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(申请人提供)：MLL融合基因是与白血病相关的染色体易位的结果。在MLL调节的已知靶点中，I型同源异型盒(HOX)基因在细胞谱系承诺和分化中发挥重要作用。Cyp33是一种亲环素，能与MLL的第三个PhD指结合，促进组蛋白脱乙酰酶(HDAC)与MLL(RD)的抑制域结合，抑制其反式激活活性。MLL融合蛋白不能结合Cyp33，因为它们缺乏PhD手指。因此，MLL融合蛋白可能作为结构性激活剂，促进HOX基因的异位表达，阻止造血祖细胞的承诺，导致它们永生化，这是白血病发生过程的第一步。特定目的1建议使用逆转录病毒载体在人细胞系或小鼠骨髓细胞中表达的带有或不带有PHD第三指的改良MLL融合基因，以测试它们对Hox基因表达和造血祖细胞永生化的影响。RNA干扰(RNAi)将被用来在相同的系统中下调CYP33的表达，以监测对相同实验终点的影响。 Cyp33可以通过其RRM结构域与RNA或MLL结合。因此，从增强子元件转录的新生非编码(NC)RNA可以滴度Cyp33从其控制释放MLL。这将为MLL蛋白复合体提供一种机制，以识别早期胚胎发育中的活性基因，并在随后的发育过程中保持其表达。因此，Cyp33可能是一个传感器，介导调控RNA对MLL功能的影响；因为它们不能与Cyp33结合，所以MLL融合蛋白对这种调节不敏感。特定目的2建议测试从转染的质粒中转录NC-RNA后在人类细胞系中诱导HOX基因的表达，并确定这种称为转导的现象是否依赖于Cyp33和MLL。Cyp33与Hox基因启动子的结合将在NC-RNA过表达之前和之后进行研究。将研究转导所需的NC-RNA序列的特征。将在SL2细胞中使用FISH来测试所预测的转基因质粒及其转录物与HOX基因位点的共定位。将用不同的方法测试Cyp33对MLL和不同RNA序列基序的相对亲和力，并通过染色质免疫沉淀法研究NC-RNA取代HOX基因启动子中Cyp33的能力。具体目的3将通过对整个果蝇胚胎中Cyp33的RNAi来研究Cyp33在果蝇发育中的作用。