Defining the Myb Transcriptional Program Controlled by via Genome-wide Chromatin Occupancy and Expression Analyses

We used the ERMYB cell line (Hogg et al., Oncogene 15, p2885-2898, 1997) as our model system to identify genes regulated by Myb using whole genome microarray expression profiling. ERMYB is a myeloid progenitor cell line derived by transformation of primary cells by an activated form of Myb (CT3-Myb) fused to the ligand binding domain of ERα. In these cells, activation of the ER-Myb fusion protein by estrogen is required to maintain a proliferative progenitor-like phenotype. The cells undergo monocytic differentiation when Myb is inactivated by withdrawal of β-estradiol (β-E2). However, re-induction of Myb within 24 hours can almost fully reverse the differentiation process. This feature enabled us to employ a novel kinetic expression profiling strategy, in that we withdrew β-E2 to inactivate Myb for 24 h, then re-added it for another 6 h and monitored the gene expression across this time course. We reasoned that this strategy may be more likely to identify true Myb target genes than conventional approaches in which differential expression is examined only at the endpoint of induction. To exclude genes that might be responsive to ERα and not Myb we used the parent CT3-Myb cell line and measured the expression changes 6hrs after β-E2 withdraw and excluded those few gene that were differentially expressed. Triplicate total RNA samples from ERMYB cells were collected at 0, 6, and 24 hr after β-E2 withdrawal and 6 hr after β-E2 re-addition. Samples were analyzed by Illumina Mouse Whole Genome arrays (WG6). Probe intensities were variance stabilised and normalized by the Lumi package (Du et al., 2008). Differentially expressed genes were identified by the LIMMA package (Smyth, 2004) and then grouped into different classes according to their kinetic profiles.