Control of Prostate-Specific Gene Expression

Probasin（PB）启动子已成为将基因靶向转基因小鼠前列腺上皮细胞的首选启动子，越来越多地用于基因治疗构建体中以将治疗基因靶向前列腺癌细胞，并作为研究雄激素受体作用的测定。由于这些原因，PB启动子是研究控制前列腺特异性基因表达的机制的理想模型。通过在转基因小鼠中测试31前列腺和非前列腺细胞中PB启动子的10个碱基对接头突变以及6个PB构建体，我们已经证实了两个元件在突变时仅在前列腺细胞系中降低生物活性，并且是转基因小鼠中前列腺特异性表达所需的。这两个DNA元素， 命名为TS 1和TS 2（TS为组织特异性）的两个基因组显示了相似的序列、相同的EMSA模式和功能可重复性。此外，TS-1紧邻雄激素受体结合位点-1（ARBS-1）的上游和邻近，TS-2紧邻ARBS-2的下游和邻近。我们已经确定了一个转录因子（TF）为HNF 3 tx，一个52 kD的叉头家族成员。TF的叉头家族在节段形成、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，HNF 3 tx作为AR功能的共激活剂发挥作用。尽管已经鉴定了40多种AR的辅助调节因子，但HNF 3 α和叉头家族的任何其他成员都没有参与AR的作用。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复合物，其中包括AR、HNF 3 α和至少两个控制前列腺特异性表达的其他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TF，并确定这些TF在前列腺发育中的作用。为了检验这一假设，三个具体目的如下：I）确定HNF 3 tx调节PB基因转录的机制; II）确定HNF 3ct在前列腺发育中的功能;和III）鉴定结合在复合物中以控制前列腺特异性基因表达的额外转录因子（TF）。

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.