The MLL PHD Fingers of MLL Block MLL Fusion Protein Mediated Transformation.

Abstract

Mixed Lineage Leukemia (MLL) is a histone H3K4 methyltransferase that is rearranged in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). MLL is required for the maintenance of Hox gene expression. Deregulation of Hox genes by MLL fusion proteins, which fuse MLL in frame to one of over 50 different translocation partners, is critical for transformation. In these translocations, the DNMT homology (CXXC) domain is always included, but the set of adjacent plant homeodomains (PHD), which includes four PHD fingers and a bromodomain, is invariably excluded. PHD fingers have recently been described to bind tri-methylated histone H3K4 and others report PHD domains binding transcriptional co-repressors, such as Mi-2a of the NuRD complex. However, the role of the PHD fingers in MLL is not well understood. To determine the function of the PHD fingers in MLL, we performed bone marrow transduction and colony assays with the MLL fusion protein MLL-AF9, engineered to contain the PHD domain region (MLL-PHD-AF9). These experiments showed that inclusion of the PHD fingers inhibited immortalization as shown by the absence of compact colonies in methylcellulose replating assays and inhibition of proliferation in liquid cultures. Initial experiments with PHD finger deletions to map the inhibiting activity suggest inclusion of any PHD fingers beyond the first PHD finger, results in inhibition of transformation. To monitor the transcriptional activity of the retrovirally infected bone marrow cells, total RNA was isolated from cells harvested after the second replating, when significant differences were seen in colony morphology and size. Consistent with the transformation inhibition, Hoxa9 gene expression was found to be significantly repressed with respect to expression detected in transformed MLL-AF9 cells as determined by qPCR. To confirm this effect is directly due to the MLL fusion proteins, we performed luciferase assays with an MLL responsive myc E-box luciferase construct in MLL −/− MEFs. We found a specific and robust activation of the reporter in the presence of MLL-AF9, which was severely compromised by the inclusion of the PHD fingers. Together, these results suggest the PHD fingers act as transcriptional repressors that inhibit transformation. Our results provide an explanation for the finding that translocations including the coding region for C terminal PHD fingers do not occur in human leukemias and suggest that this region is also involved in the regulation of wild type MLL. We are currently studying the mechanisms of transcriptional repression mediated by the PHD fingers by isolating interacting proteins and assessing their effect on transcription and transformation.