与AR的行动。我们的假设是，TS-1和TS-2分别与ARBS-1和ARBS-2相互作用，形成一个TF复合体，其中包括AR、HNF3pha和至少两个额外的调控前列腺特异性表达的TF。我们的目标是识别和表征控制前列腺特异性基因表达的转录因子，并确定这些转录因子在前列腺发育中的作用。为了验证这一假说，三个特定的目标如下：i)确定HNF3tx调控PB基因转录的机制；ii)确定HNF3ct在前列腺发育中的功能；iii)确定结合在复合体中的额外转录因子(TF)，以控制前列腺特异性基因的表达。

The Probasin (PB) promoter has become the promoter of choice to target genes to epithelial cells in the prostate of transgenic mice, increasingly used in gene therapy constructs to target therapeutic genes to prostate cancer cells, and as an assay to study androgen receptor action. For these reasons, the PB promoter is an ideal model to study the mechanism that controls prostate-specific gene expression. By testing 31 ten base pair linker-mutations of the PB promoter in prostatic and non-prostatic cells and six PB constructs in transgenic mice, we have confirmed that two elements, when mutated, reduced bioactivity only in prostatic cell lines and are required for prostate specific expression in transgenic mice. The two DNA elements, designated as TS 1 and TS2 (TS for Tissue Specific), revealed similar sequences, identical patterns in EMSA and functional interchangeability. Further, TS-1 is immediately upstream and adjacent to the Androgen Receptor Binding Site-1 (ARBS-1) and TS-2 is immediately downstream and adjacent to ARBS-2. We have identified one Transcription Factor (TF) as HNF3tx, a 52 kD forkhead family member. The forkhead family of TFs plays a fundamental role in segment formation, organ development, and cell differentiation. Furthermore, our data demonstrate that HNF3tx functions as a coactivator of AR function. Although over forty coregulators for AR have been identified, neither HNF3alpha nor any other member of theforkhead family ever been implicated in AR action. Our hypothesis is that TS-1 and TS-2 interact with ARBS-1 and ARBS-2, respectively, to form a TF complex that include AR, HNF3alpha, and at least two additional TFs that control prostate-specific expression. Our goal is to identify and characterize the TFs that control prostate-specific gene expression and determine the role of these TFs in prostate development. To test this hypothesis, the three Specific Aims are as follows: I) To determine the mechanism by which HNF3tx regulates PB gene transcription; II) To determine the function of HNF3ct in prostate development; and III) To identify the additional transcription Factors (TFs) that bind in a complex to control prostate-specific gene expression.