MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION INHUMA

16号染色体反转INHUMA的分子发病机制

• 批准号: 6829439
• 负责人: PU PAUL LIU
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6829439
• 关键词: acute myelogenous leukemia; arthritis; carcinogenesis; chromosome inversion; disease /disorder model; embryonic stem cell; fusion gene; genetic promoter element; genetic regulation; human genetic material tag; human tissue; laboratory mouse; microarray technology; molecular oncology; molecular pathology; myosins; neoplasm /cancer genetics; oncogenes; polymerase chain reaction; serial analysis of gene expression; transcription factor; transfection; tumor suppressor genes; yeast two hybrid system

Chromosome 16 inversion, inv(16), is one of the most common chromosome abnormalities in human acute myeloid leukemia (AML). A fusion gene between the core binding factor B (CBFB) gene and the myosin heavy chain 11 (MYH11) gene is generated by this inversion. CBFB encodes a transcription factor while MYH11 encodes the smooth muscle form of myosin heavy chain (SMMHC). To identify target genes of CBFb-SMMHC that are important in leukemia, we are taking two separate approaches: 1. serial analysis of gene expression (SAGE) of human leukemia samples and 2. cDNA microarray analysis of mouse embryonic stem (ES) cells with CBFB-MYH11 expression. Using SAGE, we performed genome-wide analyses of gene expression in 5 AML cases with inv(16). One SAGE library was made from each patient sample and over 50,000 tags were sequenced from each library. The results were analyzed relative to SAGE data from a normal bone marrow library. For the cDNA microarray analysis, RNA from mouse ES cells with CBFB-MYH11 turned on or off was isolated and used to generate cDNA probes to hybridize to a mouse 31K array. Potential target genes are now confirmed by quantitative PCR and promoter regions are being analyzed. These two complementary approaches will hopefully facilitate the identification of true targets of CBFB-MYH11 during leukemogenesis. Using a yeast two-hybrid screening approach we identified the c-Myc promoter binding protein (MBP-1) as a binding target of CBFB. MBP-1 binds to the promoter of the c-Myc gene and negatively regulate the expression of c-Myc, which is dysregulated in most cancer cells including leukemia, resulting in unbalanced control of cell proliferation, differentiation, or apoptosis. Subsequent studies suggest that MBP-1 may be a functional bridge between c-Myc and CBFB-MYH11 in human leukemia. We have generated mice expressing CBFB-MYH11 with various deletions of MYH11 by gene targeting in mouse ES cells. These mice are analyzed for defects in blood formation and in leukemia development. This study will tell us the functional domains of MYH11 required for leukemia development.

16号染色体倒位(inv(16))是人类急性髓系白血病(AML)最常见的染色体异常之一。这个倒位产生了核心结合因子B(Cbfb)基因和肌球蛋白重链11(MYH11)基因之间的融合基因。Cbfb编码一个转录因子，而MYH11编码肌球蛋白重链的平滑形式(SMMHC)。为了确定Cbfb-SMMHC在白血病中起重要作用的靶基因，我们采用了两种不同的方法：1.人类白血病样本的基因表达序列分析(SAGE)；2.表达Cbfb-MYH11的小鼠胚胎干细胞的基因芯片分析。使用SAGE，我们对5例inv(16)的AML患者的基因表达进行了全基因组分析。从每个患者样本中制作一个SAGE文库，并从每个文库中对超过50,000个标签进行测序。将结果与正常骨髓库中的SAGE数据进行对比分析。为进行基因芯片分析，从Cbfb-MYH11基因开启或关闭的小鼠ES细胞中提取RNA，制备与31K基因芯片杂交的cDNA微阵列。潜在的靶基因现已通过定量聚合酶链式反应确认，启动子区域正在分析中。这两种互补的方法有望有助于在白血病发生过程中识别Cbfb-MYH11的真实靶点。 通过酵母双杂交筛选方法，我们确定了c-Myc启动子结合蛋白(MBP-1)作为Cbfb的结合靶点。MBP-1与c-Myc基因启动子结合，负向调节c-Myc的表达，c-Myc的表达在包括白血病在内的大多数肿瘤细胞中都存在失调，导致对细胞增殖、分化或凋亡的不平衡控制。后续研究表明，MBP-1在人类白血病中可能是c-Myc和Cbfb-MYH11之间的功能桥梁。 我们通过在小鼠ES细胞中进行基因打靶，获得了表达不同MYH11缺失的Cbfb-MYH11的小鼠。这些小鼠被分析是否存在造血缺陷和白血病的发生。这项研究将告诉我们白血病发生所需的MYH11的功能结构域。