DPY30 KD impaired the proliferation of human HPCs. Human CD34⁺ cells were depleted of DPY30 by 2 different shRNAs (#2 and #5). (A) The KD efficiency was determined by qRT-PCR and normalized against ACTB. Averages ± standard deviation (SD) from 5 independent biological repeats (infections) are plotted. (B) Effect of DPY30 KD (short hairpin [sh]#2 and #5) on global H3K4 methylation level in cells from 2 different sources was determined by western blot analysis. H3K4me1, H3K4me2, and H3K4me3: H3K4 mono-, di-, and tri-methylation, respectively. (C) Various types of myeloid colonies were identified and quantified after the colony-forming–cell assay using control or DPY30-KD cells. Averages ± SD from 3 assays for 1 of 4 independent biological repeats are plotted (the rest are shown in supplemental Figure 2A). P < .01 (Student t test) between the control and either DPY30 shRNAs for all types of colonies. (D) Proliferation of CD34⁺-gated cells was monitored by BrdU incorporation after culture in the presence of BrdUTP for 12 hours, and the percentage of BrdU-positive cells was quantified from 3 independent biological repeats. *P < .05 (Student t test). (E) Global gene expression was examined by microarray, followed by gene ontology analysis on the genes downregulated by both shRNAs to below three-fourths of the levels in control cells (listed in supplemental Table 3). (F) Expression of selected genes from microarray assays was examined by qRT-PCR against POLR2A. Two different sets of primers were used for MYC. Averages ± SD from 4 independent biological repeats are plotted. P < .05 (Student t test) between control and either DPY30 shRNAs for all genes. (G) Chromatin immunoprecipitation assays for total H3, H3K4me3, and DPY30 binding at the transcription start sites of a negative control gene (OR2J3) and those genes examined in panel F in human CD34⁺ cells. Averages ± SD from triplicate assays are plotted.