Identification of Novel MYB Target Genes

Abstract

The MYB oncogene encodes a transcription factor, Myb, which is essential for normal haemopoiesis and also for the proliferation of most acute leukaemias (reviewed in ref. 1). While a number of Myb target genes have been reported previously, these do not completely account for key elements of MYB's activity, including its pro-leukaemic and differentiation-suppressing functions. We hypothesised that this reflects the fact that previous screens may have not been sufficiently comprehensive and/or employed the most appropriate cell systems. Thus we have embarked upon a program to identify and validate Myb targets critical for these functions. Here we report results from extensive expression profiling studies using a conditionally myb-transformed myeloid cell line, ERMYB (2). We have used ~44,000-element Illumina Beadchips in conjunction with a kinetic profiling strategy that selects candidates based on rapid, statistically-significant and consistent responses to both activation and inactivation of Myb. This has resulted in the identification of a substantial number of candidate Myb-activated and -repressed genes (381 and 502, respectively). In addition, we have used this cell system to identify candidate Myb-regulated microRNAs.Inspection of the list of candidate Myb-activated genes revealed several previously-described Myb targets including myc, bcl2, gstm1 and mpo, providing additional confidence in our approach. Our focus to date has been on novel candidates that may mediate myb's ability to enhance proliferation, suppress differentiation and possibly suppress apoptosis. Q-RT-PCR was used as an initial validation step for a number of such targets; to date 13/14 genes identified by array screening have been confirmed by this method in ERMYB cells. A second approach to validation is to confirm correlation with Myb over-expression in a second cell system (FDB-1) (3). As in primary cells, enforced Myb expression can suppress differentiation and promote proliferation of these cells in the presence of GM-CSF (4).Amongst the Myb-activated genes are gfi1 and nucleostemin/gnl3, which are involved in stem-cell functions, cellular proliferation and in the case of gfi1, lineage-specific functions. Strikingly, candidate Myb-repressed genes include several important positive regulators of haemopoietic differentiation and/or negative regulators of proliferation, namely gata3, sfpi1/pu.1, cebpb, junb, klf's-3,-6 -13 and btg1. Most of these genes have evolutionarily conserved internal or proximal candidate Myb binding sites. Our progress in validating these by chromatin immunoprecipitation will be presented.Finally, we have identified a number of microRNAs that are potentially regulated by Myb. These include members of the miR-17–92 cluster and mir-146b, which appear to be activated and repressed by Myb, respectively. These have been validated by Q-PCR for both the mature miR and the precursor pri-miR transcript. Interestingly, the mir-17–92 cluster has been strongly implicated in oncogenesis and cell cycle regulation (5), while miR-146a/b may have tumour suppressor activity.