c-Myb Functions as a Co-Activator of MLL1 through Menin, and More Globally Affects Histone Methylation.

Abstract The c-myb gene encodes a transcriptionally active protein, c-Myb, which has been demonstrated to play a critical role in both normal and malignant hematopoietic cell growth and development. c-Myb has long been postulated to play a causative role in leukemogenesis but the responsible mechanism(s) have yet to be identified. To develop a mechanistic based understanding of c-Myb function in leukemic vs non-leukemic cells, we expressed epitope-tagged c-Myb in transiently and stably transfected K562 human leukemia cells, and for comparison purposes, in transiently transfected 293T cells as well. We then carried out a series of immunoprecipitation experiments to examine proteins that c-Myb might interact with in each environment. In accord with prior reports, we found that c-Myb is associated with the tumor suppressor protein menin, a product of the MEN1 gene that is mutated in familial multiple endocrine neoplasia type 1. Menin has been reported to associate with a Trithorax family histone methyltransferase (HMT) complex, and to be an essential oncogenic cofactor for MLL1-associated leukemogenesis. Histone methylation, an epigenetic tag, is a form of chromatin modification that is known to play a fundamental role in the regulation of gene expression by helping to designate transcriptionally active from silent domains. Stable interaction between c-Myb and menin was confirmed by reciprocal immunoprecipitation in co-transfected 293T cells and untransfected K562 human leukemia cells. Of interest, menin mutants K119Del and D418N which are associated with familial multiple endocrine neoplasia type 1 formed even stronger complexes with c-Myb suggesting a possible role for this complex in endocrine neoplasms as well. We then examined whether c-Myb is associated with components of the MLL1 HMT complex. Again, using an immunoprecipitation strategy we found that c-Myb is associated with MLL1, WDR5, RbBp5, and Ash2L, which are previously identified members of a multi-protein MLL-SET1 histone H3 lysine (K) 4 methyltransferase complex. Moreover, by examining the association between in vitro-translated proteins we found that c-Myb forms a complex with MLL1 through menin. c-Myb-associated methyltransferase complexes, isolated from K562 human leukemia cells expressing epitope-tagged c-Myb, as well as untransfected K562 cells, were able to methylate histone H3 K4. Knocking down c-Myb in K562 cells using small interfering RNAs not only decreased expression of previously reported MLL1 target genes HoxA9 and Meis1 but led to a global decrease in H3 K4 methylation by (∼30–40%), suggesting that c-Myb might function generically a co-activator for H3K4 methyltransferases. In aggregate, these results suggest that in addition to regulating gene transcription by binding to DNA, c-Myb may also regulate transcription via a new paradigm, that of covalent histone modification. We hypothesize that in the case of MLL1, this interaction contributes to leukemic transformation of hematopoietic cells, and may more broadly contribute to malignant transformation in those tumors in which c-Myb has been reported to be aberrantly, or overexpressed.