Structural analysis of the AF9 C-terminus
Description
Reciprocal chromosomal translocations involving the Mixed-Lineage Leukemia (MLL) gene at locus 11q23 are common in infant acute leukemias, occurring in up to 80% of cases of AML and ALL. MLL translocations result in the in-frame fusion of MLL to a variety of different genes, which leads to the expression of a chimeric protein with transforming properties. To date, more than 50 different fusion proteins have been identified. One of the more common, AF9 fuses with MLL as a consequence of the t(9;11)(p22;q23) translocation that is detected in acute and secondary leukemias The function of AF9 in normal and diseased cells is not completely clear. It is known, however, that the ∼90 amino acids carboxy terminus of AF9 contains a putative transcription activation domain functional in reporter gene assays. Moreover, when fused to MLL, the carboxy-termini of the AF9 and ENL proteins are required for transformation of hematopoietic stem cells. In previous experiments we identified AF4, another common MLL fusion partner, as well as two other proteins involved in transcription repression, Pc3 and BCoR, as a binding partner of the AF9 carboxy terminus. Importantly, both AF4 and AF9 binding sites are retained in the MLL fusions suggesting that the interaction plays a critical role in leukemogenesis. The carboxy terminus of AF9 is comprised of approximately 90 amino acids that are predicted to adopt a structure containing two putative alpha-helices. Deletions limited to either helix prevent the binding of AF4, Pc3 and BCoR. To gain some insights into the higher order structure of AF9 C-terminus, this dissertation utilized a yeast two-hybrid selection and screen to identify the residues that mediate binding between AF9 and AF4, Pc3 and BCoR. Additionally, a method for expression and purification of the AF9 C-terminus was developed and preliminary characterization of the AF9 C-terminus by circular dichroism was initiated. Given the importance of the AF9 C-terminus in the development of acute leukemias, these studies provide the groundwork for future investigations into the overall structure of the AF9 C-terminus for the ultimate goal of drug design and targeting