PRMT4 and Mi2 are direct transcriptional regulators of c-Myb target genes in human haematopoietic cells.

<p>A–D: PRMT4 and Mi2 regulate overlapping c-Myb target genes in K562 cells. Knockdown of c-Myb, PRMT4, Mi2α and Mi2β was achieved in K562 by electroporation of the corresponding siRNAs. Cells were harvested 3 days later and protein extracts and total RNA were prepared. (A) For estimation of knockdown efficiency, protein levels of c-Myb, PRMT4 and as loading control β-Tubulin were measured by Western Blot analysis. (C) For detection of knockdown efficiency of <i>Mi2α</i> and <i>Mi2β</i> at mRNA levels, RT-qPCR was conducted using gene-specific primers for <i>Mi2α</i> and <i>Mi2β</i> and normalised to <i>GAPDH</i>. Transcript levels in si ctrl-treated cells were set to 1. (B, D) Transcript levels of the c-Myb target genes <i>Cdc7</i>, <i>c-Myc</i>, <i>Gata3</i> and <i>CyclinB1 (CycB1)</i> were measured in the various knockdown conditions by RT-qPCR and normalised to <i>GAPDH</i>. Transcript levels in si ctrl-treated cells were set to 1. E–G: Concomitant recruitment of c-Myb, PRMT4 and Mi2 to c-Myb target genes in human haematopoietic cells. K562 cells were harvested and subjected to ChIP analysis using control antibodies (IgG) and antibodies against c-Myb, PRMT4 and Mi2. Immunoprecipitated DNA was analysed by qPCR with primers for (E) <i>Cdc7</i> promoter, corresponding control region and <i>β-Tubulin</i> promoter, for (F) <i>c-Myc</i> promoter and corresponding control region and for (G) <i>Cyclin B1 (CycB1)</i> promoter and corresponding control region. Recruitment is displayed in % input of chromatin.