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.
| Xia, Zhen-Biao; Popovic, Relja; Chen, Jing et al. (2005) The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression. Proc Natl Acad Sci U S A 102:14028-33 |
