Identificazione di interattori proteici di AF4, partner di traslocazione del gene MLL (Mixed Lineage Leukaemia) nelle leucemie acute

The Mixed Lineage Leukaemia (MLL) gene on chromosome band 11q23 is a recurrent target of reciprocal translocation in human acute leukaemias. The 11q23 translocation occurs in 5-6% of patients with acute myelogenous leukaemia (AML) and 7-10% of patients with acute lymphoblastic leukemia (ALL). More than 50 partner genes are involved in MLL translocation; as a consequence, MLL is joined in-frame to different unrelated genes to form fusion proteins with oncogenic properties. The most common MLL translocation partner is the AF4 gene, on band 4q21. Translocation t(4;11)(q21;q23) is involved in a third of all MLL translocation cases and represents 95% of translocations involved in ALL, compared to only 3.3% in AML (1). AF4 is a serine/proline-rich nuclear protein with transcriptional activation domains. Gene knockout studies indicate that AF4 plays an important role in B and T lymphopoiesis (2). AF4 is a member of the AF4, LAF4, and FMR2 (ALF) family of proline- and serine-rich proteins that also includes the more recently characterized AF5Q31. Like AF4, AF5Q31 is implicated in leukemogenesis through chromosomal rearrangements, whereas inactivating mutations in FMR2 are associated with fragile site, chromosome Xq28 mental retardation (2). All proteins share a significant degree of homology over a number of regions, and it is hypothesized that they act as transcription factors; it has been demonstrated that the N-terminal region of the ALF family can activate transcription in an in vitro reporter system and that LAF4 has the ability to bind DNA nonspecifically (2). To give new insight into the understanding of the poorly known function of AF4, we will look for AF4 binding protein partners, in HEK293 cells. A yeast two-hybrid screen for AF4 interactors in HEK293 cells identified members of the ubiquitin (Ub)-proteasome pathway, Drosophila seven in absentia (sina) homologues SIAH-1 and SIAH-2 (3, 4). The Ub-proteasome pathway is an important mechanism for the regulation of protein stability. In HEK293T cells, SIAH-1 promotes ubiquitination of AF4 and therefore its degradation, whereas the normal proteasomal degradation of AF4-MLL gene was blocked, suggesting that SIAH proteins may in part regulate the oncogenic potential of the fusion protein (3). Therefore, identification of intracellular AF4 interactors would provide important insights into the partner gene function: the existence in a common macromolecular complex may be the function shared by a significant number of the gene products of MLL fusion partners (5).