c-Myb-Interacting Proteins and Their Functions.

Post-translational modifications of chromatin have fundamental roles in many biological processes including transcriptional regulation, heterochromatin organization, and x-chromosome inactivation. One of these modifications, histone methylation, is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive domains. It is possible that methylation induces alterations in chromatin architecture, either condensing or relaxing its structure. These alterations not only regulate accessibility for DNA binding proteins, but also create binding sites for regulatory proteins that contain specialized binding domains. The c-Myb gene is the normal cellular counterpart of an oncogene transduced by the avian myeloblastosis virus. It is a nuclear protein that functions as a DNA binding transcription factor and a number of complementary gene silencing strategies have demonstrated that it plays a pivotal role during hematopoietic cell development and is an obligate requirement for adult hematopoiesis. c-Myb is highly expressed in immature hematopoietic cells and down-regulated upon differentiation. It has also been shown that c-Myb expression is important in promoting the proliferation of leukemic cells.

However, the biologically relevant molecular mechanisms that regulate c-Myb activity during normal and leukemic hematopoiesis remain unclear. In order to investigate c-Myb function in leukemic vs non-leukemic cells, and ultimately in normal cells, more mechanistically, we expressed epitope-tagged c-Myb in transiently transfected 293T cells, and both transiently and stably transfected K562 human leukemia cells. We then carried out a series of immunoprecipitation experiments to examine proteins that c-Myb might interact with in both environments. 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 complex, and to be an essential oncogenic cofactor for MLL-associated leukemogenesis. Stable interaction between c-Myb and menin was confirmed by reciprocal immunoprecipitation in co-transfected 293T cells and retrovirus infected K562 human leukemia cells. We also examined whether c-Myb is associated with the histone methyltransferase complex in transfected 293T cells. Again, using an immunoprecipitation strategy we found that c-Myb is associated with both WDR5 and RbBp5, which are common components of multiple H3 K4 methyltransferase complexes. A Luciferase reporter assay revealed that transfection of 100 ng of WDR5 with 200 ng of c-Myb resulted in 2-fold activation of the reporter, as compared to signal obtained from cells transfected with 200 ng of c-Myb cDNA alone. Furthermore, c-Myb-associated methyltransferase complexes, isolated from c-Myb transfected 293T cells, K562 human leukemia cells expressing epitope-tagged c-Myb, as well as untransfected K562 cells, were able to methylate histone H3. 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.