MLL, CYP33 and non-coding RNA in leukemogenesis

MLL、CYP33 和非编码 RNA 在白血病发生中的作用

• 批准号: 6821913
• 负责人: MANUEL ORESTES DIAZ
• 金额: $ 27.31万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821913
• 关键词: Drosophilidae; RNA; RNA interference; carcinogenesis; cell line; cell transformation; chromatin immunoprecipitation; chromosome translocation; fluorescent in situ hybridization; fusion gene; gel mobility shift assay; gene expression; gene induction /repression; homeobox genes; laboratory mouse; leukemia; neoplasm /cancer genetics; nucleic acid sequence; peptidylprolyl isomerase; protein binding; protein structure function; regulatory gene

DESCRIPTION (provided by applicant): MLL fusion genes arise as the consequence of chromosome translocations associated with leukemia. Among the known targets of regulation by MLL are the type I homeobox (HOX) genes that have important roles in cell-lineage commitment and differentiation. Cyp33 is a cyclophilin that binds the third PHD finger of MLL and promotes binding of histone deacetylases (HDACs) to a repression domain of MLL (RD), inhibiting its transactivating activity. The MLL fusion proteins cannot bind Cyp33 because they lack the PHD fingers. Thus, the MLL fusion proteins may function as constitutive activators that promote ectopic expression of HOX genes and prevent commitment of hematopoietic progenitor cells, leading to their immortalization, an initial step in the leukemogenic process. Specific aim 1 proposes to use modified MLL-fusion genes with or without the 3rd PHD finger, expressed from retroviral vectors in human cell lines, or in mouse bone marrow cells, to test their effect on HOX gene expression and on immortalization of hematopoietic progenitors. RNA interference (RNAi) will be used to knock down CYP33 expression in the same systems, to monitor the effect on the same experimental endpoints. Cyp33 can bind either RNA or MLL through its RRM domain. Therefore, nascent non-coding (NC) RNAs transcribed from enhancer elements, can titer Cyp33 releasing MLL from its control. This would provide a mechanism for the MLL protein complex to recognize active genes in early embryogenesis and maintain their expression through subsequent development. Thus, Cyp33 would be a sensor mediating the effect of regulatory RNAs on MLL function; because they cannot bind Cyp33, the MLL fusion proteins would be insensitive to this type of regulation. Specific aim 2 proposes to test the induction of HOX gene expression after transcription of NC-RNA from a transfected plasmid in human cell lines, and to determine if this phenomenon, called transinduction, is dependent on Cyp33 and MLL. The binding of Cyp33 to the HOX gene promoters will be studied before and after overexpression of the NC-RNA. The features of the NC-RNA sequence necessary for transinduction will be studied. The predicted co-localization of the transfected plasmid, and its transcripts with the HOX gene loci will be tested using FISH in SL2 cells. The relative affinity of Cyp33 for MLL and for different RNA sequence motifs will be tested using different methods, and the ability of NC-RNA to displace Cyp33 from HOX gene promoters will be studied by chromatin immunoprecipitation. Specific aim 3 will study the role of Cyp33 in Drosophila development by using RNAi for Cyp33 in whole Drosophila embryos.

描述（由申请人提供）：MLL融合基因是由于与白血病相关的染色体易位而产生的。 MLL调节的已知靶标之一是I型同源物（HOX）基因，这些基因在细胞限制承诺和分化中具有重要作用。 CYP33是一种与MLL的第三个PHD手指结合并促进组蛋白脱乙酰基酶（HDAC）与MLL（RD）抑制域的结合，抑制其反式激活活性的结合。 MLL融合蛋白无法结合CYP33，因为它们缺乏博士学位的手指。因此，MLL融合蛋白可以用作促进HOX基因异位表达并防止造血祖细胞的投入，导致其永生化，这是白血病过程的第一步。具体目标1提议使用有或没有第三只PHD手指的修饰的MLL融合基因，该基因从人类细胞系中的逆转录病毒载体或小鼠骨髓细胞中表达，以测试其对HOX基因表达的影响以及对造血祖细胞的永生化。 RNA干扰（RNAI）将用于在同一系统中击倒CYP33表达，以监视对相同实验终点的影响。 CYP33可以通过其RRM域结合RNA或MLL。因此，从增强子元素转录的新生非编码（NC）RNA可以从其对照中释放MLL。这将为MLL蛋白复合物提供一种机制，以识别早期胚胎发生中的活性基因，并通过随后的发育保持其表达。因此，CYP33将是一个传感器，介导调节RNA对MLL功能的影响。由于它们无法结合CYP33，因此MLL融合蛋白对这种调节不敏感。具体目的2提议在人类细胞系中从转染的质粒转录NC-RNA后测试HOX基因表达的诱导，并确定这种现象（称为transintuction）是否取决于CYP33和MLL。在NC-RNA过表达之前和之后，将研究CYP33与HOX基因启动子的结合。将研究NC-RNA序列所需的NC-RNA序列的特征。转染的质粒的预测共定位，以及其与HOX基因位点的转录本将在SL2细胞中使用FISH测试。 CYP33对MLL和对不同RNA序列基序的相对亲和力将使用不同的方法进行测试，并且将通过染色质免疫沉淀研究NC-RNA从HOX基因启动子置换CYP33的能力。特定的目标3将通过在整个果蝇胚胎中使用RNAi进行CYP33来研究CYP33在果蝇发育中的作用。