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肿瘤之一，被认为是肌源性前体细胞生长和分化调节异常的结果。横纹肌肉瘤细胞虽然表达MyoD家族的肌源性碱性螺旋-环-螺旋蛋白，但不能退出细胞周期，不能分化为肌肉细胞。本研究的目的是探讨阻止人类胚胎横纹肌肉瘤(RD)细胞分化的分子机制。通过应用分子和生物化学相结合的方法，我们希望了解肌肉分化程序的多个步骤是如何发生调节缺陷的，并找到横纹肌肉瘤起源的线索。其具体目标是：(I)利用染色质免疫沉淀分析确定阻止MyoD激活RD细胞中的一组启动子的因素；(Ii)通过突变和蛋白质-蛋白质相互作用分析鉴定翻译后修饰及其对E2a蛋白的功能；(Iii)通过串联亲和纯化和串联质谱仪鉴定和表征MyoD相关转录复合体中的RD特异性变化。

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.