Functional study of MLL PHD finger domain in MLL-ENL induced cellular immortalization

The MLL gene encodes a protein with multiple domains including PHD fingers and SET domain that are conserved in the related Drosophila protein trithorax. Chromosomal translocations involving the MLL locus result in novel fusion proteins that induce leukemia. The PHD finger domain is absent in all fusion proteins and has been shown to interact with Cyp33, a cyclophilin that participates in the repression of HOXC8 gene in a MLL dependent manner. Cyp33 also enhances the recruitment of HDACs to the MLL repression domain. This study shows that when the third PHD finger of MLL is introduced into one of the MLL fusion, MLL-ENL, it allows the recruitment of Cyp33 as well as HDAC1 to the fusion protein. Furthermore, the fusion protein with PHD finger insertion mediates the down-regulation of HOXC8 gene when Cyp33 is induced to overexpress in 293 cells. Finally, the addition of the PHD finger domain into MLL-ENL attenuates the oncogenic capacity of the fusion protein in mouse bone marrow progenitor cells (BMPCs). These data support the hypothesis that the repression domain, Cyp33, and the PHD finger form a strong repression unit that opposes the intrinsic trans-activation potential of the wild type MLL. In the immortalizing MLL-ENL fusion protein, this balance is broken due to the loss of the PHD fingers, which makes MLL-ENL a constitutive trans-activator. The recruitment of Cyp33 by the PHD fingers is necessary for the repression of HOX genes by MLL, and the loss of this repression leads to the progenitor cell immortalization by MLL fusion proteins. The role of MLL repression domain alone in HOX gene regulation and MLL-ENL associated cellular immortalization is also investigated.; In addition, the close relationship between Cyp33, MLL and Hox genes involved in hematopoiesis and leukemogenesis led me to further explore the function of Cyp33 in primary hematopoietic cells. Cyp33 extended the proliferation potential of BMPCs in colony assay. The expression of certain Hox genes and cell cycle regulatory genes was altered in Cyp33-overexpressing progenitor cells as well as the established Cyp33-BMPC cell line. The cell line proliferated and generated colonies in a SCF and IL-3 dependent manner.