The overall objective of this proposal is to study the function of the normal MLL protein and of the fusion proteins formed by chromosomal translocations involving MLL that result in leukemia. They will use several approaches to identify downstream target genes of normal MLL and of two MLL fusions, MLL-AF9 and MLL-CBP. AF9 is the most common fusion partner of MLL in acute myeloid leukemia, both de novo and secondary to therapy with drugs that target DNA topoisomerase II. CBP is a fusion partner of MLL that has so far been observed only in therapy-related leukemias with one exception. They will identify target genes of normal MLL based on the ability of the MLL protein to bind to a target DNA sequence or structure using immunoprecipitation. From these experiments, they will identify targets of MLL to which it binds either directly or indirectly. They will also define the parameters that control binding, which may include DNA target sequence/structure, interacting proteins, MLL domains involved, and protein modification. Additionally, they will identify genes whose expression is altered by expression of either MLL-AF9 or MLL-CBP fusion proteins using transient or inducible stable expression systems followed by gene expression pattern analysis on a genome-wide scale using cDNA microarrays and oligonucleotide microarray chips. This will identify targets whose altered expression may be important in leukemogenesis, regardless of the mechanism. The direct and the downstream targets will help identify cellular pathways that are deregulated by the fusion proteins and that may be critical for the ultimate leukemia phenotype. Together, the experiments proposed in these two specific aims will provide valuable information regarding the mechanism of MLL and MLL-partner gene function in normal and in leukemic cells.
