Role of Collaborator Proteins in Hoxa9-Mediated Leukemogenesis

描述（由申请人提供）：Hoxa9是一种含有同源结构域的转录因子，在造血干细胞扩增中起关键作用，在人类急性白血病中通常不受调控。超过50%的急性髓性白血病（AML）病例显示Hoxa9过表达，几乎总是与其辅助因子Meis1过表达相关。在一项人类aml基因表达的研究中，高表达HOXA9是预后不良的唯一最具预测性的标记。大量数据表明，Hoxa9和Meis1在AML中发挥协同致病作用，尽管导致Hoxa9和Meis1转化的分子机制尚不清楚。了解hoxa9介导的白血病发生首先需要更好地了解是什么赋予Hox家族蛋白的结合特异性。所有的Hox蛋白都通过高度同源的同源结构域结合无处不在的TAAT基序，这并不能单独解释它们对转录活性的严格控制。通过与其他dna结合辅助因子（如Meis1）的结合，在体内实现了额外的序列特异性。另一个水平的调控可能是由指导Hox蛋白特异性的多种合作者赋予的，但这些蛋白质的身份和它们调节Hox结合的机制尚未阐明。通过鉴定Hoxa9和Meis1的体内结合位点以及鉴定与Hoxa9复合物相互作用的蛋白，我们在确定潜在合作者方面取得了相当大的进展。我们发现Hoxa9和Meis1结合到进化上保守的位点，这些位点包含与增强子序列一致的表观遗传特征。结合区的从头基序分析显示，C/EBP、ETS和STAT家族的转录因子（TF）基序显著富集。随后的质谱和共免疫沉淀实验证实了Hoxa9复合物与C/ebp a和Stat5的关联。我们最近已经生成了有条件表达C/ebp a、Pu.1和Stat5的白血病细胞系模型系统，我们将使用这些细胞来研究每种蛋白在靶向Hoxa9到髓系和淋巴系特定结合位点中的作用。我们还将使用这些模型来确定C/ebp a、Pu.1和Stat5对Hoxa9转录活性的影响。最后，我们将使用我们建立的有条件表达染色质重塑子Brg1的细胞系来确定Brg1和C/ebp a在调节Hoxa9复合物活性方面的功能关系。总之，这些研究将为高水平表达Hoxa9介导的白血病发生提供许多新的机制见解。

DESCRIPTION (provided by applicant): Hoxa9 is a homeodomain containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. More than 50% of acute myeloid leukemia (AML) cases show overexpression of Hoxa9, almost always in association with overexpression of its cofactor Meis1. In a study of gene expression in human AMLs, high expression HOXA9 was the single most predictive mark for poor prognosis. A wide range of data suggests that Hoxa9 and Meis1 play a synergistic causative role in AML, though the molecular mechanisms leading to transformation by Hoxa9 and Meis1 remain elusive. Understanding Hoxa9-mediated leukemogenesis first requires a better understanding of what confers binding specificity of Hox family proteins. All Hox proteins bind the ubiquitous TAAT motif through their highly homologous homeodomains, which alone cannot account for their tight control of transcriptional activity. Additional sequence specificity is achieved in vivo through association with other DNA-binding cofactors, such as Meis1. Another level of regulation is likely conferred by diverse sets of collaborators that direct Hox protein specificity, but the identity of these proteins and the mechanisms through which they regulate Hox binding have yet to be elucidated. We have made considerable progress towards determining potential collaborators by characterizing in vivo binding sites of Hoxa9 and Meis1 and by identifying proteins that interact with the Hoxa9 complex. We found that Hoxa9 and Meis1 bind to evolutionarily conserved sites that contain an epigenetic signature consistent with enhancer sequences. De novo motif analysis of the binding regions showed a marked enrichment of motifs for transcription factors (TF) in the C/EBP, ETS, and STAT families. Subsequent mass spectrometry and coimmunoprecipitation experiments have confirmed association of the Hoxa9 complex with C/ebp a and Stat5. We have recently generated leukemic cell line model systems that conditionally express C/ebp a, Pu.1 and Stat5, and we will use these cells to study the role of each protein in targeting Hoxa9 to specific binding sites in myeloid and lymphoid lineages. We will also use these models to determine the effect of C/ebp a, Pu.1 and Stat5 on the transcriptional activity of Hoxa9. Finally, we will use a cell line we have established with conditional expression of the chromatin remodeler, Brg1, to determine the functional relationship between Brg1 and C/ebp a in modulating Hoxa9 complex activity. Together these studies will provide many new mechanistic insights into leukemogenesis mediated by high-level Hoxa9 expression. ) PUBLIC HEALTH RELEVANCE: Human acute myeloid leukemia (AML) is a highly heterogeneous disease that varies significantly in prognosis and response to treatment. Understanding the molecular basis behind leukemic transformation in different subgroups will be crucial for development of targeted therapeutics needed to improve survival and outcomes. Our research will investigate the molecular mechanisms important in AML with high levels of Hoxa9 expression, a transcription factor that is upregulated in 50% of AML cases and is associated with poor prognosis.