MyoD Activity in Rhabdomyosarcomas

描述（申请人提供）：横纹肌肉瘤是儿童最常见的实体瘤之一，被认为是由于肌原性前体细胞生长和分化的调节异常而引起的。尽管MyoD家族的肌源性碱性-螺旋-环-螺旋蛋白表达，横纹肌肉瘤细胞不能退出细胞周期，不能分化为肌肉细胞。本研究的目的是研究阻止人胚胎横纹肌肉瘤（RD）细胞分化的分子机制。通过分子和生化相结合的方法，我们希望了解调控缺陷如何在肌肉分化程序的多个步骤中发生，并找到横纹肌肉瘤起源的线索。具体目的是：(I)使用染色质免疫沉淀法确定阻止MyoD激活RD细胞中启动子子集的因素；（II）利用诱变和蛋白相互作用分析表征翻译后修饰及其在E2A蛋白上的功能；（III）通过串联亲和纯化和串联质谱鉴定和表征MyoD相关转录复合物中rd特异性改变。

DESCRIPTION (provided by applicant): Rhabdomyosarcoma, one of the most common solid tumors of childhood, is believed to arise as a result of a regulatory abnormality of the growth and differentiation of myogenic precursor cells. Despite the expression of myogenic basic-helix-loop-helix proteins of the MyoD family, rhabdomyosarcoma cells fail to withdraw from the cell cycle and are unable to differentiate into muscle cells. The OBJECTIVE of this research proposal is to investigate the molecular mechanisms that prevent differentiation in human embryonal rhabdomyosarcoma (RD) cells. By applying combined molecular and biochemical approaches, we hope to understand how regulatory defects could occur at multiple steps of the muscle differentiation program and find clues to the origin of rhabdomyosarcomas. The SPECIFIC AIMS are: (I) Identify the factors that prevent MyoD from activating a subset of promoters in RD cells using chromatin immuno-precipitation assays; (II) Characterize post-translational modifications and their functionality on E2A proteins using mutagenesis and protein-protein interaction assays; (III) Identify and characterize RD-specific alterations in the MyoD- associated transcriptional complex by Tandem Affinity Purification and tandem mass spectrometry.