Alterations of the CxxC Domain Preclude Oncogenic Activation of Mixed Lineage Leukemia 2 (MLL2).

Abstract

The mixed lineage leukemia (MLL) family of histone methyltransferases has become notorious for the participation of the founding member MLL in fusion proteins that cause acute leukemia. Despite a high overall structural conservation no other MLL homolog has been found involved in leukemogenic fusion proteins. This surprising fact might be either due to a relative genomic stability of the MLL2 locus that prevents chromosomal translocations or MLL2 might be altogether incapable of contributing to oncogenic fusions because of functional constraints. Here we demonstrate the latter to be true for fusion proteins based on MLL2, the closest relative of MLL. A MLL2-ENL protein constructed in analogy to the highly leukemogenic MLL-ENL was incapable of transforming primary hematopoietic cells. Elaborate swap experiments identified the zinc binding “CxxC”- region of MLL2 and an adjacent “post-CxxC” stretch of basic amino acids as the essential determinants for the observed difference between MLL and MLL2. Surprisingly gel shift experiments indicated that the CxxC and post-CxxC domains of MLL and MLL2 conferred almost indistinguishable in vitro DNA binding properties. However, in vivo these motifs guided MLL-ENL and MLL2-ENL to a largely non-overlapping gene repertoire. Divergent nuclear localization, a distinct potency to activate a MLL model promoter in reporter assays, and measurements of homeobox gene levels in primary cells expressing MLL and MLL2 fusion proteins all argued for a separate target site selection of the two fusions. Therefore, the CxxC domain appears to be largely responsible for target finding and as a consequence this domain is a promising object for therapies aimed at MLL fusion proteins without affecting the general function of other MLL family members.