Conditional inhibition of erythroid differentiation by c-Myb/oestrogen receptor fusion proteins

Abstract

The c- myb proto-oncogene encodes a transcription factor that has been implicated in the regulation of haemopoietic cell differentiation and appears also to be required for cell proliferation in a number of different lineages. Typically, transcription of c- myb is down-regulated during haemopoietic cell differentiation, and it has been found in several erythroid and myeloid cell lines that constitutive c- myb expression, from a transfected plasmid, blocks this differentiation process. To investigate further the activity of c- myb in haemopoietic cell differentiation, we have transfected Friend murine erythroleukaemia (F-MEL) cells with plasmids encoding conditionally active c-Myb/oestrogen receptor (Myb/ER) fusion proteins. Transcriptional activity of the Myb/ER fusion proteins was found to be strictly hormone-dependent, and this property was correlated with the ability of these proteins to inhibit erythroid differentiation. From analysis of a Myb/ER protein that lacks the c-Myb transactivation domain, it was apparent that the C-terminal ER transactivation domain could substitute for that of c-Myb in inhibition of differentiation. Activation of Myb/ER in F-MEL cells had no effect upon the early and transient inhibition of entry into S phase associated with dimethyl sulfoxide (DMSO) induction. Further analyses of α-globin and PU.1 gene transcription suggested that c-Myb is unable to influence gene expression immediately following DMSO-induction and that inhibition of F-MEL cell differentiation must therefore result from the function of c-Myb in the post-commitment period. Nonetheless, c-Myb had effects on the erythroid differentiation programme that were clearly dissociated from its role in cell proliferation. The potential use of this system to identify c-Myb target genes involved in F-MEL cell differentiation is discussed.