Construction of inducible cell lines for the transcription factor c-Myb and its viral counterpart v-Myb : Model systems for the identification of target genes

Abstract

The hematopoietic transcription factor c-Myb has been reported to be involved in diverse and important cellular functions such as differentiation, proliferation and apoptosis. It has also been implicated in tumorigenesis, and its viral counterpart v-Myb has been known to cause acute myelogenous leukemia in chickens. The biological mechanisms behind this oncogenic activation of c-Myb are not very well understood, and the list of target genes proposed for this transcription factor is at present too short to properly explain its biological function.

In an effort to reveal some of the secrets of this elusive transcription factor we have established a new model system based on stable transfection of a human cell line with plasmids encoding three different versions of c-Myb: Full-length human c-Myb, a C-terminally truncated version of human c-Myb designated c-Myb1-443 and v-MybAMV. By use of the Tet-On and Tet-Off gene regulation systems for the generation of these stably transfected cell lines the expression of c-Myb could be induced in a Dox-dependent fashion, allowing for superior reproducibility and control of gene expression compared to what can be obtained in model systems based on transient transfections.

We succeeded in generating two Tet-On HEK293 cell lines with inducible expression of c-Myb and v-MybAMV. These cell lines were employed in microarray experiments for the determination of potential target genes for these transcription factors. The preliminary results obtained from these experiments were in keeping with observations from other recent studies regarding microarray analyses of c-Myb: The activity of this transcription factor appears to be exceptionally context-specific, indicating that c-Myb is entirely dependent on the correct composition of cooperating factors and post-translational modifications in order to be able to fully exert its potential as a transcriptional activator. In addition it would appear that the truncations and mutations found in v-MybAMV result in a transcription factor whose activity differs from its cellular counterpart both in a quantitative and qualitative manner.