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. 2013 Mar 7;9(3):e1003343. doi: 10.1371/journal.pgen.1003343

Figure 6. PRMT4 and Mi2 are direct transcriptional regulators of c-Myb target genes in human haematopoietic cells.

Figure 6

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 Mi2α and Mi2β at mRNA levels, RT-qPCR was conducted using gene-specific primers for Mi2α and Mi2β and normalised to GAPDH. Transcript levels in si ctrl-treated cells were set to 1. (B, D) Transcript levels of the c-Myb target genes Cdc7, c-Myc, Gata3 and CyclinB1 (CycB1) were measured in the various knockdown conditions by RT-qPCR and normalised to GAPDH. 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) Cdc7 promoter, corresponding control region and β-Tubulin promoter, for (F) c-Myc promoter and corresponding control region and for (G) Cyclin B1 (CycB1) promoter and corresponding control region. Recruitment is displayed in % input of chromatin.