MLL, CYP33 and non-coding RNA in leukemogenesis

描述（申请人提供）：MLL融合基因是白血病相关染色体易位的结果。MLL调控的已知靶点是I型同源框（HOX）基因，其在细胞谱系定型和分化中具有重要作用。 Cyp 33是亲环蛋白，其结合MLL的第三PHD指并促进组蛋白脱乙酰酶（HDAC）与MLL的阻遏结构域（RD）结合，抑制其反式激活活性。MLL融合蛋白无法结合Cyp 33，因为它们缺乏PHD指。因此，MLL融合蛋白可作为组成型激活剂发挥作用，其促进HOX基因的异位表达并防止造血祖细胞定型，从而导致其永生化，这是白血病发生过程中的初始步骤。具体目的1提出使用在人细胞系或小鼠骨髓细胞中从逆转录病毒载体表达的具有或不具有第三PHD指的修饰的ML融合基因，以测试它们对HOX基因表达和对造血祖细胞永生化的影响。RNA干扰（RNAi）将用于敲低相同系统中的CYP 33表达，以监测对相同实验终点的影响。 Cyp 33可以通过其RRM结构域结合RNA或MLL。因此，从增强子元件转录的新生非编码（NC）RNA可以滴定Cyp 33，从而从其控制中释放MLL。这将为MLL蛋白复合物在早期胚胎发生中识别活性基因并在随后的发育中维持其表达提供机制。因此，Cyp 33将是介导调节RNA对MLL功能的影响的传感器;因为它们不能结合Cyp 33，所以MLL融合蛋白将对这种类型的调节不敏感。具体目标2提出在人细胞系中检测NC-RNA从转染质粒转录后HOX基因表达的诱导，并确定这种称为转诱导的现象是否依赖于Cyp 33和MLL。将在NC-RNA过表达之前和之后研究Cyp 33与HOX基因启动子的结合。将研究转诱导所需的NC-RNA序列的特征。将在SL 2细胞中使用FISH检测转染质粒及其转录物与HOX基因座的预测共定位。Cyp 33对MLL和不同RNA序列基序的相对亲和力将使用不同的方法进行测试，NC-RNA从HOX基因启动子置换Cyp 33的能力将通过染色质免疫沉淀进行研究。具体目标3将通过在整个果蝇胚胎中使用针对Cyp 33的RNAi来研究Cyp 33在果蝇发育中的作用。

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.