Regulation of the Pax-5 Proto-Oncogene

描述（由申请人提供）：DNA结合蛋白的Pax家族包括人类和其他高等真核生物中组织特异性基因表达的基本调节因子。Pax蛋白对于分化细胞和组织的形成是必需的，然而，DNA结合活性水平的增加导致肿瘤转化和肿瘤发生。例如，导致Pax-5过表达的染色体异常和基因重排与人类B谱系淋巴瘤相关。Pax-5介导的淋巴瘤发生的机制尚不清楚，但多条证据表明，Pax-5的剂量在正常B细胞中通过转录和翻译后机制进行精确调节。作为Pax-5的翻译后调控的一种机制，我们提出Pax- 5 DNA结合的调节，在一定程度上，由其配对的DNA结合结构域中高度保守的半胱氨酸残基的氧化还原状态。因此，Pax-5（和其他Pax家族成员）的DNA结合可能会在氧化应激反应中减少。到目前为止，这一假设只进行了测试，使用有限的体外模型系统，不能充分反映体内转录活性的稳态机制的复杂性。此外，尚未确定Pax-5的S-硫醇化（谷胱甘肽化）是否是控制其体内活性的重要机制。在本申请中，我们提出了一种遗传方法，绕过了以前遇到的缺陷与转染试验和其他体外实验方案。我们的实验将解决一个相对未探索的分子生物学领域，对理解细胞如何保持关键调控因子的精确水平具有深远的意义。我们的研究最终将有助于设计治疗Pax相关癌症（淋巴瘤、星形细胞瘤和横纹肌肉瘤）的新疗法。

DESCRIPTION (provided by applicant): The Pax family of DNA-binding proteins includes essential regulators of tissue-specific gene expression in humans and other higher eukaryotes. Pax proteins are essential for the formation of differentiated cells and tissues, however, increasing levels of DNA binding activity results in neoplastic transformation and tumorigenesis. For example, chromosomal abnormalities and gene rearrangements resulting in overexpression of Pax-5 are associated with B lineage lymphomas in humans. Mechanisms contributing to Pax-5-mediated lymphomagenesis are not understood, but multiple lines of evidence suggest that the dosage of Pax-5 is exquisitely regulated in normal B cells by transcriptional and post-translational mechanisms. As one mechanism contributing to post-translational regulation of Pax-5, we propose that Pax- 5 DNA binding is regulated, in part, by the redox status of highly conserved cysteine residues in its paired DNA-binding domain. Thus, DNA binding by Pax-5 (and other Pax family members) may be reduced in response to oxidative stress. To date, this hypothesis has only been tested using limited in vitro model systems that do not adequately reflect the complexity of homeostatic mechanisms governing transcriptional activity in vivo. Moreover, it has not been determined whether S-thiolation (glutathionylation) of Pax-5 is an important mechanism for controlling its activity in vivo. In this application, we propose a genetic approach that bypasses previously encountered deficiencies associated with transfection assays and other in vitro experimental protocols. Our experiments will address a relatively unexplored area of molecular biology with profound implications for understanding how cells maintain precise levels of key regulatory factors. Our studies will eventually aid in devising new therapeutics for treating Pax-related cancers (lymphoma, astrocytoma, and rhabdomyosarcoma).